US20200085916A1 - Polynucleotides encoding porphobilinogen deaminase for the treatment of acute intermittent porphyria - Google Patents

Polynucleotides encoding porphobilinogen deaminase for the treatment of acute intermittent porphyria Download PDF

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US20200085916A1
US20200085916A1 US16/302,339 US201716302339A US2020085916A1 US 20200085916 A1 US20200085916 A1 US 20200085916A1 US 201716302339 A US201716302339 A US 201716302339A US 2020085916 A1 US2020085916 A1 US 2020085916A1
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pbgd
subject
hours
sequence
group
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Inventor
Paolo Martini
Stephen Hoge
Kerry Benenato
Vladimir Presnyak
Lei Jiang
Iain McFadyen
Ellalahewage Sathyajith Kumarasinghe
Antonio Fontanellas Roma
Pedro Berraondo Lopez
Matias Antonio Avila Zaragoza
Lin Tung GUEY
Staci Sabnis
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Fundacion para la Investigacion Medica Aplicada
ModernaTx Inc
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Fundacion para la Investigacion Medica Aplicada
ModernaTx Inc
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Priority to US16/302,339 priority Critical patent/US20200085916A1/en
Priority claimed from PCT/US2017/033418 external-priority patent/WO2017201346A1/en
Assigned to MODERNATX, INC. reassignment MODERNATX, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: SABNIS, Staci, JIANG, LEI, HOGE, STEPHEN, GUEY, Lin Tung, MARTINI, PAOLO, BENENATO, Kerry, KUMARASINGHE, ELLALAHEWAGE SATHYAJITH, MCFADYEN, IAIN, PRESNYAK, Vladimir
Assigned to FUNDACION PARA LA INVESTIGACION MEDICA APLICADA reassignment FUNDACION PARA LA INVESTIGACION MEDICA APLICADA ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: AVILA ZARAGOZA, MATIAS ANTONIO, BERRAONDO LOPEZ, PEDRO, FONTANELLAS ROMA, ANTONIO
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/45—Transferases (2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0033—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being non-polymeric
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/08—Plasma substitutes; Perfusion solutions; Dialytics or haemodialytics; Drugs for electrolytic or acid-base disorders, e.g. hypovolemic shock
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y205/00—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5)
    • C12Y205/01—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5) transferring alkyl or aryl groups, other than methyl groups (2.5.1)
    • C12Y205/01061—Hydroxymethylbilane synthase (2.5.1.61)

Definitions

  • AIP Acute intermittent porphyria
  • PBGD porphobilinogen deaminase
  • HMBS hydroxymethylbilane synthase
  • uroporphyrinogen I synthase uroporphyrinogen I synthase
  • PBGD's biological function is to catalyze the head to tail condensation of four porphobilinogen molecules into the linear hydroxymethylbilane.
  • PBGD peripheral blood vessel glycoprotein
  • the ubiquitous PBGD isoform 1 is 361 amino acid residues, while the erythrocyte-specific variant (PBGD isoform 2) is 344 amino acids. Id.
  • Mutations within the PBGD gene can result in the complete or partial loss of PBGD function, resulting in impaired heme production and the abnormal accumulation of aminolevulinic acid (ALA) and porphobilinogen (PBG) in cytoplasm of cells, plasma and urine. Id.
  • ALA aminolevulinic acid
  • PBG porphobilinogen
  • neurological e.g., agitation, delirium, seizures, loss of motor function, respiratory paralysis
  • gastrointestinal e.g., extreme abdominal pain, vomiting, painful urination
  • AIP has an estimate prevalence of about 5.9 per million people worldwide (Elder G et al., J. Inherit. Metab. Dis. 36:849-57 (2012)). While AIP patients from all ethnic groups have been reported, the disorder is much more prevalent in both the Dutch and Swedish populations (1 in 10,000 to 8 in 10,000). Tjensvoll K et al., Dis Markers. 19: 41-6 (2003-2004).
  • SOC Standard of Care
  • Panhematin therapy also known as “hemin” therapy.
  • the SOC therapy is based on a down-regulation of hepatic heme synthesis using heme administration.
  • heme therapy is indicated only if an acute attack of porphyria is proven by a marked increase in urine PBG.
  • there remain several unmet medical needs including ineffectiveness in chronic AIP, and short-acting efficacy (lasts only 1-2 days).
  • recurrent hyper-activation of the hepatic heme synthesis pathway can be associated with neurological and metabolic manifestations and long-term complications including chronic kidney disease and increased risk of hepatocellular carcinoma in certain AIP patients.
  • Prophylactic heme infusion can be an effective strategy in some of these patients, but it induces tolerance and its frequent application may be associated with thromboembolic disease and hepatic siderosis.
  • ERT enzyme replacement therapy
  • HMBS-gene transfer gene therapy
  • ALASI-gene expression inhibition a potential therapy based on ALASI-gene expression inhibition
  • the present invention provides mRNA therapeutics for the treatment of acute intermittent porphyria (AIP).
  • AIP acute intermittent porphyria
  • the mRNA therapeutics of the invention are particularly well-suited for the treatment of AIP as the technology provides for the intracellular delivery of mRNA encoding PBGD followed by de novo synthesis of functional PBGD protein within target cells.
  • the instant invention features the incorporation of modified nucleotides within therapeutic mRNAs to (1) minimize unwanted immune activation (e.g., the innate immune response associated with the in vivo introduction of foreign nucleic acids) and (2) optimize the translation efficiency of mRNA to protein.
  • Exemplary aspects of the invention feature a combination of nucleotide modifications to reduce the innate immune response and sequence optimization, in particular, within the open reading frame (ORF) of therapeutic mRNAs encoding PBGD to enhance protein expression.
  • ORF open reading frame
  • the mRNA therapeutic technology of the instant invention also features delivery of mRNA encoding PBGD via a lipid nanoparticle (LNP) delivery system.
  • LNP lipid nanoparticle
  • the instant invention features novel ionizable lipid-based LNPs which have improved properties when combined with mRNA encoding PBGD and administered in vivo, for example, cellular uptake, intracellular transport and/or endosomal release or endosomal escape.
  • the LNP formulations of the invention also demonstrate reduced immunogenicity associated with the in vivo administration of LNPs.
  • compositions and delivery formulations comprising a polynucleotide, e.g., a ribonucleic acid (RNA), e.g., a messenger RNA (mRNA), encoding porphobilinogen deaminase and methods for treating acute intermittent porphyria (AIP) in a subject in need thereof by administering the same.
  • a polynucleotide e.g., a ribonucleic acid (RNA), e.g., a messenger RNA (mRNA), encoding porphobilinogen deaminase and methods for treating acute intermittent porphyria (AIP) in a subject in need thereof by administering the same.
  • RNA ribonucleic acid
  • mRNA messenger RNA
  • AIP acute intermittent porphyria
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising a lipid nanoparticle encapsulated mRNA that comprises an open reading frame (ORF) encoding an porphobilinogen deaminase (PBGD) polypeptide, wherein the composition is suitable for administration to a human subject in need of treatment for acute intermittent porphyria (AIP).
  • ORF open reading frame
  • PBGD porphobilinogen deaminase
  • the present disclosure further provides a pharmaceutical composition
  • a pharmaceutical composition comprising: (a) a mRNA that comprises (i) an open reading frame (ORF) encoding an porphobilinogen deaminase (PBGD) polypeptide, wherein the ORF comprises at least one chemically modified nucleobase, sugar, backbone, or any combination thereof, (ii) an untranslated region (UTR) comprising a microRNA (miRNA) binding site; and (b) a delivery agent, wherein the pharmaceutical composition is suitable for administration to a human subject in need of treatment for acute intermittent porphyria (AIP).
  • ORF open reading frame
  • PBGD porphobilinogen deaminase
  • UTR untranslated region
  • miRNA microRNA
  • the present disclosure further provides a pharmaceutical composition
  • a pharmaceutical composition comprising an mRNA comprising an open reading frame (ORF) encoding a human porphobilinogen deaminase (PBGD) polypeptide
  • the composition when administered to a subject in need thereof as a single intravenous dose is sufficient to reduce urinary excretion of: (i) aminolevulinate acid (ALA) at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold or at least 50-fold as compared to a reference ALA excretion level (e.g., during an acute porphyria attack), for at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours post-administration, (ii) porphobilinogen (PBG) at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold or at least 50-fold as compared to a reference PBG excretion level (e.g., during an acute porphyria attack), for at
  • the present disclosure further provides a pharmaceutical composition
  • a pharmaceutical composition comprising an mRNA comprising an open reading frame (ORF) encoding a human porphobilinogen deaminase (PBGD) polypeptide
  • the composition when administered to a subject in need thereof as a single intravenous dose is sufficient to reduce urinary excretion of: (i) aminolevulinate acid (ALA) by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 98%, at least 99%, or 100% as compared to the subject's baseline level or a reference ALA excretion level (e.g., in a subject with AIP or during an acute porphyria attack), for at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours post-administration, (ii) porphobilinogen (PBG) by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least
  • the present disclosure further provides a pharmaceutical composition
  • a pharmaceutical composition comprising an mRNA comprising an open reading frame (ORF) encoding a human porphobilinogen deaminase (PBGD) polypeptide, wherein the composition when administered to a subject in need thereof as a single intravenous dose is sufficient to reduce serum levels of: (i) alanine transaminase (ALT) to at least within 10-fold, at least within 5-fold, at least within 2-fold, or at least within 1.5-fold or to within at least 50%, at least 40%, at least 30%, at least 20%, or at least 10% of a reference ALT serum level within at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours post-administration, (ii) aspartate transaminase (AST) to at least within 10-fold, at least within 5-fold, at least within 2-fold, or at least within 1.5-fold or within at least 50%, at least 40%, at least 30%, at least 20%, or at least 10% of
  • the present disclosure further provides a pharmaceutical composition
  • a pharmaceutical composition comprising an mRNA comprising an open reading frame (ORF) encoding a human porphobilinogen deaminase (PBGD) polypeptide
  • the composition when administered to a subject in need thereof as a single intravenous dose is sufficient to reduce serum levels of: (i) alanine transaminase (ALT) by at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, or at least 30% as compared to the subject's baseline level or a reference ALT serum level (e.g., in a subject with AIP or during an acute porphyria attack), within at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours post-administration, (ii) aspartate transaminase (AST) by at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, or at least 30% as compared to the
  • the present disclosure further provides a pharmaceutical composition
  • a pharmaceutical composition comprising an mRNA comprising an open reading frame (ORF) encoding a human porphobilinogen deaminase (PBGD) polypeptide, wherein the composition when administered to a subject in need thereof as a single intravenous dose is sufficient to: (i) maintain hepatic PBGD activity levels at or above a reference physiological level or at a supraphysiological level for at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours post-administration, and/or (ii) maintain hepatic PBGD activity levels at 50% or more of a reference hepatic PBGD activity level for at least 24 hours, at least 48 hours, at least 72 hours, or at least 96 hours post-administration.
  • ORF open reading frame
  • compositions disclosed herein further comprise a delivery agent.
  • the present disclosure provides a polynucleotide comprising an open reading frame (ORF) encoding a porphobilinogen deaminase (PBGD) polypeptide, wherein the uracil or thymine content of the ORF relative to the theoretical minimum uracil or thymine content of a nucleotide sequence encoding the PBGD polypeptide (% U TM or % T TM ), is between about 100% and about 150%.
  • ORF open reading frame
  • PBGD porphobilinogen deaminase
  • the % U TM or % T TM is between about 105% and about 145%, between about 105% and about 140%, between about 110% and about 140%, between about 110% and about 145%, between about 115% and about 135%, between about 105% and about 135%, between about 110% and about 135%, between about 115% and about 145%, or between about 115% and about 140%.
  • the uracil or thymine content of the ORF relative to the uracil or thymine content of the corresponding wild-type ORF is less than 100%.
  • the % U WT or % T WT is less than about 95%, less than about 90%, less than about 85%, less than 80%, less than 79%, less than 78%, less than 77%, less than 76%, less than 75%, less than 74%, or less than 73%. In some embodiments, the % U WT or % T WT is between 65% and 73%. In some embodiments, the uracil or thymine content in the ORF relative to the total nucleotide content in the ORF (% U TL or % TTL) is less than about 50%, less than about 40%, less than about 30%, or less than about 19%.
  • the % U TL or % T TL is less than about 19%. In some embodiments, the % U TL or % T TL is between about 13% and about 15%. In some embodiments, the guanine content of the ORF with respect to the theoretical maximum guanine content of a nucleotide sequence encoding the PBGD polypeptide (% G TMX ) is at least 69%, at least 70%, at least 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%. In some embodiments, the % G TMX is between about 70% and about 80%, between about 71% and about 79%, between about 71% and about 78%, or between about 71% and about 77%.
  • the cytosine content of the ORF relative to the theoretical maximum cytosine content of a nucleotide sequence encoding the PBGD polypeptide is at least 59%, at least 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%.
  • the % C TMX is between about 60% and about 80%, between about 62% and about 80%, between about 63% and about 79%, or between about 68% and about 76%.
  • the guanine and cytosine content (G/C) of the ORF relative to the theoretical maximum G/C content in a nucleotide sequence encoding the PBGD polypeptide (% G/C TMX ) is at least about 81%, at least about 85%, at least about 90%, at least about 95%, or about 100%. In some embodiments, the % G/C TMX is between about 80% and about 100%, between about 85% and about 99%, between about 90% and about 97%, or between about 91% and about 96%.
  • the G/C content in the ORF relative to the G/C content in the corresponding wild-type ORF is at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 110%, at least 115%, or at least 120%.
  • the average G/C content in the 3 rd codon position in the ORF is at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, or at least 30% higher than the average G/C content in the 3 rd codon position in the corresponding wild-type ORF.
  • the ORF has at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 9 to 33, and 89 to 117.
  • the ORF has at least 85%, 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 identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 9 to 33, and 89 to 117.
  • the ORF has at least 85%, 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 identity to the nucleic acid sequence of SEQ ID NO: 104, 112, or 114.
  • the ORF has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 104, 112, or 114. In some embodiments, the ORF comprises the nucleic acid sequence of SEQ ID NO: 104, 112, or 114.
  • the PBGD polypeptide comprises an amino acid sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to (i) the polypeptide sequence of wild type PBGD, isoform 1 (SEQ ID NO: 1), (ii) the polypeptide sequence of wild type PBGD, isoform 2 (SEQ ID NO: 3), the polypeptide sequence of wild type PBGD, isoform 3 (SEQ ID NO: 5), or the polypeptide sequence of wild type PBGD, isoform 4 (SEQ ID NO: 7), and wherein the PBGD polypeptide has porphobilinogen deaminase activity.
  • the PBGD polypeptide is a variant, derivative, or mutant having a porphobilinogen deaminase activity (e.g., the SM gain of function variant, SEQ ID NO: 152).
  • the gain-of-function mutant PBGD comprises an 1291M mutation, an N340S mutation, or a combination thereof.
  • the gain-of-function mutant PBGD comprises the polypeptide sequence of SEQ ID NO: 152.
  • the PBGD polypeptide is a PBGD fusion protein.
  • the PBGD fusion protein comprises heterologous protein moiety.
  • the heterologous protein moiety is an apolipoprotein.
  • the apolipoprotein is human apolipoprotein A1. In some embodiments, the human apolipoprotein A1 is mature human apolipoprotein A1. In some embodiments, a fusion protein comprising PBGD and mature human apolipoprotein A1 comprises the polypeptide sequence of SEQ ID NO: 154.
  • the polynucleotide sequence further comprises a nucleotide sequence encoding a transit peptide.
  • the polynucleotide is single stranded. In some embodiments, the polynucleotide is double stranded. In some embodiments, the polynucleotide is DNA. In some embodiments, the polynucleotide is RNA. In some embodiments, the polynucleotide is mRNA. In some embodiments, the polynucleotide comprises at least one chemically modified nucleobase, sugar, backbone, or any combination thereof.
  • the at least one chemically modified nucleobase is selected from the group consisting of pseudouracil ( ⁇ ), N1-methylpseudouracil (m1 ⁇ ), 2-thiouracil (s2U), 4′-thiouracil, 5-methylcytosine, 5-methyluracil, and any combination thereof.
  • the at least one chemically modified nucleobase is selected from the group consisting of pseudouracil (W), N1-methylpseudouracil (m1 ⁇ ), 1-ethylpseudouracil, 2-thiouracil (s2U), 4′-thiouracil, 5-methylcytosine, 5-methyluracil, 5-methoxyuracil, and any combination thereof.
  • the at least one chemically modified nucleobase is 5-methoxyuracil.
  • at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or 100% of the uracils are 5-methoxyuracils.
  • at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or 100% of the uracils or thymines are chemically modified.
  • At least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or 100% of the guanines are chemically modified.
  • at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or 100% of the cytosines are chemically modified.
  • At least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or 100% of the adenines are chemically modified.
  • the polynucleotide further comprises a miRNA binding site.
  • the polynucleotide comprises at least two different microRNA (miR) binding sites.
  • the microRNA is expressed in an immune cell of hematopoietic lineage or a cell that expresses TLR7 and/or TLR8 and secretes pro-inflammatory cytokines and/or chemokines, and wherein the polynucleotide (e.g., mRNA) comprises one or more modified nucleobases.
  • the polynucleotide e.g., mRNA
  • the mRNA comprises at least one first microRNA binding site of a microRNA abundant in an immune cell of hematopoietic lineage and at least one second microRNA binding site is of a microRNA abundant in endothelial cells.
  • the mRNA comprises multiple copies of a first microRNA binding site and at least one copy of a second microRNA binding site.
  • the mRNA comprises first and second microRNA binding sites of the same microRNA.
  • the microRNA binding sites are of the 3p and 5p arms of the same microRNA.
  • the microRNA binding site comprises one or more nucleotide sequences selected from Table 3 or Table 4.
  • the microRNA binding site binds to miR-126, miR-142, miR-144, miR-146, miR-150, miR-155, miR-16, miR-21, miR-223, miR-24, miR-27 or miR-26a, or any combination thereof.
  • the microRNA binding site binds to miR126-3p, miR-142-3p, miR-142-5p, or miR-155, or any combination thereof.
  • the microRNA binding site is a miR-126 binding site. In some embodiments, at least one microRNA binding site is a miR-142 binding site. In some embodiments, one microRNA binding site is a miR-126 binding site and the second microRNA binding site is for a microRNA selected from the group consisting of miR-142-3p, miR-142-5p, miR-146-3p, miR-146-5p, miR-155, miR-16, miR-21, miR-223, miR-24 and miR-27.
  • the mRNA comprises at least one miR-126-3p binding site and at least one miR-142-3p binding site. In some embodiments, the mRNA comprises at least one miR-142-3p binding site and at least one 142-5p binding site.
  • the microRNA binding sites are located in the 5′ UTR, 3′ UTR, or both the 5′ UTR and 3′ UTR of the mRNA. In some embodiments, the microRNA binding sites are located in the 3′ UTR of the mRNA. In some embodiments, the microRNA binding sites are located in the 5′ UTR of the mRNA. In some embodiments, the microRNA binding sites are located in both the 5′ UTR and 3′ UTR of the mRNA. In some embodiments, at least one microRNA binding site is located in the 3′ UTR immediately adjacent to the stop codon of the coding region of the mRNA.
  • At least one microRNA binding site is located in the 3′ UTR 70-80 bases downstream of the stop codon of the coding region of the mRNA. In some embodiments, at least one microRNA binding site is located in the 5′ UTR immediately preceding the start codon of the coding region of the mRNA. In some embodiments, at least one microRNA binding site is located in the 5′ UTR 15-20 nucleotides preceding the start codon of the coding region of the mRNA. In some embodiments, at least one microRNA binding site is located in the 5′ UTR 70-80 nucleotides preceding the start codon of the coding region of the mRNA.
  • the mRNA comprises multiple copies of the same microRNA binding site positioned immediately adjacent to each other or with a spacer of less than 5, 5-10, 10-15, or 15-20 nucleotides.
  • the mRNA comprises multiple copies of the same microRNA binding site located in the 3′ UTR, wherein the first microRNA binding site is positioned immediately adjacent to the stop codon and the second and third microRNA binding sites are positioned 30-40 bases downstream of the 3′ most residue of the first microRNA binding site.
  • the microRNA binding site comprises one or more nucleotide sequences selected from SEQ ID NO:36 and SEQ ID NO:38. In some embodiments, the miRNA binding site binds to miR-142. In some embodiments, the miRNA binding site binds to miR-142-3p or miR-142-5p. In some embodiments, the miR-142 comprises SEQ ID NO: 34.
  • the microRNA binding site comprises one or more nucleotide sequences selected from SEQ ID NO:158 and SEQ ID NO:160. In some embodiments, the miRNA binding site binds to miR-126. In some embodiments, the miRNA binding site binds to miR-126-3p or miR-126-5p. In some embodiments, the miR-126 comprises SEQ ID NO: 156.
  • the mRNA comprises a 3′ UTR comprising a microRNA binding site that binds to miR-142, miR-126, or a combination thereof.
  • the polynucleotide e.g., mRNA
  • the polynucleotide further comprises a 3′ UTR.
  • the 3′ UTR comprises a nucleic acid sequence at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a 3′UTR sequence selected from the group consisting of SEQ ID NOs: 57 to 81, 84, 149 to 151, 161 to 172, 192 to 199, or any combination thereof.
  • the miRNA binding site is located within the 3′ UTR.
  • the 3′ UTR comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 57 to 81, 84, 149 to 151, 161 to 172, 192 to 199, and any combination thereof.
  • the mRNA comprises a 3′ UTR comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 149 to 151, or any combination thereof.
  • the mRNA comprises a 3′ UTR comprising a nucleic acid sequence of SEQ ID NO: 150.
  • the mRNA comprises a 3′ UTR comprising a nucleic acid sequence of SEQ ID NO: 151.
  • the polynucleotide e.g., mRNA
  • the polynucleotide further comprises a 5′ UTR.
  • the 5′ UTR comprises a nucleic acid sequence at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to a 5′UTR sequence selected from the group consisting of SEQ ID NO: 39 to 56, 83, 189 to 191, or any combination thereof.
  • the 5′ UTR comprises a sequence selected from the group consisting of SEQ ID NO: 39 to 56, 83, 189 to 191, and any combination thereof.
  • the mRNA comprises a 5′ UTR comprising the nucleic acid sequence of SEQ ID NO: 39.
  • the polynucleotide e.g., mRNA
  • the polynucleotide further comprises a 5′ terminal cap.
  • the 5′ terminal cap comprises a Cap0, Cap1, 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.
  • the 5′ terminal cap comprises a Cap1.
  • the polynucleotide e.g., mRNA
  • the poly-A region is at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, or at least about 90 nucleotides in length.
  • the poly-A region has about 10 to about 200, about 20 to about 180, about 50 to about 160, about 70 to about 140, about 80 to about 120 nucleotides in length.
  • the polynucleotide e.g., mRNA
  • the one or more heterologous polypeptides increase a pharmacokinetic property of the PBGD polypeptide.
  • the polynucleotide upon administration to a subject, has (i) a longer plasma half-life; (ii) increased expression of a PBGD polypeptide encoded by the ORF; (iii) a lower frequency of arrested translation resulting in an expression fragment; (iv) greater structural stability; or (v) any combination thereof, relative to a corresponding polynucleotide comprising SEQ ID NO: 2, 4, 6, 8, or 153.
  • the polynucleotide encodes a PBGD polypeptide that is fused to human apolipoprotein A1 (e.g., SEQ ID NO: 155).
  • the polynucleotide e.g., mRNA, comprises (i) a 5′-terminal cap; (ii) a 5′-UTR; (iii) an ORF encoding a PBGD polypeptide; (iv) a 3′-UTR; and (v) a poly-A region.
  • the 3′-UTR comprises a miRNA binding site.
  • the polynucleotide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 118-148, for example, SEQ ID NO: 133, 141, 144, or 145.
  • the polynucleotide further comprises a 5′-terminal cap (e.g., Cap1) and a poly-A-tail region (e.g., about 100 nucleotides in length).
  • the present disclosure also provides a method of producing a polynucleotide, e.g., mRNA, of the present invention, the method comprising modifying an ORF encoding a PBGD polypeptide by substituting at least one uracil nucleobase with an adenine, guanine, or cytosine nucleobase, or by substituting at least one adenine, guanine, or cytosine nucleobase with a uracil nucleobase, wherein all the substitutions are synonymous substitutions.
  • the method further comprises replacing at least about 90%, at least about 95%, at least about 99%, or about 100% of uracils with 5-methoxyuracils.
  • the present disclosure also provides a composition
  • a composition comprising (a) a polynucleotide, e.g., mRNA, of the invention; and (b) a delivery agent.
  • the delivery agent comprises a lipidoid, a liposome, a lipoplex, a lipid nanoparticle, a polymeric compound, a peptide, a protein, a cell, a nanoparticle mimic, a nanotube, or a conjugate.
  • the delivery agent comprises a lipid nanoparticle.
  • the lipid nanoparticle comprises a lipid selected from the group consisting of 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate
  • the delivery agent comprises a compound having the Formula (I)
  • R 1 is selected from the group consisting of C 5-30 alkyl, C 5-20 alkenyl, —R*YR′′, —YR′′, and —R′′M′R′;
  • R 2 and R 3 are independently selected from the group consisting of H, C 1-14 alkyl, C 2-14 alkenyl, —R*YR′′, —YR′′, and —R*OR′′, or R 2 and R 3 , together with the atom to which they are attached, form a heterocycle or carbocycle;
  • R 4 is selected from the group consisting of a C 3-6 carbocycle, —(CH 2 ) n Q, —(CH 2 ) n CHQR,
  • each R 5 is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • each R 6 is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O) 2 —, —S—S—, an aryl group, and a heteroaryl group;
  • R 7 is selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • R 8 is selected from the group consisting of C 3-6 carbocycle and heterocycle
  • R 9 is selected from the group consisting of H, CN, NO 2 , C 1-6 alkyl, —OR, —S(O) 2 R, —S(O) 2 N(R) 2 , C 2-6 alkenyl, C 3-6 carbocycle and heterocycle;
  • each R is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • each R′ is independently selected from the group consisting of C 1-18 alkyl, C 2-18 alkenyl, —R*YR′′, —YR′′, and H;
  • each R′′ is independently selected from the group consisting of C 3-14 alkyl and C 3-14 alkenyl
  • each R* is independently selected from the group consisting of C 1-12 alkyl and C 2-12 alkenyl;
  • each Y is independently a C 3-6 carbocycle
  • each X is independently selected from the group consisting of F, Cl, Br, and I;
  • n is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13;
  • R 4 is —(CH 2 ) n Q, —(CH 2 ) n CHQR, —CHQR, or —CQ(R) 2 , then (i) Q is not —N(R) 2 when n is 1, 2, 3, 4 or 5, or (ii) Q is not 5, 6, or 7-membered heterocycloalkyl when n is 1 or 2.
  • the present disclosure also provides a composition comprising a nucleotide sequence encoding a PBGD polypeptide and a delivery agent, wherein the delivery agent comprises a compound having the Formula (I)
  • R 1 is selected from the group consisting of C 5-30 alkyl, C 5-20 alkenyl, —R*YR′′, —YR′′, and —R′′M′R′;
  • R 2 and R 3 are independently selected from the group consisting of H, C 1-14 alkyl, C 2-14 alkenyl, —R*YR′′, —YR′′, and —R*OR′′, or R 2 and R 3 , together with the atom to which they are attached, form a heterocycle or carbocycle;
  • R 4 is selected from the group consisting of a C 3-6 carbocycle, —(CH 2 ) n Q, —(CH 2 ) n CHQR, —CHQR, —CQ(R) 2 , and unsubstituted C 1-6 alkyl, where Q is selected from a carbocycle, heterocycle, —OR, —O(CH 2 ) n N(R) 2 , —C(O)OR, —OC(O)R, —CX 3 , —CX 2 H, —CXH 2 , —CN, —N(R) 2 , —C(O)N(R) 2 , —N(R)C(O)R, —N(R)S(O) 2 R, —N(R)C(O)N(R) 2 , —N(R)C(S)N(R) 2 , —N(R)R 8 , —O(CH 2 ) n OR,
  • each R 5 is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • each R 6 is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O) 2 —, —S—S—, an aryl group, and a heteroaryl group;
  • R 7 is selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • R 8 is selected from the group consisting of C 3-6 carbocycle and heterocycle
  • R 9 is selected from the group consisting of H, CN, NO 2 , C 1-6 alkyl, —OR, —S(O) 2 R, —S(O) 2 N(R) 2 , C 2-6 alkenyl, C 3-6 carbocycle and heterocycle;
  • each R is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • each R′ is independently selected from the group consisting of C 1-18 alkyl, C 2-18 alkenyl, —R*YR′′, —YR′′, and H;
  • each R′′ is independently selected from the group consisting of C 3-14 alkyl and C 3-14 alkenyl
  • each R* is independently selected from the group consisting of C 1-12 alkyl and C 2-12 alkenyl;
  • each Y is independently a C 3-6 carbocycle
  • each X is independently selected from the group consisting of F, Cl, Br, and I;
  • n is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13;
  • R 4 is —(CH 2 ) n Q, —(CH 2 ) n CHQR, —CHQR, or —CQ(R) 2 , then (i) Q is not —N(R) 2 when n is 1, 2, 3, 4 or 5, or (ii) Q is not 5, 6, or 7-membered heterocycloalkyl when n is 1 or 2.
  • the delivery agent comprises a compound having the Formula (I), or a salt or stereoisomer thereof, wherein
  • R 1 is selected from the group consisting of C 5-20 alkyl, C 5-20 alkenyl, —R*YR′′, —YR′′, and —R′′M′R′;
  • R 2 and R 3 are independently selected from the group consisting of H, C 1-14 alkyl, C 2-14 alkenyl, —R*YR′′, —YR′′, and —R*OR′′, or R 2 and R 3 , together with the atom to which they are attached, form a heterocycle or carbocycle;
  • R 4 is selected from the group consisting of a C 3-6 carbocycle, —(CH 2 ) n Q, —(CH 2 ) n CHQR,
  • n is independently selected from 1, 2, 3, 4, and 5;
  • each R 5 is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • each R 6 is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O) 2 —, an aryl group, and a heteroaryl group;
  • R 7 is selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • each R is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • each R′ is independently selected from the group consisting of C 1-18 alkyl, C 2-18 alkenyl, —R*YR′′, —YR′′, and H;
  • each R′′ is independently selected from the group consisting of C 3-14 alkyl and C 3-14 alkenyl
  • each R* is independently selected from the group consisting of C 1-12 alkyl and C 2-12 alkenyl;
  • each Y is independently a C 3-6 carbocycle
  • each X is independently selected from the group consisting of F, Cl, Br, and I;
  • n is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13;
  • R 4 is —(CH 2 ) n Q, —(CH 2 ) n CHQR, —CHQR, or —CQ(R) 2 , then (i) Q is not —N(R) 2 when n is 1, 2, 3, 4 or 5, or (ii) Q is not 5, 6, or 7-membered heterocycloalkyl when n is 1 or 2.
  • the compound is of Formula (IA):
  • l is selected from 1, 2, 3, 4, and 5;
  • n is selected from 5, 6, 7, 8, and 9;
  • M 1 is a bond or M′
  • R 4 is unsubstituted C 1-3 alkyl, or —(CH 2 ) n Q, in which n is 1, 2, 3, 4, or 5 and Q is OH, —NHC(S)N(R) 2 , —NHC(O)N(R) 2 , —N(R)C(O)R, —N(R)S(O) 2 R, —N(R)R 8 , —NHC( ⁇ NR 9 )N(R) 2 , —NHC( ⁇ CHR 9 )N(R) 2 , —OC(O)N(R) 2 , —N(R)C(O)OR, heteroaryl, or heterocycloalkyl;
  • M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —P(O)(OR′)O—, —S—S—, an aryl group, and a heteroaryl group; and
  • R 2 and R 3 are independently selected from the group consisting of H, C 1-14 alkyl, and C 2-14 alkenyl.
  • m is 5, 7, or 9.
  • the compound is of Formula (IA), or a salt or stereoisomer thereof, wherein
  • l is selected from 1, 2, 3, 4, and 5;
  • n is selected from 5, 6, 7, 8, and 9;
  • M 1 is a bond or M′
  • R 4 is unsubstituted C 1-3 alkyl, or —(CH 2 ) n Q, in which n is 1, 2, 3, 4, or 5 and Q is OH, —NHC(S)N(R) 2 , or —NHC(O)N(R) 2 ;
  • M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —P(O)(OR′)O—, an aryl group, and a heteroaryl group; and
  • R 2 and R 3 are independently selected from the group consisting of H, C 1-14 alkyl, and C 2-14 alkenyl.
  • m is 5, 7, or 9.
  • the compound is of Formula (II):
  • l is selected from 1, 2, 3, 4, and 5;
  • M 1 is a bond or M′
  • R 4 is unsubstituted C 1-3 alkyl, or —(CH 2 ) n Q, in which n is 2, 3, or 4 and Q is OH, —NHC(S)N(R) 2 , —NHC(O)N(R) 2 , —N(R)C(O)R, —N(R)S(O) 2 R, —N(R)R 8 , —NHC( ⁇ NR 9 )N(R) 2 , —NHC( ⁇ CHR 9 )N(R) 2 , —OC(O)N(R) 2 , —N(R)C(O)OR, heteroaryl, or heterocycloalkyl;
  • M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —P(O)(OR′)O—, —S—S—, an aryl group, and a heteroaryl group; and
  • R 2 and R 3 are independently selected from the group consisting of H, C 1-14 alkyl, and C 2-14 alkenyl.
  • the compound is of Formula (II), or a salt or stereoisomer thereof, wherein
  • l is selected from 1, 2, 3, 4, and 5;
  • M 1 is a bond or M′
  • R 4 is unsubstituted C 1-3 alkyl, or —(CH 2 ) n Q, in which n is 2, 3, or 4 and Q is OH, —NHC(S)N(R) 2 , or —NHC(O)N(R) 2 ;
  • M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —P(O)(OR′)O—, an aryl group, and a heteroaryl group; and
  • R 2 and R 3 are independently selected from the group consisting of H, C 1-14 alkyl, and C 2-14 alkenyl.
  • M 1 is M′.
  • M and M′ are independently —C(O)O— or —OC(O)—.
  • l is 1, 3, or 5.
  • the compound is selected from the group consisting of Compound 1 to Compound 232, salts and stereoisomers thereof, and any combination thereof.
  • the compound is selected from the group consisting of Compound 1 to Compound 147, salts and stereoisomers thereof, and any combination thereof.
  • the compound is of the Formula (IIa),
  • the compound is of the Formula (IIb),
  • the compound is of the Formula (IIc) or (IIe),
  • R 4 is as described herein. In some embodiments, R 4 is selected from —(CH 2 ) n Q and —(CH 2 ) n CHQR.
  • the compound is of the Formula (IId),
  • n is selected from 2, 3, and 4, and m, R′, R′′, and R 2 through R 6 are as described herein.
  • each of R 2 and R 3 may be independently selected from the group consisting of C 5-14 alkyl and C 5-14 alkenyl.
  • the compound is of the Formula (IId), or a salt or stereoisomer thereof,
  • R 2 and R 3 are independently selected from the group consisting of C 5-14 alkyl and C 5-14 alkenyl, n is selected from 2, 3, and 4, and R′, R′′, R 5 , R 6 and m are as defined herein.
  • R 2 is C 8 alkyl.
  • R 3 is C 5 alkyl, C 6 alkyl, C 7 alkyl, C 8 alkyl, or C 9 alkyl.
  • m is 5, 7, or 9.
  • each R 5 is H.
  • each R 6 is H.
  • the delivery agent comprises a compound having the Formula (III)
  • t 1 or 2;
  • a 1 and A 2 are each independently selected from CH or N;
  • Z is CH 2 or absent wherein when Z is CH 2 , the dashed lines (1) and (2) each represent a single bond; and when Z is absent, the dashed lines (1) and (2) are both absent;
  • R 1 , R 2 , R 3 , R 4 , and R 5 are independently selected from the group consisting of C 5-20 alkyl, C 5-20 alkenyl, —R′′MR′, —R*YR′′, —YR′′, and —R*OR′′;
  • each M is independently selected from the group consisting of —C(O)O—, —OC(O)—, —OC(O)O—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O) 2 —, an aryl group, and a heteroaryl group;
  • X 1 , X 2 , and X 3 are independently selected from the group consisting of a bond, —CH 2 —, —(CH 2 ) 2 —, —CHR—, —CHY—, —C(O)—, —C(O)O—, —OC(O)—, —C(O)—CH 2 —, —CH 2 —C(O)—, —C(O)O—CH 2 —, —OC(O)—CH 2 —, —CH 2 —C(O)O—, —CH 2 —OC(O)—, —CH(OH)—, —C(S)—, and —CH(SH)—;
  • each Y is independently a C 3-6 carbocycle
  • each R* is independently selected from the group consisting of C 1-12 alkyl and C 2-12 alkenyl;
  • each R is independently selected from the group consisting of C 1-3 alkyl and a C 3-6 carbocycle;
  • each R′ is independently selected from the group consisting of C 1-12 alkyl, C 2-12 alkenyl, and H; and each R′′ is independently selected from the group consisting of C 3-12 alkyl and C 3-12 alkenyl,
  • R 1 , R 2 , R 3 , R 4 , and R 5 is —R′′MR′.
  • the compound is of any of Formulae (IIIa1)-(IIIa6):
  • the compounds of Formula (III) or any of (IIIa1)-(IIIa6) include one or more of the following features when applicable.
  • ring A is
  • ring A is
  • ring A is
  • ring A is
  • ring A is
  • ring A is
  • Z is CH 2 .
  • Z is absent.
  • At least one of A 1 and A 2 is N.
  • each of A 1 and A 2 is N.
  • each of A 1 and A 2 is CH.
  • a 1 is N and A 2 is CH.
  • a 1 is CH and A 2 is N.
  • At least one of X 1 , X 2 , and X 3 is not —CH 2 —.
  • X 1 is not —CH 2 —.
  • at least one of X 1 , X 2 , and X 3 is —C(O)—.
  • X 2 is —C(O)—, —C(O)O—, —OC(O)—, —C(O)—CH 2 —, —CH 2 —C(O)—, —C(O)O—CH 2 —, —OC(O)—CH 2 —, —CH 2 —C(O)O—, or —CH 2 —OC(O)—.
  • X 3 is —C(O)—, —C(O)O—, —OC(O)—, —C(O)—CH 2 —, —CH 2 —C(O)—, —C(O)O—CH 2 —, —OC(O)—CH 2 —, —CH 2 —C(O)O—, or —CH 2 —OC(O)—.
  • X 3 is —CH 2 —.
  • X 3 is a bond or —(CH 2 ) 2 —.
  • R 1 and R 2 are the same. In certain embodiments, R 1 , R 2 , and R 3 are the same. In some embodiments, R 4 and R 5 are the same. In certain embodiments, R 1 , R 2 , R 3 , R 4 , and R 5 are the same.
  • At least one of R 1 , R 2 , R 3 , R 4 , and R 5 is —R′′MR′. In some embodiments, at most one of R 1 , R 2 , R 3 , R 4 , and R 5 is —R′′MR′. For example, at least one of R 1 , R 2 , and R 3 may be —R′′MR′, and/or at least one of R 4 and R 5 is —R′′MR′.
  • at least one M is —C(O)O—. In some embodiments, each M is —C(O)O—. In some embodiments, at least one M is —OC(O)—. In some embodiments, each M is —OC(O)—.
  • At least one M is —OC(O)O—. In some embodiments, each M is —OC(O)O—. In some embodiments, at least one R′′ is C 3 alkyl. In certain embodiments, each R′′ is C 3 alkyl. In some embodiments, at least one R′′ is C 5 alkyl. In certain embodiments, each R′′ is C 5 alkyl. In some embodiments, at least one R′′ is C 6 alkyl. In certain embodiments, each R′′ is C 6 alkyl. In some embodiments, at least one R′′ is C 7 alkyl. In certain embodiments, each R′′ is C 7 alkyl. In some embodiments, at least one R′ is C 5 alkyl.
  • each R′ is C 5 alkyl. In other embodiments, at least one R′ is C 1 alkyl. In certain embodiments, each R′ is C 1 alkyl. In some embodiments, at least one R′ is C 2 alkyl. In certain embodiments, each R′ is C 2 alkyl.
  • At least one of R 1 , R 2 , R 3 , R 4 , and R 5 is C 12 alkyl. In certain embodiments, each of R 1 , R 2 , R 3 , R 4 , and R 5 are C 12 alkyl.
  • the delivery agent comprises a compound having the Formula (IV)
  • a 1 and A 2 are each independently selected from CH or N and at least one of A 1 and A 2 is N;
  • Z is CH 2 or absent wherein when Z is CH 2 , the dashed lines (1) and (2) each represent a single bond; and when Z is absent, the dashed lines (1) and (2) are both absent;
  • R 1 , R 2 , R 3 , R 4 , and R 5 are independently selected from the group consisting of C 6-20 alkyl and C 6-20 alkenyl;
  • R 1 , R 2 , R 3 , R 4 , and R 5 are the same, wherein R 1 is not C 12 alkyl, C 18 alkyl, or C 18 alkenyl;
  • R 1 , R 2 , R 3 , R 4 , and R 5 is selected from C 6-20 alkenyl
  • R 1 , R 2 , R 3 , R 4 , and R 5 have a different number of carbon atoms than at least one other of R 1 , R 2 , R 3 , R 4 , and R 5 ;
  • R 1 , R 2 , and R 3 are selected from C 6-20 alkenyl, and R 4 and R 5 are selected from C 6-20 alkyl; or
  • R 1 , R 2 , and R 3 are selected from C 6-20 alkyl, and R 4 and R 5 are selected from C 6-20 alkenyl.
  • the compound is of Formula (IVa):
  • the compounds of Formula (IV) or (IVa) include one or more of the following features when applicable.
  • Z is CH 2 .
  • Z is absent.
  • At least one of A 1 and A 2 is N.
  • each of A 1 and A 2 is N.
  • each of A 1 and A 2 is CH.
  • a 1 is N and A 2 is CH.
  • a 1 is CH and A 2 is N.
  • R 1 , R 2 , R 3 , R 4 , and R 5 are the same, and are not C 12 alkyl, C 18 alkyl, or C 18 alkenyl. In some embodiments, R 1 , R 2 , R 3 , R 4 , and R 5 are the same and are C 9 alkyl or C 14 alkyl.
  • R 1 , R 2 , R 3 , R 4 , and R 5 are selected from C 6-20 alkenyl. In certain such embodiments, R 1 , R 2 , R 3 , R 4 , and R 5 have the same number of carbon atoms. In some embodiments, R 4 is selected from C 5-20 alkenyl. For example, R 4 may be C 12 alkenyl or C 18 alkenyl.
  • At least one of R 1 , R 2 , R 3 , R 4 , and R 5 have a different number of carbon atoms than at least one other of R 1 , R 2 , R 3 , R 4 , and R 5 .
  • R 1 , R 2 , and R 3 are selected from C 6-20 alkenyl, and R 4 and R 5 are selected from C 6-20 alkyl. In other embodiments, R 1 , R 2 , and R 3 are selected from C 6-20 alkyl, and R 4 and R 5 are selected from C 6-20 alkenyl. In some embodiments, R 1 , R 2 , and R 3 have the same number of carbon atoms, and/or R 4 and R 5 have the same number of carbon atoms. For example, R 1 , R 2 , and R 3 , or R 4 and R 5 , may have 6, 8, 9, 12, 14, or 18 carbon atoms.
  • R 1 , R 2 , and R 3 , or R 4 and R 5 are C 18 alkenyl (e.g., linoleyl). In some embodiments, R 1 , R 2 , and R 3 , or R 4 and R 5 , are alkyl groups including 6, 8, 9, 12, or 14 carbon atoms.
  • R 1 has a different number of carbon atoms than R 2 , R 3 , R 4 , and R 5 .
  • R 3 has a different number of carbon atoms than R 1 , R 2 , R 4 , and R 5 .
  • R 4 has a different number of carbon atoms than R 1 , R 2 , R 3 , and R 5 .
  • the delivery agent comprises a compound having the Formula (V)
  • a 3 is CH or N
  • a 4 is CH 2 or NH; and at least one of A 3 and A 4 is N or NH;
  • Z is CH 2 or absent wherein when Z is CH 2 , the dashed lines (1) and (2) each represent a single bond; and when Z is absent, the dashed lines (1) and (2) are both absent;
  • R 1 , R 2 , and R 3 are independently selected from the group consisting of C 5-20 alkyl, C 5-20 alkenyl, —R′′MR′, —R*YR′′, —YR′′, and —R*OR′′;
  • each M is independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O) 2 —, an aryl group, and a heteroaryl group;
  • X 1 and X 2 are independently selected from the group consisting of —CH 2 —, —(CH 2 ) 2 —, —CHR—, —CHY—, —C(O)—, —C(O)O—, —OC(O)—, —C(O)—CH 2 —, —CH 2 —C(O)—, —C(O)O—CH 2 —, —OC(O) —CH 2 —, —CH 2 —C(O)O—, —CH 2 —OC(O)—, —CH(OH)—, —C(S)—, and —CH(SH)—;
  • each Y is independently a C 3-6 carbocycle
  • each R* is independently selected from the group consisting of C 1-12 alkyl and C 2-12 alkenyl;
  • each R is independently selected from the group consisting of C 1-3 alkyl and a C 3-6 carbocycle;
  • each R′ is independently selected from the group consisting of C 1-12 alkyl, C 2-12 alkenyl, and H;
  • each R′′ is independently selected from the group consisting of C 3-12 alkyl and C 3-12 alkenyl.
  • the compound is of Formula (Va):
  • the compounds of Formula (V) or (Va) include one or more of the following features when applicable.
  • Z is CH 2 .
  • Z is absent.
  • At least one of A 3 and A 4 is N or NH.
  • a 3 is N and A 4 is NH.
  • a 3 is N and A 4 is CH 2 .
  • a 3 is CH and A 4 is NH.
  • At least one of X 1 and X 2 is not —CH 2 —.
  • X 1 is not —CH 2 —.
  • at least one of X 1 and X 2 is —C(O)—.
  • X 2 is —C(O)—, —C(O)O—, —OC(O)—, —C(O)—CH 2 —, —CH 2 —C(O)—, —C(O)O—CH 2 —, —OC(O)—CH 2 —, —CH 2 —C(O)O—, or —CH 2 —OC(O)—.
  • R 1 , R 2 , and R 3 are independently selected from the group consisting of C 5-20 alkyl and C 5-20 alkenyl. In some embodiments, R 1 , R 2 , and R 3 are the same. In certain embodiments, R 1 , R 2 , and R 3 are C 6 , C 9 , C 12 , or C 14 alkyl. In other embodiments, R 1 , R 2 , and R 3 are C 18 alkenyl. For example, R 1 , R 2 , and R 3 may be linoleyl.
  • the delivery agent comprises a compound having the Formula (VI):
  • a 6 and A 7 are each independently selected from CH or N, wherein at least one of A 6 and A 7 is N;
  • Z is CH 2 or absent wherein when Z is CH 2 , the dashed lines (1) and (2) each represent a single bond; and when Z is absent, the dashed lines (1) and (2) are both absent;
  • X 4 and X 5 are independently selected from the group consisting of —CH 2 —, —(CH 2 ) 2 —, —CHR—, —CHY—, —C(O)—, —C(O)O—, —OC(O)—, —C(O)—CH 2 —, —CH 2 —C(O)—, —C(O)O—CH 2 —, —OC(O)—CH 2 —, —CH 2 —C(O)O—, —CH 2 —OC(O)—, —CH(OH)—, —C(S)—, and —CH(SH)—;
  • R 1 , R 2 , R 3 , R 4 , and R 5 each are independently selected from the group consisting of C 5-20 alkyl, C 5-20 alkenyl, —R′′MR′, —R*YR′′, —YR′′, and —R*OR′′;
  • each M is independently selected from the group consisting of —C(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O) 2 —, an aryl group, and a heteroaryl group;
  • each Y is independently a C 3-6 carbocycle
  • each R* is independently selected from the group consisting of C 1-12 alkyl and C 2-12 alkenyl;
  • each R is independently selected from the group consisting of C 1-3 alkyl and a C 3-6 carbocycle;
  • each R′ is independently selected from the group consisting of C 1-12 alkyl, C 2-12 alkenyl, and H;
  • each R′′ is independently selected from the group consisting of C 3-12 alkyl and C 3-12 alkenyl.
  • R 1 , R 2 , R 3 , R 4 , and R 5 each are independently selected from the group consisting of C 6-20 alkyl and C 6-20 alkenyl.
  • R 1 and R 2 are the same. In certain embodiments, R 1 , R 2 , and R 3 are the same. In some embodiments, R 4 and R 5 are the same. In certain embodiments, R 1 , R 2 , R 3 , R 4 , and R 5 are the same.
  • At least one of R 1 , R 2 , R 3 , R 4 , and R 5 is C 9-12 alkyl. In certain embodiments, each of R 1 , R 2 , R 3 , R 4 , and R 5 independently is C 9 , C 12 or C 14 alkyl. In certain embodiments, each of R 1 , R 2 , R 3 , R 4 , and R 5 is C 9 alkyl.
  • a 6 is N and A 7 is N. In some embodiments, A 6 is CH and A 7 is N.
  • X 4 is —CH 2 — and X 5 is —C(O)—. In some embodiments, X 4 and X 5 are —C(O)—.
  • At least one of X 4 and X 5 is not —CH 2 —, e.g., at least one of X 4 and X 5 is —C(O)—. In some embodiments, when A 6 is N and A 7 is N, at least one of R 1 , R 2 , R 3 , R 4 , and R 5 is —R′′MR′.
  • At least one of R 1 , R 2 , R 3 , R 4 , and R 5 is not —R′′MR′.
  • the composition disclosed herein is a nanoparticle composition.
  • the delivery agent further comprises a phospholipid.
  • the phospholipid is selected from the group consisting of
  • the delivery agent further comprises a structural lipid.
  • the structural lipid is selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and any mixtures thereof.
  • the delivery agent further comprises a PEG lipid.
  • the PEG lipid is selected from the group consisting of a PEG-modified phosphatidylethanolamnine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and any mixtures thereof.
  • the PEG lipid has the Formula:
  • the PEG lipid is Compound 428.
  • the delivery agent further comprises an ionizable lipid selected from the group consisting of
  • the delivery agent further comprises a phospholipid, a structural lipid, a PEG lipid, or any combination thereof.
  • the delivery agent comprises Compound 18, DSPC, Cholesterol, and Compound 428, e.g., with a mole ratio of about 50:10:38.5:1.5.
  • the composition is formulated for in vivo delivery. In some embodiments, the composition is formulated for intramuscular, subcutaneous, or intradermal delivery.
  • the present disclosure further provides a polynucleotide comprising an mRNA comprising: (i) a 5′ UTR, (ii) an open reading frame (ORF) encoding a human porphobilinogen deaminase (PBGD) polypeptide, wherein the ORF comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 9 to 33 and 89 to 117, and (iii) a 3′ UTR comprising a microRNA binding site selected from miR-142, miR-126, or a combination thereof, wherein the mRNA comprises at least one chemically modified nucleobase.
  • ORF open reading frame
  • PBGD human porphobilinogen deaminase
  • the present disclosure further provides a polynucleotide comprising an mRNA comprising: (i) a 5′-terminal cap; (ii) a 5′ UTR comprising a sequence selected from the group consisting of SEQ ID NO: 39 to 56, 83, 189 to 191, and any combination thereof; (iii) an open reading frame (ORF) encoding a human porphobilinogen deaminase (PBGD) polypeptide, wherein the ORF comprises a sequence selected from the group consisting of SEQ ID NOs: 9 to 33 and 89 to 117, wherein the mRNA comprises at least one chemically modified nucleobase selected from the group consisting of pseudouracil ( ⁇ ), N1-methylpseudouracil (m1 ⁇ ), 1-ethylpseudouracil, 2-thiouracil (s2U), 4′-thiouracil, 5-methylcytosine, 5-methyluracil, 5-methoxyuracil, and any combination thereof;
  • the polynucleotide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 133, 141, 144, and 145.
  • the present disclosure further provides a pharmaceutical composition
  • a pharmaceutical composition comprising the polynucleotide, e.g., an mRNA, and a delivery agent.
  • the delivery agent is a lipid nanoparticle comprising Compound 18, Compound 236, a salt or a stereoisomer thereof, or any combination thereof.
  • the polynucleotide comprising a nucleotide sequence encoding a PBGD polypeptide disclosed herein is formulated with a delivery agent comprising, e.g., a compound having the Formula (I), e.g., any of Compounds 1-232, e.g., Compound 18; a compound having the Formula (III), (IV), (V), or (VI), e.g., any of Compounds 233-342, e.g., Compound 236; or a compound having the Formula (VIII), e.g., any of Compounds 419-428, e.g., Compound 428, or any combination thereof.
  • the delivery agent comprises Compound 18, DSPC, Cholesterol, and Compound 428, e.g., with a mole ratio of about 50:10:38.5:1.5.
  • the subject is a human subject in need of treatment or prophylaxis for acute intermittent porphyria (AIP) and/or an acute porphyria attack.
  • AIP acute intermittent porphyria
  • AIP acute porphyria
  • the mRNA upon administration to the subject, has: (i) a longer plasma half-life; (ii) increased expression of a PBGD polypeptide encoded by the ORF; (iii) a lower frequency of arrested translation resulting in an expression fragment; (iv) greater structural stability; or (v) any combination thereof, relative to a corresponding mRNA having the nucleic acid sequence of SEQ ID NO: 2, 4, 6, or 8 and/or administered as naked mRNA.
  • a pharmaceutical composition or polynucleotide disclosed herein is suitable for administration as a single unit dose or a plurality of single unit doses.
  • a pharmaceutical composition or polynucleotide disclosed herein is suitable for reducing the level of one or more biomarkers of AIP in the subject.
  • a pharmaceutical composition or polynucleotide disclosed herein is for use in treating, preventing or delaying the onset of AIP signs or symptoms in the subject.
  • the signs or symptoms include pain, seizures, paralysis, neuropathy, death, or a combination thereof.
  • the present disclosure also provides a host cell comprising a polynucleotide of the invention.
  • the host cell is a eukaryotic cell.
  • the present disclosure also provides a vector comprising a polynucleotide of the invention. Also provided is a method of making a polynucleotide of the invention comprising synthesizing the polynucleotide enzymatically or chemically.
  • the present disclosure also provides a polypeptide encoded by a polynucleotide of the invention, a composition comprising a polynucleotide of the invention, a host cell comprising a polynucleotide of the invention, a vector comprising a polynucleotide of the invention, or produced by the method of making disclosed herein.
  • the present disclosure also provides a method of expressing in vivo an active PBGD polypeptide in a subject in need thereof comprising administering to the subject an effective amount of the polynucleotide of the invention, a composition comprising a polynucleotide of the invention, a host cell comprising a polynucleotide of the invention, a vector comprising a polynucleotide of the invention.
  • AIP acute intermittent porphyria
  • a method of treating acute intermittent porphyria (AIP) in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the polynucleotide of the invention, a composition comprising a polynucleotide of the invention, a host cell comprising a polynucleotide of the invention, a vector comprising a polynucleotide of the invention, wherein the administration alleviates the signs or symptoms of AIP in the subject.
  • AIP acute intermittent porphyria
  • the present disclosure also provides a method to prevent or delay the onset of AIP signs or symptoms in a subject in need thereof comprising administering to the subject a prophylactically effective amount of the polynucleotide of the invention, a composition comprising a polynucleotide of the invention, a host cell comprising a polynucleotide of the invention, a vector comprising a polynucleotide of the invention before AIP signs or symptoms manifest, wherein the administration prevents or delays the onset of AIP signs or symptoms in the subject.
  • Also provided is a method to ameliorate the signs or symptoms of AIP in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the polynucleotide of the invention, a composition comprising a polynucleotide of the invention, a host cell comprising a polynucleotide of the invention, a vector comprising a polynucleotide of the invention before AIP signs or symptoms manifest, wherein the administration ameliorates AIP signs or symptoms in the subject.
  • the present disclosure further provides a method of expressing a porphobilinogen deaminase (PBGD) polypeptide in a human subject in need thereof comprising administering to the subject an effective amount of a pharmaceutical composition or a polynucleotide, e.g., an mRNA, described herein, wherein the pharmaceutical composition or polynucleotide is suitable for administrating as a single dose or as a plurality of single unit doses to the subject.
  • PBGD porphobilinogen deaminase
  • the present disclosure further provides a method of treating, preventing or delaying the onset of acute intermittent porphyria (AIP) signs or symptoms in a human subject in need thereof comprising administering to the subject an effective amount of a pharmaceutical composition or a polynucleotide, e.g., an mRNA, described herein, wherein the administration treats, prevents or delays the onset of one or more of the signs or symptoms of AIP in the subject.
  • AIP acute intermittent porphyria
  • the present disclosure further provides a method for the treatment of acute intermittent porphyria (AIP), comprising administering to a human subject suffering from AIP a single intravenous dose of a pharmaceutical composition or a polynucleotide, e.g., an mRNA, described herein.
  • AIP acute intermittent porphyria
  • the present disclosure further provides a method of reducing an aminolevulinate acid (ALA), a porphobilinogen (PBG) and/or a porphyrin urinary excretion level in a human subject comprising administering to the subject an effective amount of a pharmaceutical composition or a polynucleotide, e.g., an mRNA, described herein, wherein the administration reduces the ALA, PBG and/or porphyrin urinary excretion level in the subject.
  • ALA aminolevulinate acid
  • PBG porphobilinogen
  • a porphyrin urinary excretion level in a human subject comprising administering to the subject an effective amount of a pharmaceutical composition or a polynucleotide, e.g., an mRNA, described herein, wherein the administration reduces the ALA, PBG and/or porphyrin urinary excretion level in the subject.
  • ALA urinary excretion level is reduced at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold or at least 50-fold as compared to a reference ALA excretion level during an acute porphyria attack, for at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours post-administration,
  • PBG urinary excretion level is reduced at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold or at least 50-fold as compared to a reference PBG excretion level during an acute porphyria attack, for at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours post-administration, and/or
  • porphyrin urinary excretion level is reduced at least at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold or at least 50-fold as compared to a reference porphyrin excretion level during an acute porphyria attack, for at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours post-administration.
  • the present disclosure further provides a method of reducing an aminolevulinate acid (ALA), a porphobilinogen (PBG) and/or a porphyrin urinary excretion level in a human subject comprising administering to the subject an effective amount of a pharmaceutical composition or a polynucleotide described herein, wherein the administration reduces the ALA, PBG and/or porphyrin urinary excretion level in the subject.
  • ALA aminolevulinate acid
  • PBG porphobilinogen
  • a porphyrin urinary excretion level in a human subject comprising administering to the subject an effective amount of a pharmaceutical composition or a polynucleotide described herein, wherein the administration reduces the ALA, PBG and/or porphyrin urinary excretion level in the subject.
  • ALA urinary excretion level is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% as compared to the subject's baseline level or a reference ALA excretion level (e.g., in a subject with AIP or during an acute porphyria attack), for at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours post-administration,
  • PBG urinary excretion level is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% as compared to the subject's baseline level or a reference PBG excretion level (e.g., in a subject with AIP or during an acute porphyria attack), for at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours post-administration, and/or
  • porphyrin urinary excretion level is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% as compared to the subject's baseline level or a reference porphyrin excretion level (e.g., in a subject with AIP or during an acute porphyria attack), for at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours post-administration.
  • a reference porphyrin excretion level e.g., in a subject with AIP or during an acute porphyria attack
  • the present disclosure further provides a method of reducing an alanine transaminase (ALT), a aspartate transaminase (AST) and/or a bilirubin serum level in a human subject comprising administering to the subject an effective amount of a pharmaceutical composition or a polynucleotide, e.g., an mRNA, described herein, wherein the administration reduces the ALT, AST and/or bilirubin serum level in the subject.
  • a pharmaceutical composition or a polynucleotide e.g., an mRNA, described herein
  • ALT serum level is reduced to at least within 10-fold, at least within 5-fold, at least within 2-fold, or at least within 1.5-fold as compared to a reference ALT serum level within at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours post-administration,
  • AST serum level is reduced to at least within 10-fold, at least within 5-fold, at least within 2-fold, or at least within 1.5-fold, as compared to a reference AST serum level, for at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours post-administration, and/or
  • bilirubin serum level is reduced to at least within 10-fold, at least within 5-fold, at least within 2-fold, or at least within 1.5 fold as compared to a reference bilirubin serum level, for at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours post-administration.
  • the present disclosure further provides a method of reducing an alanine transaminase (ALT), a aspartate transaminase (AST) and/or a bilirubin serum level in a human subject comprising administering to the subject an effective amount of a pharmaceutical composition or a polynucleotide described herein, wherein the administration reduces the ALT, AST and/or bilirubin serum level in the subject.
  • ALT alanine transaminase
  • AST aspartate transaminase
  • a bilirubin serum level in a human subject comprising administering to the subject an effective amount of a pharmaceutical composition or a polynucleotide described herein, wherein the administration reduces the ALT, AST and/or bilirubin serum level in the subject.
  • ALT serum level is reduced by at least 90%, at least 80%, at least 70%, at least 60% at least 50%, at least 40%, or at least 30% as compared to the subject's baseline level or a reference ALT serum level (e.g., in a subject with AIP or during an acute porphyria attack) within at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours post-administration,
  • AST serum level is reduced by at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, or at least 30% as compared to the subject's baseline level or a reference AST serum level (e.g., in a subject with AIP or during an acute porphyria attack), for at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours post-administration, and/or
  • bilirubin serum level is reduced by at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, or at least 30% as compared to the subject's baseline level or a reference bilirubin serum level (e.g., in a subject with AIP or during an acute porphyria attack), for at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours post-administration.
  • the PBGD activity in the subject is increased at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400 %, at least 500%, or at least 600% compared to the subject's baseline PBGD activity.
  • the PBGD activity is increased in the liver of the subject.
  • the increased PBGD activity persists for greater than 24, 36, 48, 60, 72, or 96 hours.
  • the pharmaceutical composition or polynucleotide is administered to the subject during an acute porphyria attack.
  • the level of ALA in the subject is reduced by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% compared to the subject's baseline ALA.
  • the level of ALA is reduced in one or more of the urine, plasma, serum, and/or liver of the subject.
  • the level of ALA in the subject is reduced compared to the baseline level in the subject for at least one day, at least two days, at least three days, at least four days, at least five days, at least one week, at least two weeks, at least three weeks, or at least one month.
  • the level of PBG in the subject is reduced by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% compared to the subject's baseline ALA.
  • the level of PBG is reduced in one or more of the urine, plasma, serum, and/or liver of the subject.
  • the level of PBG in the subject is reduced compared to the baseline level in the subject for at least one day, at least two days, at least three days, at least four days, at least five days, at least one week, at least two weeks, at least three weeks, or at least one month.
  • the level of porphyrin in the subject is reduced by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% compared to the subject's baseline ALA.
  • the level of porphyrin is reduced in one or more of the urine, plasma, serum, and/or liver of the subject.
  • the level of porphyrin in the subject is reduced compared to the baseline level in the subject for at least one day, at least two days, at least three days, at least four days, at least five days, at least one week, at least two weeks, at least three weeks, or at least one month.
  • the level of ALT in the subject is reduced by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% compared to the subject's baseline ALT.
  • the level of ALT is reduced in one or more of the urine, plasma, serum, and/or liver of the subject.
  • the level of ALT in the subject is reduced compared to the baseline level in the subject for at least one day, at least two days, at least three days, at least four days, at least five days, at least one week, at least two weeks, at least three weeks, or at least one month.
  • the level of AST in the subject is reduced by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% compared to the subject's baseline AST.
  • the level of AST is reduced in one or more of the urine, plasma, serum, and/or liver of the subject.
  • the level of AST in the subject is reduced compared to the baseline level in the subject for at least one day, at least two days, at least three days, at least four days, at least five days, at least one week, at least two weeks, at least three weeks, or at least one month.
  • the level of bilirubin in the subject is reduced by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% compared to the subject's baseline bilirubin.
  • the level of bilirubin is reduced in one or more of the urine, plasma, serum, and/or liver of the subject.
  • the level of bilirubin in the subject is reduced compared to the baseline level in the subject for at least one day, at least two days, at least three days, at least four days, at least five days, at least one week, at least two weeks, at least three weeks, or at least one month.
  • the AIP is clinically manifest (overt) AIP.
  • the AIP is clinically presymptomatic (latent) AIP.
  • the level the PBGD polypeptide activity level is sufficient to reduce the risk of or prevent the onset of an acute attack and/or sufficient to treat an acute attack.
  • the pharmaceutical composition or polynucleotide is administered as a single dose of less than 1.5 mg/kg, less than 1.25 mg/kg, less than 1 mg/kg, or less than 0.75 mg/kg.
  • the administration to the subject is about once a week, about once every two weeks, or about once a month.
  • the pharmaceutical composition or polynucleotide is administered intravenously.
  • FIG. 1A-D shows the protein sequence ( FIG. 1A ), table with domain features ( FIG. 1B ), graphic representation of domain structure ( FIG. 1C ), and nucleic acid sequence ( FIG. 1D ) of isoform 1 of PBGD.
  • FIG. 2A-D shows the protein sequence ( FIG. 2A ), table with domain features ( FIG. 2B ), graphic representation of domain structure ( FIG. 2C ), and nucleic acid sequence ( FIG. 2D ) of isoform 2 of PBGD.
  • FIG. 3A-D shows the protein sequence ( FIG. 3A ), table with domain features ( FIG. 3B ), graphic representation of domain structure ( FIG. 3C ), and nucleic acid sequence ( FIG. 3D ) of isoform 3 of PBGD.
  • FIG. 4A-D shows the protein sequence ( FIG. 4A ), table with domain features ( FIG. 4B ), graphic representation of domain structure ( FIG. 4C ), and nucleic acid sequence ( FIG. 4D ) of isoform 4 of PBGD.
  • FIG. 5 shows uracil (U) metrics corresponding to wild type isoform 1 of PBGD and 25 sequence optimized PBGD polynucleotides.
  • the column labeled “U content (%)” corresponds to the % U TL parameter.
  • the column labeled “U Content v. WT (%)” corresponds to % U WT .
  • the column labeled “U Content v. Theoretical Minimum (%)” corresponds to % U TM .
  • the column labeled “UU pairs v. WT (%)” corresponds to % UU WT .
  • FIG. 6 shows guanine (G) metrics corresponding to wild type isoform 1 of PBGD and 25 sequence optimized PBGD polynucleotides.
  • G Content (%) corresponds to % G TL .
  • G Content v. WT (%) corresponds to % G WT .
  • G Content v. Theoretical Maximum (%)” corresponds to % G TMX .
  • FIG. 7 shows cytosine (C) metrics corresponding to wild type isoform 1 of PBGD and 25 sequence optimized PBGD polynucleotides.
  • C Content (%) corresponds to % C TL .
  • C Content v. WT (%) corresponds to % C WT .
  • C Content v. Theoretical Maximum (%)” corresponds to % C TMX .
  • FIG. 8 shows guanine plus cytosine (G/C) metrics corresponding to wild type isoform 1 of PBGD and 25 sequence optimized PBGD polynucleotides.
  • the column labeled “G/C Content (%)” corresponds to % G/C TL .
  • the column labeled “G/C Content v. WT (%)” corresponds to % G/C WT .
  • the column labeled “G/C Content v. Theoretical Maximum (%)” corresponds to % G/C TMX .
  • FIG. 9 shows a comparison between the G/C compositional bias for codon positions 1, 2, 3 corresponding to the wild type isoform 1 of PBGD and 25 sequence optimized PBGD polynucleotides.
  • FIG. 10A-C shows the protein sequence ( FIG. 10A ), table with domain features ( FIG. 10B ), graphic representation of domain structure ( FIG. 10C ) of the I291M/N340S gain of function mutant of isoform 1 of PBGD.
  • FIG. 11 shows the nucleic acid sequence of the I291M/N340S gain of function mutant of isoform 1 of PBGD.
  • FIG. 12A-D shows the protein sequence ( FIG. 12A ), table with domain features ( FIG. 12B ), graphic representation of domain structure ( FIG. 12C ), and nucleic acid sequence ( FIG. 12D ) of a fusion construct comprising the mature form of apolipoprotein A1 (sequence without signal peptide and propeptide) and isoform 1 of PBGD.
  • FIG. 13 shows the levels of hepatic PBGD activity after IV administration of chemically modified mRNAs encoding wild type PBGD (COV1 and COV2), PBGD-SM protein variant, and ApoAI-PBGD-SM conjugate to AIP mice.
  • a single IV dose of mRNA construct at 1 nmol/kg was administered.
  • PBGD activity in liver was quantitated as pmol of uroporphyrin produced per mg of protein per hour.
  • Levels of hepatic PBGD activity observed in wild type mice and AIP mice are also shown for comparison. Data are expressed as mean ⁇ SD.
  • FIG. 14 shows the design of a pharmacodynamics study to evaluate the effects of the administration of a single IV dose of modified mRNA encoding wild type or SM variant of PBGD to AIP mice subjected to three acute porphyria attacks triggered by phenobarbital challenges. Intraperitoneal injections of phenobarbital and intravenous administration of PBGD mRNA are indicated by arrows above the time line of the study.
  • FIG. 15 shows urinary ALA excretion levels (micrograms of ALA per mg of creatinine) in AIP mice subjected to porphyria attacks caused by intraperitoneal phenobarbital challenges.
  • AIP mice were administered a single intravenous injection of a chemically modified mRNA encoding PBGD, SM variant of PBGD, or luciferase, all formulated in MC3 and at 0.5 mg/kg. Data are expressed as mean ⁇ SD.
  • FIG. 16 shows urinary PBG excretion levels (micrograms of PBG per mg of creatinine) in AIP mice subjected to porphyria attacks caused by intraperitoneal phenobarbital challenges.
  • AIP mice were administered a single intravenous injection of a chemically modified mRNA encoding PBGD, SM variant of PBGD, or luciferase, all formulated in MC3 and at 0.5 mg/kg. Data are expressed as mean ⁇ SD.
  • FIG. 17 shows pain measurements in AIP mice subjected to porphyria attacks caused by intraperitoneal phenobarbital challenges.
  • AIP mice were administered a single intravenous injection of a chemically modified mRNA encoding PBGD, SM variant of PBGD, or luciferase, all formulated in MC3 and at 0.5 mg/kg. Pain levels were measured after the first, second, and third phenobarbital challenge (pain scale was measured using the method reported in Langford et al., Nat. Methods. 7(6): 447-9 (2010); Matsumiya et al., J. Am. Assoc. Lab. Anim. Sci. 51(1): 42-9 (2012)). Data are expressed as mean ⁇ SD.
  • FIGS. 18A and 18B present assessments of peripheral neuropathy as determined by rotarod ( FIG. 18A ) and footprint measurements (gait patterns) ( FIG. 18B ) following a single IV administration of PBGD, SM variant of PBGD, or luciferase mRNA (0.5 mg/kg) according to the study design shown in FIG. 14 . Baseline measurements and measurements after each phenobarbital challenge are shown. Data are expressed as mean ⁇ SD.
  • FIGS. 19A, 19B, 19C, 19D and 19E show correction of sciatic nerve dysfunction following a single IV administration of PBGD or SM variant of PBGD mRNA (0.5 mg/kg) compared to luciferase mRNA (0.5 mg/kg) in AIP mice induced by three consecutive phenobarbital challenges according to the study design shown in FIG. 14 .
  • FIG. 19A shows sciatic nerve conduction data corresponding to AIP mice administered mRNA encoding luciferase.
  • FIG. 19B and FIG. 19C shows sciatic nerve conduction data corresponding to AIP mice administered modified mRNA encoding wild type PBGD ( FIG. 19B ) and modified mRNA encoding PBGD-SM protein variant ( FIG. 19C ).
  • FIG. 19D and FIG. 19E respectively, show latency and amplitude values from sciatic nerve conduction data corresponding to AIP mice administered mRNA encoding luciferase or modified mRNA encoding wild type PBGD or modified mRNA encoding PBGD-SM variant.
  • FIGS. 20A, 20B, 20C, 20D, and 20E show hepatic expression of PBGD protein observed after IV administration of modified mRNA encoding wild type PBGD, or an AAV-PBGD vector.
  • FIG. 20A shows hepatic PBGD protein expression after administration of AAV-PBGD.
  • FIG. 20B shows basal PBGD protein expression levels.
  • FIG. 20C , FIG. 20D and FIG. 20E shows hepatic PBGD protein levels on day 1, day 2 and day 4, respectively, after administration of modified mRNA encoding wild type PBGD.
  • FIG. 21A and FIG. 21B show urinary ALA excretion levels (micrograms of ALA per mg of creatinine) ( FIG. 21A ) and urinary PBG excretion levels (micrograms of PBG per mg of creatinine) ( FIG. 21B ) in AIP mice subjected to porphyria attacks caused by intraperitoneal phenobarbital challenges.
  • AIP mice were administered a single intravenous injection of a chemically modified mRNA encoding PBGD or mRNA encoding luciferase. mRNAs were formulated in MC3 or Compound 18 lipid nanoparticles.
  • the mRNA encoding luciferase was administered at 0.5 mg/kg.
  • the mRNA encoding PBGD was administered at 0.5 mg/kg and 0.1 mg/kg. Data are expressed as mean ⁇ SD.
  • FIG. 22A shows pain measurements in AIP mice subjected to porphyria attacks caused by intraperitoneal phenobarbital challenges. Pain levels were measured using a method reported in Langford et al., Nature Methods. 7(6): 447-9 (2010) after the first, second and third phenobarbital challenges.
  • AIP mice were administered a single intravenous injection of a chemically modified mRNA encoding PBGD or mRNA encoding luciferase. mRNAs were formulated in MC3 or Compound 18 lipid nanoparticles. The mRNA encoding luciferase was administered at 0.5 mg/kg. The mRNA encoding PBGD was administered at 0.5 mg/kg and 0.1 mg/kg. Data are expressed as mean ⁇ SD.
  • FIG. 22B shows rotarod measurements in AIP mice subjected to porphyria attacks caused by intraperitoneal phenobarbital challenges. Time spent on rotarod was measured before the phenobarbital challenge as baseline, and after the first, second and third phenobarbital challenges.
  • AIP mice were administered a single intravenous injection of a chemically modified mRNA encoding PBGD or an mRNA encoding luciferase at the beginning of the study (day 1). mRNAs were formulated in MC3 or Compound 18 lipid nanoparticles. The mRNA encoding luciferase was administered at 0.5 mg/kg. The mRNA encoding PBGD was administered at 0.5 mg/kg and 0.1 mg/kg. Data are expressed as mean ⁇ SD.
  • FIGS. 23A-B shows in vivo hepatic PBGD activity (expressed as nM uroporphyrin 1 concentration) ( FIG. 23A ) and in vivo hepatic PBGD protein expression ( FIG. 23B ) in wild type CD-1 mice administered modified mRNA constructs encoding wild type PBGD or luciferase as a control.
  • FIG. 24 shows the design of a multi-dose pharmacodynamics/dose-response study to evaluate the effects of the administration of a multiple doses of modified mRNA encoding human wild type PBGD to AIP mice subjected to three acute porphyria attacks triggered by phenobarbital challenges. Intraperitoneal injections of phenobarbital and intravenous administration of PBGD mRNA are indicated by arrows above the time line of the study.
  • the AIP mice were administered intravenous injections of a chemically modified mRNA encoding PBGD (Construct #14) formulated in Compound 18 lipid nanoparticles, which was administered at a dose of 0.5 mg/kg, 0.2 mg/kg, or 0.05 mg/kg every other week during each attack.
  • PBS and an mRNA encoding luciferase were used as controls.
  • FIG. 25 shows urinary ALA excretion levels (micrograms of ALA per mg of creatinine) in AIP mice subjected to multiple porphyria attacks induced by intraperitoneal phenobarbital challenges according to the study design shown in FIG. 24 . Data are expressed as mean ⁇ SD.
  • FIG. 26 shows urinary PBG excretion levels (micrograms of PBG per mg of creatinine) in AIP mice subjected to multiple porphyria attacks induced by intraperitoneal phenobarbital challenges according to the study design shown in FIG. 24 . Data are expressed as mean ⁇ SD.
  • FIG. 27 shows urinary porphyrin excretion levels (micrograms of porphyrin per mg of creatinine) in AIP mice subjected to multiple porphyria attacks induced by intraperitoneal phenobarbital challenges according to the study design shown in FIG. 24 . Data are expressed as mean ⁇ SD.
  • FIG. 28 shows pain measurements in AIP mice subjected to multiple porphyria attacks induced by intraperitoneal phenobarbital challenges according to the study design shown in FIG. 24 . Pain levels were measured using a method reported in Langford et al., Nature Methods, 7(6): 447-9 (2010) after the first, second and third phenobarbital challenges. Data are expressed as mean ⁇ SD. P-values obtained from repeated measures ANOVA. **p ⁇ 0.01, ***p ⁇ 0.001
  • FIG. 29 present assessments of peripheral neuropathy as determined by rotarod. Measurement at baseline and measurements after each phenobarbital challenge are shown. AIP mice were subjected to three porphyria attacks induced by intraperitoneal phenobarbital challenges according to the study design shown in FIG. 24 . The figure also shows data corresponding to untreated wild type animals (WT). Data are expressed as mean with SD. In case of the statistical analysis (see asterisks), data were log transformed prior to repeated measures ANOVA analysis to equalize variances and comparisons between baseline and marks obtained after each induction were made using Bonferroni's multiple comparisons. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001
  • FIG. 30 present assessments of peripheral neuropathy as determined by gait pattern analysis. Measurements at baseline and measurements after each phenobarbital challenge are shown. AIP mice were subjected to three porphyria attacks induced by intraperitoneal phenobarbital challenges according to the study design shown in FIG. 24 . The figure also shows data corresponding to untreated wild type animals (WT). The stride length was measured in the two hind legs of each of the animals. The bars represent mean with S.D. Data were log transformed prior to repeated measures ANOVA analysis to equalize variances and comparisons between baseline and footprint marks obtained after each induction were made using Bonferroni's multiple comparisons. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001
  • FIG. 31 present assessments of sciatic nerve dysfunction due to recurrent acute porphyria attacks in AIP mice induced by phenobarbital challenge according to the study design shown in FIG. 24 .
  • Amplitude values from sciatic nerve conduction data correspond to mice under control conditions or after administration of a modified mRNA encoding wild type human PBGD (at doses, e.g., 0.05 mg/kg, 0.2 mg/kg, and 0.5 mg/kg).
  • Control measurements correspond to: AIP mice treated with buffer, AIP mice administered mRNA encoding luciferase, untreated wild type mice, and wild type mice treated with phenobarbital. The bars represent mean with S.D. P-values were obtained from a one-way ANOVA. *p ⁇ 0.05, ***p ⁇ 0.001
  • FIGS. 32A, 32B, and 32C present assessments of serum transaminases and bilirubin levels in AIP mice according to the study design shown in FIG. 24 .
  • Each drawing presents measurements from mice under control conditions or after 3 IV administrations of modified mRNA encoding human wild type PBGD (at doses of 0.05 mg/kg, 0.2 mg/kg, and 0.5 mg/kg).
  • Control measurements correspond to: AIP mice treated with buffer, AIP mice treated with mRNA encoding luciferase, untreated wild type mice, and wild type mice treated with phenobarbital. The bars represent mean with S.D.
  • FIG. 32A shows levels of serum ALT transaminase (I.U./L) at sacrifice.
  • FIG. 32B shows levels of serum AST transaminase (I.U./L) at sacrifice.
  • FIG. 32C shows serum bilirubin levels (mg/dL).
  • FIGS. 33A, 33B, and 33C show the effect of the administration of a modified mRNA encoding wild type PBGD on AIP urine biomarker levels (ALA, PBG, and porphyrin, respectively) during a porphyria attack triggered by phenobarbital challenges.
  • AIP urine biomarker levels ALA, PBG, and porphyrin, respectively
  • FIG. 33A shows the effect on urine ALA levels
  • FIG. 33B shows the effect on urine PBG levels
  • FIG. 33C shows the effect on urine porphyrin levels.
  • FIG. 34 shows the pharmacokinetic profile of hepatic PBGD activity in WT CD-1 mice after IV administration of either a control mRNA encoding luciferase, or a modified mRNA encoding human wild type PBGD (0.5 mg/kg). The error bars represent S.D. Hepatic PBGD activity was quantitated based on uroporphyrin levels (nM). Splenic PBGD activity did not differ between mice administered luciferase (vehicle control) mRNA and hPBGD mRNA (data not shown).
  • FIG. 35A shows the decay in hepatic PBGD activity in WT CD-1 mice after IV administration of a modified mRNA encoding human wild type PBGD (0.5 mg/kg).
  • FIG. 35B shows the decay in hepatic PBGD activity in AIP mice after IV administration of a modified mRNA encoding human wild type PBGD. In both cases, the terminal t1/2 of the PBGD activity, determined by noncompartmental analysis, was 8 days.
  • FIG. 36 shows the pharmacokinetic of hepatic human PBGD protein concentration in WT CD-1 mice after IV administration of either a control mRNA encoding luciferase, or a modified mRNA encoding human wild type PBGD (0.5 mg/kg).
  • the error bars represent S.D.
  • Human PBGD protein levels in liver were quantified by LC-MS/MS using a human specific peptide (ASYPGLQFEIIAMSTTGDK; SEQ ID NO: 85).
  • FIG. 37 shows hepatic PBGD activity (measured as uroporphyrin production, nM; left axis) and hPBGD mRNA levels in liver (pg/uL; right axis) in WT CD1 mice administered a single IV bolus of human PBGD mRNA (0.5 mg/kg).
  • FIGS. 38A, 38B, 38C, 38D, 38E, and 38F show hepatic expression of PBGD protein observed in WT CD1 mice after IV administration of modified mRNA encoding human wild type PBGD (0.5 mg/kg).
  • FIGS. 38A, 38B, 38C, 38D, and 38E show hepatic PBGD protein levels at 2 hours, 6 hours, 10 hours, 16 hours, and 24 hours, respectively, after administration of modified mRNA encoding human wild type PBGD.
  • FIG. 38F shows basal PBGD protein expression levels detected by Novus anti-PBGD antibody which doesn't cross-react with endogenous mouse PBGD protein.
  • FIG. 39 shows hepatic PBGD activity levels after a single IV administration of 5-methoxyuracil comprising chemically modified mRNAs encoding wild type PBGD to AIP mice.
  • a single IV dose of mRNA construct at 0.2 mg/kg or 0.5 mg/kg was administered.
  • Levels of hepatic PBGD activity observed in wild type mice and AIP mice are also shown for comparison. Data are expressed as mean ⁇ SD.
  • FIGS. 40A and 40B shows a decrease in systolic ( FIG. 40A ) and diastolic ( FIG. 40B ) blood pressure in AIP mice administered a single IV injection of human PBGD mRNA (0.5 mg/kg).
  • a porphyric attack was induced in AIP mice by daily intraperitoneal phenobarbital injections. Blood pressure in WT and AIP mice that were not administered phenobarbital are shown as control groups.
  • FIG. 41 shows the hepatic PBGD activity 1 and 2 days after a single IV administration of human PBGD mRNA or luciferase vehicle control mRNA (0.5 mg/kg) to Sprague Dawley rats. Data are presented as mean ⁇ SD. In contrast, PBGD activity levels in spleen did not differ between treatment arms 1-2 days post-injection in these rats.
  • FIG. 42A shows hepatic expression of PBGD protein observed in Sprague Dawley rats at 24 hours after IV administration of modified mRNA encoding human wild type PBGD (1 mg/kg).
  • FIG. 42B shows basal PBGD protein expression levels detected by Novus anti-PBGD antibody which doesn't cross-react with endogenous rat PBGD protein significantly.
  • FIGS. 43A-B shows in vivo hepatic PBGD activity (expressed as expressed as pmol uroporphyrinogen/mg protein/hour) ( FIG. 43A ) and in vivo hepatic PBGD protein expression ( FIG. 43B ) in Cynomolgus macaque liver following administration of modified mRNA constructs encoding wild type PBGD.
  • the present invention provides mRNA therapeutics for the treatment of acute intermittent porphyria (AIP).
  • AIP acute intermittent porphyria
  • AIP is a genetic metabolic disorder affecting the production of heme, the oxygen-binding prosthetic group of hemoglobin.
  • AIP is caused by mutations in the HMBS gene, which codes for the enzyme porphobilinogen deaminase (PBGD).
  • PBGD porphobilinogen deaminase
  • PBGD porphobilinogen deaminase
  • PBGD porphobilinogen deaminase
  • mRNA therapeutics are particularly well-suited for the treatment of AIP as the technology provides for the intracellular delivery of mRNA encoding PBGD followed by de novo synthesis of functional PBGD protein within target cells. After delivery of mRNA to the target cells, the desired PBGD protein is expressed by the cells' own translational machinery, and hence, fully functional PBGD protein replaces the defective or missing protein.
  • nucleic acid-based therapeutics e.g., mRNA therapeutics
  • mRNA therapeutics e.g., mRNA therapeutics
  • TLRs toll-like receptors
  • ssRNA single-stranded RNA
  • RAG-1 retinoic acid-inducible gene 1
  • Immune recognition of foreign mRNAs can result in unwanted cytokine effects including interleukin-1 ⁇ (IL-1 ⁇ ) production, tumor necrosis factor- ⁇ (TNF- ⁇ ) distribution and a strong type I interferon (type I IFN) response.
  • the instant invention features the incorporation of different modified nucleotides within therapeutic mRNAs to minimize the immune activation and optimize the translation efficiency of mRNA to protein.
  • Particular aspects of the invention feature a combination of nucleotide modification to reduce the innate immune response and sequence optimization, in particular, within the open reading frame (ORF) of therapeutic mRNAs encoding PBGD to enhance protein expression.
  • ORF open reading frame
  • Certain embodiments of the mRNA therapeutic technology of the instant invention also feature delivery of mRNA encoding PBGD via a lipid nanoparticle (LNP) delivery system.
  • LNPs lipid nanoparticles
  • LNPs are an ideal platform for the safe and effective delivery of mRNAs to target cells.
  • LNPs have the unique ability to deliver nucleic acids by a mechanism involving cellular uptake, intracellular transport and endosomal release or endosomal escape.
  • the instant invention features novel ionizable lipid-based LNPs combined with mRNA encoding PBGD which have improved properties when administered in vivo.
  • novel ionizable lipid-based LNP formulations of the invention have improved properties, for example, cellular uptake, intracellular transport and/or endosomal release or endosomal escape.
  • LNPs administered by systemic route e.g., intravenous (IV) administration
  • IV intravenous
  • LNPs administered by systemic route can accelerate the clearance of subsequently injected LNPs, for example, in further administrations.
  • This phenomenon is known as accelerated blood clearance (ABC) and is a key challenge, in particular, when replacing deficient enzymes (e.g., PBGD) in a therapeutic context.
  • LNPs can be engineered to avoid immune sensing and/or recognition and can thus further avoid ABC upon subsequent or repeat dosing.
  • Exemplary aspect of the invention feature novel LNPs which have been engineered to have reduced ABC.
  • Porphobilinogen deaminase (PBGD; EC 4.3.1.8) is the third enzyme of the biosynthetic pathway leading to the production of heme. It catalyzes the synthesis of hydroxymethylbilane by stepwise condensation of 4 porphobilinogen units. Hydroxymethylbilane is then converted to uroporphyrinogen III by uroporphyrinogen III synthetase.
  • the structure of 40-42 kDa porphobilinogen deaminase which is highly conserved amongst organisms, consists of three domains. Domains 1 and 2 are structurally very similar: each consisting of five beta-sheets and three alpha helices in humans.
  • Domain 3 is positioned between the other two and has a flattened beta-sheet geometry.
  • a dipyrrole a cofactor of this enzyme consisting of two condensed porphobilinogen molecules, is covalently attached to domain 3 and extends into the active site, the cleft between domains 1 and 2.
  • Several positively charged arginine residues positioned to face the active site from domains 1 and 2, have been shown to stabilize the carboxylate functionalities on the incoming porphobilinogen as well as the growing pyrrole chain. These structural features presumably favor the formation of the final hydroxymethylbilane product.
  • Porphobilinogen deaminase usually exists in dimer units in the cytoplasm of the cell.
  • porphobilinogen deaminase The most well-known health issue involving porphobilinogen deaminase is acute intermittent porphyria (AIP), an autosomal dominant genetic disorder where insufficient hydroxymethylbilane is produced, leading to a build-up of porphobilinogen in the cytoplasm as well as elevation in ALA and PBG levels in plasma and urine. This is caused by a gene mutation that, in 90% of cases, causes decreased amounts of enzyme. However, mutations where less-active enzymes and/or different isoforms have been described. See Grandchamp et al. (1989) Nucleic Acids Res. 17:6637-49; and Astrin et al. (1994) Hum. Mut. 4:243-52.
  • AIP acute intermittent porphyria
  • the coding sequence (CDS) for wild type PBGD canonical mRNA sequence, corresponding to isoform 1, is described at the NCBI Reference Sequence database (RefSeq) under accession number NM_000109.3 (“ Homo sapiens hydroxymethylbilane synthase (HBMS), transcript variant 1, mRNA”).
  • the wild type PBGD canonical protein sequence, corresponding to isoform 1, is described at the RefSeq database under accession number NP_000181.2 (“Porphobilinogen deaminase isoform 1 [ Homo sapiens ]”).
  • the PBGD isoform 1 protein is 361 amino acids long. It is noted that the specific nucleic acid sequences encoding the reference protein sequence in the Ref Seq sequences are the coding sequence (CDS) as indicated in the respective RefSeq database entry.
  • Isoforms 2, 3, and 4 are produced by alternative splicing.
  • the RefSeq protein and mRNA sequences for isoform 2 of PBGD are NP_001019553.1 and NM_001024382.1, respectively.
  • the RefSeq protein and mRNA sequences for isoform 3 of PBGD are NP_001245137.1 and NM_001258208.1, respectively.
  • the RefSeq protein and mRNA sequences for isoform 4 of PBGD are NP_001245138.1 and NM_001258209.1, respectively.
  • Isoforms 2, 3, and 4 PBGD are encoded by the CDS disclosed in each one of the above mentioned mRNA RefSeq entries.
  • the isoform 2 polynucleotide contains an alternate, in-frame exon in the 5′ coding region and uses a downstream start codon, compared to variant 1. It encodes a PBGD isoform 2 polypeptide, which has a shorter N-terminus compared to isoform 1.
  • the PBGD isoform 2 protein is 344 amino acids long and lacks the amino acids corresponding to positions 1-17 in isoform 1.
  • the isoform 3 polynucleotide lacks an alternate in-frame exon compared to variant 1.
  • the resulting PBGD isoform 3 polypeptide has the same N- and C-termini but is shorter compared to isoform 1.
  • the PBGD isoform 3 protein is 321 amino acids long and lacks the amino acids corresponding to positions 218-257 in isoform 1.
  • the isoform 4 polynucleotide uses an alternate splice junction at the 3′ end of the first exon and lacks an alternate in-frame exon compared to variant 1.
  • the resulting PBGD isoform 4 polypeptide is shorter at the N-terminus and lacks an alternate internal segment compared to isoform 1.
  • the PBGD isoform 4 protein is 304 amino acids long and lacks the amino acids corresponding to positions 1-17 and positions 218-257 in isoform 1.
  • the invention provides a polynucleotide (e.g., a ribonucleic acid (RNA), e.g., a messenger RNA (mRNA)) comprising a nucleotide sequence (e.g., an open reading frame (ORF)) encoding a PBGD polypeptide.
  • a polynucleotide e.g., a ribonucleic acid (RNA), e.g., a messenger RNA (mRNA)
  • mRNA messenger RNA
  • ORF open reading frame
  • the PBGD polypeptide of the invention is a wild type PBGD isoform 1, 2, 3, or 4 protein.
  • the PBGD polypeptide of the invention is a variant, a peptide or a polypeptide containing a substitution, and insertion and/or an addition, a deletion and/or a covalent modification with respect to a wild-type PBGD isoform 1, 2, 3, or 4 sequence.
  • sequence tags or amino acids can be added to the sequences encoded by the polynucleotides of the invention (e.g., at the N-terminal or C-terminal ends), e.g., for localization.
  • amino acid residues located at the carboxy, amino terminal, or internal regions of a polypeptide of the invention can optionally be deleted providing for fragments.
  • the polynucleotide e.g., a RNA, e.g., an mRNA
  • a nucleotide sequence e.g., an ORF
  • the substitutional variant can comprise one or more conservative amino acids substitutions.
  • the variant is an insertional variant.
  • the variant is a deletional variant.
  • PBGD isoform 1, 2, 3, or 4 protein fragments, functional protein domains, variants, and homologous proteins (orthologs) are also considered to be within the scope of the PBGD polypeptides of the invention.
  • Nonlimiting examples of polypeptides encoded by the polynucleotides of the invention are shown in FIGS. 1 to 4 .
  • FIG. 1 shows the amino acid sequence of human PBGD wild type isoform 1.
  • compositions and methods presented in this disclosure refer to the protein or polynucleotide sequences of PBGD isoform 1. A person skilled in the art will understand that such disclosures are equally applicable to any other isoforms of PBGD known in the art.
  • the instant invention features mRNAs for use in treating (i.e., prophylactically and/or therapeutically treating) AIP.
  • the mRNAs featured for use in the invention are administered to subjects and encode human porphobilinogen deaminase (PBGD) proteins(s) in vivo.
  • PBGD porphobilinogen deaminase
  • the invention relates to polynucleotides, e.g., mRNA, comprising an open reading frame of linked nucleosides encoding human porphobilinogen deaminase (PBGD), isoforms thereof, functional fragments thereof, and fusion proteins comprising PBGD.
  • the open reading frame is sequence-optimized.
  • the invention provides sequence-optimized polynucleotides comprising nucleotides encoding the polypeptide sequence of isoforms 1, 2, 3 or 4 of human PBGD, or sequence having high sequence identity with those sequence optimized polynucleotides.
  • the invention provides polynucleotides (e.g., a RNA, e.g., an mRNA) that comprise a nucleotide sequence (e.g., an ORF) encoding one or more PBGD polypeptides.
  • a RNA e.g., an mRNA
  • a nucleotide sequence e.g., an ORF
  • the encoded PBGD polypeptide of the invention can be selected from:
  • PBGD polypeptide e.g., having the same or essentially the same length as wild-type PBGD isoform 1, 2, 3 or 4;
  • a functional fragment of any of the PBGD isoforms described herein e.g., a truncated (e.g., deletion of carboxy, amino terminal, or internal regions) sequence shorter than one of wild-type isoforms 1, 2, 3 or 4; but still retaining PBGD enzymatic activity);
  • a variant thereof e.g., full length or truncated isoform 1, 2, 3, or 4 protein in which one or more amino acids have been replaced, e.g., variants that retain all or most of the PBGD activity of the polypeptide with respect to a reference isoform (such as, e.g., T59I, D178N, or any other natural or artificial variants known in the art, or a variant comprising the I291M and N340S mutations); or
  • a fusion protein comprising (i) a full length PBGD isoform 1, 2, 3, or 4 protein, a functional fragment or a variant thereof, and (ii) at least one heterologous protein (e.g., Apolipoprotein A1).
  • the encoded PBGD polypeptide is a mammalian PBGD polypeptide, such as a human PBGD polypeptide, a functional fragment or a variant thereof.
  • the polynucleotide e.g., a RNA, e.g., an mRNA
  • PBGD protein expression levels and/or PBGD enzymatic activity can be measured according to methods know in the art.
  • the polynucleotide is introduced to the cells in vitro. In some embodiments, the polynucleotide is introduced to the cells in vivo.
  • the polynucleotides (e.g., a RNA, e.g., an mRNA) of the invention comprise a nucleotide sequence (e.g., an ORF) that encodes a wild-type human PBGD, e.g., wild-type isoform 1 of human PBGD (SEQ ID NO: 1, see FIG. 1 ), wild-type isoform 2 of human PBGD (SEQ ID NO: 3, see FIG. 2 ), wild-type isoform 3 of human PBGD (SEQ ID NO: 5, see FIG. 3 ), or wild-type isoform 4 of human PBGD (SEQ ID NO: 7, see FIG. 4 ).
  • a nucleotide sequence e.g., an ORF
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprises a sequence optimized nucleic acid sequence, wherein the open reading frame (ORF) of the sequence optimized nucleic acid sequence is derived from a wild-type PBGD sequence (e.g., wild-type isoforms 1, 2, 3 or 4).
  • ORF open reading frame
  • the corresponding wild type sequence is the native PBGD isoform 2.
  • the corresponding wild type sequence is the corresponding fragment from PBGD isoform 1.
  • the polynucleotides (e.g., a RNA, e.g., an mRNA) of the invention comprise a nucleotide sequence encoding PBGD isoform 1 having the full length sequence of human PBGD isoform 1 (i.e., including the initiator methionine).
  • the initiator methionine can be removed to yield a “mature PBGD” comprising amino acid residues of 2-361 of the translated product.
  • the teachings of the present disclosure directed to the full sequence of human PBGD are also applicable to the mature form of human PBGD lacking the initiator methionine (amino acids 2-361).
  • the polynucleotides (e.g., a RNA, e.g., an mRNA) of the invention comprise a nucleotide sequence encoding PBGD isoform 1 having the mature sequence of human PBGD isoform 1 (i.e., lacking the initiator methionine).
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprising a nucleotide sequence encoding PBGD isoform 1 having the full length or mature sequence of human PBGD isoform 1 is sequence optimized.
  • the polynucleotides e.g., a RNA, e.g., an mRNA
  • the polynucleotides of the invention comprise a nucleotide sequence (e.g., an ORF) encoding a mutant PBGD polypeptide, e.g., a double mutant I291M/N340S PBGD.
  • the protein (SEQ ID NO: 152) and polynucleotide (SEQ ID NO: 153) of double mutant I291M/N340S human PBGD isoform 1 (“SM PBGD”) are shown in FIG. 10A and FIG. 11 .
  • the polynucleotides of the invention comprise an ORF encoding a PBGD polypeptide that comprises at least one point mutation in the PBGD sequence and retains PBGD enzymatic activity.
  • the mutant PBGD polypeptide has a PBGD activity which is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% of the PBGD activity of the corresponding wild-type PBGD (i.e., the same PBGD isoform but without the mutation(s)).
  • the polynucleotide e.g., a RNA, e.g., an mRNA
  • the polynucleotide comprising an ORF encoding a mutant PBGD polypeptide
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprises a nucleotide sequence (e.g., an ORF) that encodes a PBGD polypeptide with mutations that do not alter PBGD enzymatic activity.
  • a mutant PBGD polypeptides can be referred to as function-neutral.
  • the polynucleotide comprises an ORF that encodes a mutant PBGD polypeptide comprising one or more function-neutral point mutations.
  • the mutant PBGD polypeptide has higher PBGD enzymatic activity than the corresponding wild-type PBGD. In some embodiments, the mutant PBGD polypeptide has a PBGD activity that is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% higher than the activity of the corresponding wild-type PBGD (i.e., the same PBGD isoform but without the mutation(s)).
  • the polynucleotides e.g., a RNA, e.g., an mRNA
  • the polynucleotides of the invention comprise a nucleotide sequence (e.g., an ORF) encoding a functional PBGD fragment, e.g., where one or more fragments correspond to a polypeptide subsequence of a wild type PBGD polypeptide and retain PBGD enzymatic activity.
  • the PBGD fragment has a PBGD activity which is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% of the PBGD activity of the corresponding full length PBGD.
  • the polynucleotide e.g., a RNA, e.g., an mRNA
  • the polynucleotide e.g., a RNA, e.g., an mRNA
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprises a nucleotide sequence (e.g., an ORF) encoding a PBGD fragment that has higher PBGD enzymatic activity than the corresponding full length PBGD.
  • a nucleotide sequence e.g., an ORF
  • the PBGD fragment has a PBGD activity which is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% higher than the PBGD activity of the corresponding full length PBGD.
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprises a nucleotide sequence (e.g., an ORF) encoding a PBGD fragment that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24% or 25% shorter than wild-type isoform 1, 2, 3, or 4 of PBGD.
  • a nucleotide sequence e.g., an ORF
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprises a nucleotide sequence (e.g., an ORF) encoding a PBGD polypeptide (e.g., the wild-type sequence, functional fragment, or variant thereof), wherein the nucleotide sequence is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of SEQ ID NO:2, 4, 6 or 8 (see, e.g., panel D in FIGS. 1, 2, 3 and 4 , respectively).
  • a nucleotide sequence e.g., an ORF
  • PBGD polypeptide e.g., the wild-type sequence, functional fragment, or variant thereof
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprises a nucleotide sequence (e.g., an ORF) encoding a PBGD polypeptide (e.g., the wild-type sequence, functional fragment, or variant thereof), wherein the nucleotide sequence has at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprises a nucleotide sequence (e.g., an ORF) encoding a PBGD polypeptide (e.g., the wild-type sequence, functional fragment, or variant thereof), wherein the nucleotide sequence has 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 70% to 95%, 80% to 95%, 70% to 85%, 75% to 90%, 80% to 95%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100%, sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 9 to 33, and 89 to 117. See TABLE 2.
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprises an ORF encoding a PBGD polypeptide (e.g., the wild-type sequence, functional fragment, or variant thereof), wherein the polynucleotide comprises a nucleic acid sequence having 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 70% to 95%, 80% to 95%, 70% to 85%, 75% to 90%, 80% to 95%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100%, sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 118-148. See TABLE 5.
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprises a nucleotide sequence (e.g., an ORF) encoding a PBGD polypeptide (e.g., the wild-type sequence, functional fragment, or variant thereof), wherein the nucleotide sequence is at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of SEQ ID NO:2, 4, 6 or 8 (see, e.g., panel D FIGS. 1, 2, 3 and
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprises a nucleotide sequence (e.g., an ORF) encoding a PBGD polypeptide (e.g., the wild-type sequence, functional fragment, or variant thereof), wherein the nucleotide sequence is between 70% and 90% identical; between 75% and 85% identical; between 76% and 84% identical; between 77% and 83% identical, between 77% and 82% identical, or between 78% and 81% identical to the sequence of SEQ ID NO:2, 4, 6 or 8 (see, e.g., panel D in FIGS. 1, 2, 3, and 4 , respectively).
  • a nucleotide sequence e.g., an ORF
  • PBGD polypeptide e.g., the wild-type sequence, functional fragment, or variant thereof
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprises from about 900 to about 100,000 nucleotides (e.g., from 900 to 1,000, from 900 to 1,100, from 900 to 1,200, from 900 to 1,300, from 900 to 1,400, from 900 to 1,500, from 1,000 to 1,100, from 1,000 to 1,100, from 1,000 to 1,200, from 1,000 to 1,300, from 1,000 to 1,400, from 1,000 to 1,500, from 1,083 to 1,200, from 1,083 to 1,400, from 1,083 to 1,600, from 1,083 to 1,800, from 1,083 to 2,000, from 1,083 to 3,000, from 1,083 to 5,000, from 1,083 to 7,000, from 1,083 to 10,000, from 1,083 to 25,000, from 1,083 to 50,000, from 1,083 to 70,000, or from 1,083 to 100,000).
  • nucleotides e.g., from 900 to 1,000, from 900
  • the polynucleotide of the invention (e.g., a RNA, e.g., an mRNA) comprises a nucleotide sequence (e.g., an ORF) encoding a PBGD polypeptide (e.g., the wild-type sequence, functional fragment, or variant thereof), wherein the length of the nucleotide sequence (e.g., an ORF) is at least 500 nucleotides in length (e.g., at least or greater than about 500, 600, 700, 80, 900, 1,000, 1,050, 1,083, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,100, 2,200, 2,300, 2,400, 2,500, 2,600, 2,700, 2,800, 2,900, 3,000, 3,100, 3,200, 3,300, 3,400, 3,500, 3,600, 3,700, 3,800, 3,900, 4,000, 4,100, 4,200, 4,300, 4,400, 4,
  • the polynucleotide of the invention (e.g., a RNA, e.g., an mRNA) comprises a nucleotide sequence (e.g., an ORF) encoding a PBGD polypeptide (e.g., the wild-type sequence, functional fragment, or variant thereof), and further comprises at least one nucleic acid sequence that is noncoding, e.g., a miRNA binding site.
  • a nucleotide sequence e.g., an ORF
  • PBGD polypeptide e.g., the wild-type sequence, functional fragment, or variant thereof
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention further comprises a 5′-UTR (e.g., selected from the sequences of SEQ ID NOs: 39 to 56, 83, 189 to 191) and a 3′UTR (e.g., selected from the sequences of SEQ ID NOs: 57 to 81, 84, 149 to 151, 161 to 172, 192 to 199).
  • a 5′-UTR e.g., selected from the sequences of SEQ ID NOs: 39 to 56, 83, 189 to 191
  • a 3′UTR e.g., selected from the sequences of SEQ ID NOs: 57 to 81, 84, 149 to 151, 161 to 172, 192 to 199.
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprises a sequence selected from the group consisting of SEQ ID NO: 118-148, e.g., SEQ ID NO: 133, 141, 144 or 145.
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) comprises a 5′ terminal cap (e.g., Cap0, Cap1, 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) and a poly-A-tail region (e.g., about 100 nucleotides in length).
  • a 5′ terminal cap e.g., Cap0, Cap1, ARCA, inosine, N1-methyl-guanosine, 2′-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) a comprises a 3′ UTR comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 149 to 151, or any combination thereof.
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) comprises a 3′ UTR comprising a nucleic acid sequence of SEQ ID NO: 150.
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) comprises a 3′ UTR comprising a nucleic acid sequence of SEQ ID NO: 151.
  • the polynucleotide of the invention e.g., a RNA, e.g., an mRNA
  • a nucleotide sequence e.g., an ORF
  • the polynucleotide of the invention comprising a nucleotide sequence (e.g., an ORF) encoding a PBGD polypeptide (e.g., the wild-type sequence, functional fragment, or variant thereof) is DNA or RNA.
  • the polynucleotide of the invention is RNA.
  • the polynucleotide of the invention is, or functions as, a messenger RNA (mRNA).
  • the mRNA comprises a nucleotide sequence (e.g., an ORF) that encodes at least one PBGD polypeptide, and is capable of being translated to produce the encoded PBGD polypeptide in vitro, in vivo, in situ, or ex vivo.
  • a nucleotide sequence e.g., an ORF
  • the polynucleotide of the invention (e.g., a RNA, e.g., an mRNA) comprises a sequence-optimized nucleotide sequence (e.g., an ORF) encoding a PBGD polypeptide (e.g., the wild-type sequence, functional fragment, or variant thereof), wherein the polynucleotide comprises at least one chemically modified nucleobase, e.g., 5-methoxyuracil.
  • the polynucleotide further comprises a miRNA binding site, e.g., a miRNA binding site that binds to miR-142 and/or a miRNA binding site that binds to miR-126.
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) disclosed herein is formulated with a delivery agent comprising, e.g., a compound having the Formula (I), e.g., any of Compounds 1-232, e.g., Compound 18; a compound having the Formula (III), (IV), (V), or (VI), e.g., any of Compounds 233-342, e.g., Compound 236; or a compound having the Formula (VIII), e.g., any of Compounds 419-428, e.g., Compound 428, or any combination thereof.
  • the delivery agent comprises Compound 18, DSPC, Cholesterol, and Compound 428, e.g., with a mole ratio of about 50:10:38.5:1.5.
  • the polynucleotides e.g., a RNA, e.g., an mRNA
  • One such feature that aids in protein trafficking is the signal sequence, or targeting sequence.
  • the peptides encoded by these signal sequences are known by a variety of names, including targeting peptides, transit peptides, and signal peptides.
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) comprises a nucleotide sequence (e.g., an ORF) that encodes a signal peptide operably linked to a nucleotide sequence that encodes a PBGD polypeptide described herein.
  • a nucleotide sequence e.g., an ORF
  • the “signal sequence” or “signal peptide” is a polynucleotide or polypeptide, respectively, which is from about 9 to 200 nucleotides (3-70 amino acids) in length that, optionally, is incorporated at the 5′ (or N-terminus) of the coding region or the polypeptide, respectively. Addition of these sequences results in trafficking the encoded polypeptide to a desired site, such as the endoplasmic reticulum or the mitochondria through one or more targeting pathways. Some signal peptides are cleaved from the protein, for example by a signal peptidase after the proteins are transported to the desired site.
  • the polynucleotide of the invention comprises a nucleotide sequence encoding a PBGD polypeptide, wherein the nucleotide sequence further comprises a 5′ nucleic acid sequence encoding a heterologous signal peptide.
  • the polynucleotide of the invention e.g., a RNA, e.g., an mRNA
  • polynucleotides of the invention comprise a single ORF encoding a PBGD polypeptide, a functional fragment, or a variant thereof.
  • the polynucleotide of the invention can comprise more than one ORF, for example, a first ORF encoding a PBGD polypeptide (a first polypeptide of interest), a functional fragment, or a variant thereof, and a second ORF expressing a second polypeptide of interest.
  • two or more polypeptides of interest can be genetically fused, i.e., two or more polypeptides can be encoded by the same ORF.
  • the polynucleotide can comprise a nucleic acid sequence encoding a linker (e.g., a G4S peptide linker or another linker known in the art) between two or more polypeptides of interest.
  • a polynucleotide of the invention e.g., a RNA, e.g., an mRNA
  • a polynucleotide of the invention can comprise two, three, four, or more ORFs, each expressing a polypeptide of interest.
  • the polynucleotide of the invention e.g., a RNA, e.g., an mRNA
  • a first nucleic acid sequence e.g., a first ORF
  • a second nucleic acid sequence e.g., a second ORF
  • the polynucleotide of the invention comprises a nucleic acid encoding a PBGD fusion protein, wherein said fusion protein comprises an apolipoprotein A1 fused to PBGD.
  • the apolipoprotein A1 (ApoA1) fusion component is a mature form of human apolipoprotein A1 without the native signal peptide and propeptide sequence. See FIGS. 12A-D .
  • the ApoA1-PBGD fusion protein comprises, consists, or consists essentially of the sequence of SEQ ID NO: 154 (see FIG. 12A ).
  • the polypeptide of the invention is encoded by a nucleic acid sequence encoding an ApoA1-PBGD fusion protein, wherein said nucleic acid comprises, consists, or consists essentially of the sequence of SEQ ID NO: 155 (see FIG. 12D ).
  • a polynucleotide of the invention can comprise a portion encoding PBGD (e.g., a wild type PBGD or a variant such as the SM gain of function variant), and, in some embodiments, an ApoA1 component.
  • PBGD e.g., a wild type PBGD or a variant such as the SM gain of function variant
  • ApoA1 component e.g., an ApoA1 component
  • the polynucleotides of the invention can comprise, for example, polynucleotides encoding (i) human PBGD isoform 1 (human housekeeping PBGD), (ii) human PBGD isoform 2 (human erythroid-specific PBGD), (iii) SM variant (I291M and N340S double mutant) of PBGD1, (iv) SM variant of PBGD2, (v) human apolipoprotein A 1 fused to human PBGD1, (vi) human apolipoprotein A1 fused to PBGD2, (vii) human apolipoprotein A1 fused to SM variant of PBGD1, (viii) human apolipoprotein A1 fused to SM variant of PBGD2, or (ix) combinations thereof.
  • the polynucleotides have been sequence optimized (e.g., see TABLE 2 and the sequences disclosed in International Publication WO2010/0361
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention is sequence optimized.
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention comprises a nucleotide sequence (e.g., an ORF) encoding a PBGD polypeptide, a nucleotide sequence (e.g., an ORF) encoding another polypeptide of interest, a 5′-UTR, a 3′-UTR, a miRNA, a nucleotide sequence encoding a linker, or any combination thereof, that is sequence optimized.
  • a sequence optimized nucleotide sequence e.g., a codon optimized mRNA sequence encoding a PBGD polypeptide, is a sequence comprising at least one synonymous nucleobase substitution with respect to a reference sequence (e.g., a wild type nucleotide sequence encoding a PBGD polypeptide).
  • a sequence optimized nucleotide sequence can be partially or completely different in sequence from the reference sequence.
  • a reference sequence encoding polyserine uniformly encoded by TCT codons can be sequence optimized by having 100% of its nucleobases substituted (for each codon, T in position 1 replaced by A, C in position 2 replaced by G, and T in position 3 replaced by C) to yield a sequence encoding polyserine which would be uniformly encoded by AGC codons.
  • the percentage of sequence identity obtained from a global pairwise alignment between the reference polyserine nucleic acid sequence and the sequence optimized polyserine nucleic acid sequence would be 0%.
  • the protein products from both sequences would be 100% identical.
  • sequence optimization also sometimes referred to codon optimization
  • results can include, e.g., matching codon frequencies in certain tissue targets and/or host organisms to ensure proper folding; biasing G/C content to increase mRNA stability or reduce secondary structures; minimizing tandem repeat codons or base runs that can impair gene construction or expression; customizing transcriptional and translational control regions; inserting or removing protein trafficking sequences; removing/adding post translation modification sites in an encoded protein (e.g., glycosylation sites); adding, removing or shuffling protein domains; inserting or deleting restriction sites; modifying ribosome binding sites and mRNA degradation sites; adjusting translational rates to allow the various domains of the protein to fold properly; and/or reducing or eliminating problem secondary structures within the polynucleotide.
  • Sequence optimization tools, algorithms and services are known in the art, non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (
  • a polynucleotide e.g., a RNA, e.g., an mRNA
  • a sequence-optimized nucleotide sequence e.g., an ORF
  • the PBGD polypeptide, functional fragment, or a variant thereof encoded by the sequence-optimized nucleotide sequence has improved properties (e.g., compared to a PBGD polypeptide, functional fragment, or a variant thereof encoded by a reference nucleotide sequence that is not sequence optimized), e.g., improved properties related to expression efficacy after administration in vivo.
  • Such properties include, but are not limited to, improving nucleic acid stability (e.g., mRNA stability), increasing translation efficacy in the target tissue, reducing the number of truncated proteins expressed, improving the folding or prevent misfolding of the expressed proteins, reducing toxicity of the expressed products, reducing cell death caused by the expressed products, increasing and/or decreasing protein aggregation.
  • nucleic acid stability e.g., mRNA stability
  • increasing translation efficacy in the target tissue reducing the number of truncated proteins expressed, improving the folding or prevent misfolding of the expressed proteins, reducing toxicity of the expressed products, reducing cell death caused by the expressed products, increasing and/or decreasing protein aggregation.
  • the sequence optimized nucleotide sequence is codon optimized for expression in human subjects, having structural and/or chemical features that avoid one or more of the problems in the art, for example, features which are useful for optimizing formulation and delivery of nucleic acid-based therapeutics while retaining structural and functional integrity; overcoming a threshold of expression; improving expression rates; half-life and/or protein concentrations; optimizing protein localization; and avoiding deleterious bio-responses such as the immune response and/or degradation pathways.
  • the polynucleotides of the invention comprise a nucleotide sequence (e.g., a nucleotide sequence (e.g., an ORF) encoding a PBGD polypeptide, a nucleotide sequence (e.g., an ORF) encoding another polypeptide of interest, a 5′-UTR, a 3′-UTR, a microRNA binding site, a nucleic acid sequence encoding a linker, or any combination thereof) that is sequence-optimized according to a method comprising:
  • the sequence optimized nucleotide sequence (e.g., an ORF encoding a PBGD polypeptide) has at least one improved property with respect to the reference nucleotide sequence.
  • the sequence optimization method is multiparametric and comprises one, two, three, four, or more methods disclosed herein and/or other optimization methods known in the art.
  • features which can be considered beneficial in some embodiments of the invention, can be encoded by or within regions of the polynucleotide and such regions can be upstream (5′) to, downstream (3′) to, or within the region that encodes the PBGD polypeptide. These regions can be incorporated into the polynucleotide before and/or after sequence-optimization of the protein encoding region or open reading frame (ORF). Examples of such features include, but are not limited to, untranslated regions (UTRs), microRNA sequences, Kozak sequences, oligo(dT) sequences, poly-A tail, and detectable tags and can include multiple cloning sites that can have XbaI recognition.
  • UTRs untranslated regions
  • microRNA sequences Kozak sequences
  • oligo(dT) sequences poly-A tail
  • detectable tags can include multiple cloning sites that can have XbaI recognition.
  • the polynucleotide of the invention comprises a 5′ UTR, a 3′ UTR and/or a miRNA binding site. In some embodiments, the polynucleotide comprises two or more 5′ UTRs and/or 3′ UTRs, which can be the same or different sequences. In some embodiments, the polynucleotide comprises two or more miRNA, which can be the same or different sequences. Any portion of the 5′ UTR, 3′ UTR, and/or miRNA binding site, including none, can be sequence-optimized and can independently contain one or more different structural or chemical modifications, before and/or after sequence optimization.
  • the polynucleotide is reconstituted and transformed into a vector such as, but not limited to, plasmids, viruses, cosmids, and artificial chromosomes.
  • a vector such as, but not limited to, plasmids, viruses, cosmids, and artificial chromosomes.
  • the optimized polynucleotide can be reconstituted and transformed into chemically competent E. coli , yeast, neurospora, maize, drosophila, etc. where high copy plasmid-like or chromosome structures occur by methods described herein.
  • the polynucleotide of the invention comprises a sequence optimized nucleotide sequence encoding a PBGD polypeptide disclosed herein. In some embodiments, the polynucleotide of the invention comprises an open reading frame (ORF) encoding a PBGD polypeptide, wherein the ORF has been sequence optimized.
  • ORF open reading frame
  • Exemplary sequence optimized nucleotide sequences encoding human PBGD isoform 1 are set forth as SEQ ID Nos: 9-33 (PBGD-CO01, PBGD-CO02, PBGD-CO03, PBGD-CO04, PBGD-CO05, PBGD-CO06, PBGD-CO07, PBGD-CO08, PBGD-CO09, PBGD-CO10, PBGD-CO11, PBGD-CO12, PBGD-CO13, PBGD-CO14, PBGD-CO15, PBGD-CO16, PBGD-CO17, PBGD-CO18, PBGD-CO19, PBGD-CO20, PBGD-CO21, PBGD-CO22, PBGD-CO23, PBGD-CO24, and PBGD-CO25, respectively.
  • sequence optimized nucleotide sequences encoding human PBGD isoform 1 are shown in TABLE 2.
  • sequence optimized PBGD sequences set forth as SEQ ID Nos: 9-33 or shown in TABLE 2 fragments, and variants thereof are used to practice the methods disclosed herein.
  • sequence optimized PBGD sequences set forth as SEQ ID Nos: 9-33 or shown in TABLE 2 fragments and variants thereof are combined with or alternatives to the wild-type sequences disclosed in FIGS. 1-4
  • Exemplary sequence optimized nucleotide sequences encoding human PBGD isoform 1 are set forth as SEQ ID Nos: 89 to 117 (PBGD-CO30, PBGD-CO31, PBGD-CO32, PBGD-CO33, PBGD-CO34, PBGD-CO35, PBGD-CO36, PBGD-CO37, PBGD-CO38, PBGD-CO39, PBGD-CO40A, PBGD-CO41A, PBGD-CO42A, PBGD-CO43A, PBGD-CO44A, PBGD-CO45A, PBGD-CO46A, PBGD-CO47A, PBGD-CO40B, PBGD-CO41B, PBGD-CO42B, PBGD-CO43B, PBGD-CO44B, PBGD-CO45B, PBGD-CO46B, PBGD-CO47B, PBGD-CO48, PBGD-CO49, PBGD-CO
  • sequence optimized nucleotide sequences encoding human PBGD isoform 1 are shown in TABLE 2.
  • sequence optimized PBGD sequences set forth as SEQ ID Nos: 89 to 117, or shown in TABLE 2 fragments, and variants thereof are used to practice the methods disclosed herein.
  • sequence optimized PBGD sequences set forth as SEQ ID Nos: 89 to 117, or shown in TABLE 2 fragments and variants thereof are combined with or alternatives to the wild-type sequences disclosed in FIGS. 1-4 .
  • a polynucleotide of the present disclosure for example a polynucleotide comprising an mRNA nucleotide sequence encoding a PBGD polypeptide, comprises from 5′ to 3′ end:
  • a 5′ UTR such as the sequences provided herein, for example, SEQ ID NO: 39;
  • PBGD polypeptide e.g., a sequence optimized nucleic acid sequence encoding PBGD set forth as SEQ ID Nos: 9 to 33 and 89 to 117, or shown in TABLE 2;
  • a 3′ UTR such as the sequences provided herein, for example, SEQ ID NOs 149 to 151;
  • sequence optimized nucleotide sequences disclosed herein are distinct from the corresponding wild type nucleotide acid sequences and from other known sequence optimized nucleotide sequences, e.g., these sequence optimized nucleic acids have unique compositional characteristics.
  • the percentage of uracil or thymine nucleobases in a sequence optimized nucleotide sequence is modified (e.g., reduced) with respect to the percentage of uracil or thymine nucleobases in the reference wild-type nucleotide sequence.
  • a sequence optimized nucleotide sequence e.g., encoding a PBGD polypeptide, a functional fragment, or a variant thereof
  • is modified e.g., reduced
  • a sequence is referred to as a uracil-modified or thymine-modified sequence.
  • the percentage of uracil or thymine content in a nucleotide sequence can be determined by dividing the number of uracils or thymines in a sequence by the total number of nucleotides and multiplying by 100.
  • the sequence optimized nucleotide sequence has a lower uracil or thymine content than the uracil or thymine content in the reference wild-type sequence.
  • the uracil or thymine content in a sequence-optimized nucleotide sequence of the invention is greater than the uracil or thymine content in the reference wild-type sequence and still maintain beneficial effects, e.g., increased expression and/or reduced Toll-Like Receptor (TLR) response when compared to the reference wild-type sequence.
  • beneficial effects e.g., increased expression and/or reduced Toll-Like Receptor (TLR) response when compared to the reference wild-type sequence.
  • the uracil or thymine content of wild-type PBGD isoform 1 is about 20%. In some embodiments, the uracil or thymine content of a uracil- or thymine-modified sequence encoding a PBGD polypeptide is less than 20%. In some embodiments, the uracil or thymine content of a uracil- or thymine-modified sequence encoding a PBGD polypeptide of the invention is less than 19%, less that 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, or less than 10%.
  • the uracil or thymine content is not less than 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, or 10%.
  • the uracil or thymine content of a sequence disclosed herein, i.e., its total uracil or thymine content is abbreviated herein as % U TL or % T TL .
  • a uracil- or thymine-modified sequence encoding a PBGD polypeptide of the invention can also be described according to its uracil or thymine content relative to the uracil or thymine content in the corresponding wild-type nucleic acid sequence (% U WT or % T WT ), or according to its uracil or thymine content relative to the theoretical minimum uracil or thymine content of a nucleic acid encoding the wild-type protein sequence (% U TM or (% T TM ).
  • uracil or thymine content relative to the uracil or thymine content in the wild type nucleic acid sequence refers to a parameter determined by dividing the number of uracils or thymines in a sequence-optimized nucleic acid by the total number of uracils or thymines in the corresponding wild-type nucleic acid sequence and multiplying by 100. This parameter is abbreviated herein as % U WT or % T WT .
  • the % U WT or % T WT of a uracil- or thymine-modified sequence encoding a PBGD polypeptide of the invention is above 50%, above 55%, above 60%, above 65%, above 70%, above 75%, above 80%, above 85%, above 90%, or above 95%.
  • Uracil- or thymine-content relative to the uracil or thymine theoretical minimum refers to a parameter determined by dividing the number of uracils or thymines in a sequence-optimized nucleotide sequence by the total number of uracils or thymines in a hypothetical nucleotide sequence in which all the codons in the hypothetical sequence are replaced with synonymous codons having the lowest possible uracil or thymine content and multiplying by 100.
  • This parameter is abbreviated herein as % U TM or % T TM .
  • the % U TM of a uracil-modified sequence encoding a PBGD polypeptide of the invention is between about 118% and about 132%.
  • a uracil-modified sequence encoding a PBGD polypeptide of the invention has a reduced number of consecutive uracils with respect to the corresponding wild-type nucleic acid sequence.
  • two consecutive leucines can be encoded by the sequence CUUUUG, which includes a four uracil cluster.
  • Such a subsequence can be substituted, e.g., with CUGCUC, which removes the uracil cluster.
  • Phenylalanine can be encoded by UUC or UUU. Thus, even if phenylalanines encoded by UUU are replaced by UUC, the synonymous codon still contains a uracil pair (UU). Accordingly, the number of phenylalanines in a sequence establishes a minimum number of uracil pairs (UU) that cannot be eliminated without altering the number of phenylalanines in the encoded polypeptide.
  • the polypeptide e.g., wild type PBGD isoform 1
  • the absolute minimum number of uracil pairs (UU) in that a uracil-modified sequence encoding the polypeptide (e.g., wild type PBGD isoform 1) can contain is 7, 8, or 9, respectively.
  • Wild type PBGD isoform 1 contains 32 uracil pairs (UU), and four uracil triplets (UUU).
  • a uracil-modified sequence encoding a PBGD polypeptide of the invention has a reduced number of uracil triplets (UUU) with respect to the wild-type nucleic acid sequence.
  • a uracil-modified sequence encoding a PBGD polypeptide of the invention contains 4, 3, 2, 1 or no uracil triplets (UU).
  • a uracil-modified sequence encoding a PBGD polypeptide has a reduced number of uracil pairs (UU) with respect to the number of uracil pairs (UU) in the wild-type nucleic acid sequence.
  • a uracil-modified sequence encoding a PBGD polypeptide of the invention has a number of uracil pairs (UU) corresponding to the minimum possible number of uracil pairs (UU) in the wild-type nucleic acid sequence, e.g., 9 uracil pairs in the case of wild type PBGD isoform 1.
  • a uracil-modified sequence encoding a PBGD polypeptide of the invention has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 24 uracil pairs (UU) less than the number of uracil pairs (UU) in the wild-type nucleic acid sequence. In some embodiments, a uracil-modified sequence encoding a PBGD polypeptide of the invention has between 8 and 16 uracil pairs (UU).
  • uracil pairs (UU) relative to the uracil pairs (UU) in the wild type nucleic acid sequence refers to a parameter determined by dividing the number of uracil pairs (UU) in a sequence-optimized nucleotide sequence by the total number of uracil pairs (UU) in the corresponding wild-type nucleotide sequence and multiplying by 100. This parameter is abbreviated herein as % UU wt .
  • a uracil-modified sequence encoding a PBGD polypeptide of the invention has a % UU wt less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 65%, less than 60%, less than 55%, less than 50%, less than 40%, less than 30%, or less than 20%.
  • a uracil-modified sequence encoding a PBGD polypeptide has a % UU wt between 20% and 55%. In a particular embodiment, a uracil-modified sequence encoding a PBGD polypeptide of the invention has a % UU wt between 25% and 55%.
  • the polynucleotide of the invention comprises a uracil-modified sequence encoding a PBGD polypeptide disclosed herein.
  • the uracil-modified sequence encoding a PBGD polypeptide comprises at least one chemically modified nucleobase, e.g., 5-methoxyuracil.
  • at least 95% of a nucleobase (e.g., uracil) in a uracil-modified sequence encoding a PBGD polypeptide of the invention are modified nucleobases.
  • At least 95% of uracil in a uracil-modified sequence encoding a PBGD polypeptide is 5-methoxyuracil.
  • the polynucleotide comprising a uracil-modified sequence further comprises a miRNA binding site, e.g., a miRNA binding site that binds to miR-142 and/or a miRNA binding site that binds to miR-126.
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) comprising a uracil-modified sequence disclosed herein is formulated with a delivery agent comprising, e.g., a compound having the Formula (I), e.g., any of Compounds 1-232, e.g., Compound 18; a compound having the Formula (III), (IV), (V), or (VI), e.g., any of Compounds 233-342, e.g., Compound 236; or a compound having the Formula (VIII), e.g., any of Compounds 419-428, e.g., Compound 428, or any combination thereof.
  • the delivery agent comprises Compound 18, DSPC, Cholesterol, and Compound 428, e.g., with a mole ratio of about 50:10:38.5:1.5.
  • the “guanine content of the sequence optimized ORF encoding PBGD with respect to the theoretical maximum guanine content of a nucleotide sequence encoding the PBGD polypeptide,” abbreviated as % G TMX is at least 69%, at least 70%, at least 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%. In some embodiments, the % G TMX is between about 70% and about 80%, between about 71% and about 79%, between about 71% and about 78%, or between about 71% and about 77%.
  • the “cytosine content of the ORF relative to the theoretical maximum cytosine content of a nucleotide sequence encoding the PBGD polypeptide,” abbreviated as % C TMX , is at least 59%, at least 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%.
  • the % C TMX is between about 60% and about 80%, between about 62% and about 80%, between about 63% and about 79%, or between about 68% and about 76%.
  • the “guanine and cytosine content (G/C) of the ORF relative to the theoretical maximum G/C content in a nucleotide sequence encoding the PBGD polypeptide,” abbreviated as % G/C TMX is at least about 81%, at least about 85%, at least about 90%, at least about 95%, or about 100%.
  • the % G/C TMX is between about 80% and about 100%, between about 85% and about 99%, between about 90% and about 97%, or between about 91% and about 96%.
  • the “G/C content in the ORF relative to the G/C content in the corresponding wild-type ORF,” abbreviated as % G/C WT is at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 110%, at least 115%, or at least 120%.
  • the average G/C content in the 3rd codon position in the ORF is at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, or at least 30% higher than the average G/C content in the 3rd codon position in the corresponding wild-type ORF.
  • the polynucleotide of the invention comprises an open reading frame (ORF) encoding a PBGD polypeptide, wherein the ORF has been sequence optimized, and wherein each of % U TL , % U WT , % U TM , % G TL , % G WT , % G TMX , % C TL , % C WT , % C TMX , % G/C TL , % G/C WT , or % G/C TMX , alone or in a combination thereof is in a range between (i) a maximum corresponding to the parameter's maximum value (MAX) plus about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 standard deviations (STD DEV), and (ii) a minimum corresponding to the parameter's minimum value (MIN) less 0.5, 1, 1.5,
  • a polynucleotide, e.g., mRNA, of the invention is sequence optimized.
  • a sequence optimized nucleotide sequence comprises at least one codon modification with respect to a reference sequence (e.g., a wild-type sequence encoding a PBGD polypeptide).
  • a reference sequence e.g., a wild-type sequence encoding a PBGD polypeptide.
  • at least one codon is different from a corresponding codon in a reference sequence (e.g., a wild-type sequence).
  • sequence optimized nucleic acids are generated by at least a step comprising substituting codons in a reference sequence with synonymous codons (i.e., codons that encode the same amino acid).
  • substitutions can be effected, for example, by applying a codon substitution map (i.e., a table providing the codons that will encode each amino acid in the codon optimized sequence), or by applying a set of rules (e.g., if glycine is next to neutral amino acid, glycine would be encoded by a certain codon, but if it is next to a polar amino acid, it would be encoded by another codon).
  • compositions and formulations comprising these sequence optimized nucleic acids (e.g., a RNA, e.g., an mRNA) can be administered to a subject in need thereof to facilitate in vivo expression of functionally active PBGD.
  • sequence optimized nucleic acids e.g., a RNA, e.g., an mRNA
  • RNA e.g., an mRNA
  • a RNA e.g., an mRNA
  • PBGD a functionally active PBGD
  • compositions or formulations comprising the same
  • codon usage i.e., the frequency with which different organisms use codons for expressing a polypeptide sequence
  • codon usage differs among organisms (for example, recombinant production of human or humanized therapeutic antibodies frequently takes place in hamster cell cultures).
  • a reference nucleic acid sequence can be sequence optimized by applying a codon map.
  • T bases are present in DNA, whereas the T bases would be replaced by U bases in corresponding RNAs.
  • a sequence optimized nucleic acid disclosed herein in DNA form e.g., a vector or an in-vitro translation (IVT) template, would have its T bases transcribed as U based in its corresponding transcribed mRNA.
  • IVT in-vitro translation
  • a TTC codon (DNA map) would correspond to a UUC codon (RNA map), which in turn can correspond to a ⁇ C codon (RNA map in which U has been replaced with pseudouridine).
  • a reference sequence encoding PBGD can be optimized by replacing all the codons encoding a certain amino acid with only one of the alternative codons provided in a codon map. For example, all the valines in the optimized sequence would be encoded by GTG or GTC or GTT.
  • Sequence optimized polynucleotides of the invention can be generated using one or more optimization methods, or a combination thereof. Sequence optimization methods which can be used to sequence optimize nucleic acid sequences are described in detail herein. This list of methods is not comprehensive or limiting.
  • sequence optimization methods can be, for example, dependent on the specific chemistry used to produce a synthetic polynucleotide. Such a choice can also depend on characteristics of the protein encoded by the sequence optimized nucleic acid, e.g., a full sequence, a functional fragment, or a fusion protein comprising PBGD, etc. In some embodiments, such a choice can depend on the specific tissue or cell targeted by the sequence optimized nucleic acid (e.g., a therapeutic synthetic mRNA).
  • the mechanisms of combining the sequence optimization methods or design rules derived from the application and analysis of the optimization methods can be either simple or complex.
  • the combination can be:
  • Sequential Each sequence optimization method or set of design rules applies to a different subsequence of the overall sequence, for example reducing uridine at codon positions 1 to 30 and then selecting high frequency codons for the remainder of the sequence.
  • Hierarchical Several sequence optimization methods or sets of design rules are combined in a hierarchical, deterministic fashion. For example, use the most GC-rich codons, breaking ties (which are common) by choosing the most frequent of those codons.
  • Multifactorial/Multiparametric Machine learning or other modeling techniques are used to design a single sequence that best satisfies multiple overlapping and possibly contradictory requirements. This approach would require the use of a computer applying a number of mathematical techniques, for example, genetic algorithms.
  • each one of these approaches can result in a specific set of rules which in many cases can be summarized in a single codon table, i.e., a sorted list of codons for each amino acid in the target protein (i.e., PBGD), with a specific rule or set of rules indicating how to select a specific codon for each amino acid position.
  • a specific set of rules which in many cases can be summarized in a single codon table, i.e., a sorted list of codons for each amino acid in the target protein (i.e., PBGD), with a specific rule or set of rules indicating how to select a specific codon for each amino acid position.
  • uridine in a nucleic acid sequence can have detrimental effects on translation, e.g., slow or prematurely terminated translation, especially when modified uridine analogs are used in the production of synthetic mRNAs.
  • high uridine content can also reduce the in vivo half-life of synthetic mRNAs due to TLR activation.
  • a nucleic acid sequence can be sequence optimized using a method comprising at least one uridine content optimization step.
  • a step comprises, e.g., substituting at least one codon in the reference nucleic acid with an alternative codon to generate a uridine-modified sequence, wherein the uridine-modified sequence has at least one of the following properties:
  • the sequence optimization process comprises optimizing the global uridine content, i.e., optimizing the percentage of uridine nucleobases in the sequence optimized nucleic acid with respect to the percentage of uridine nucleobases in the reference nucleic acid sequence. For example, 30% of nucleobases can be uridines in the reference sequence and 10% of nucleobases can be uridines in the sequence optimized nucleic acid.
  • the sequence optimization process comprises reducing the local uridine content in specific regions of a reference nucleic acid sequence, i.e., reducing the percentage of uridine nucleobases in a subsequence of the sequence optimized nucleic acid with respect to the percentage of uridine nucleobases in the corresponding subsequence of the reference nucleic acid sequence.
  • the reference nucleic acid sequence can have a 5′-end region (e.g., 30 codons) with a local uridine content of 30%, and the uridine content in that same region could be reduced to 10% in the sequence optimized nucleic acid.
  • codons can be replaced in the reference nucleic acid sequence to reduce or modify, for example, the number, size, location, or distribution of uridine clusters that could have deleterious effects on protein translation.
  • codons can be replaced in the reference nucleic acid sequence to reduce or modify, for example, the number, size, location, or distribution of uridine clusters that could have deleterious effects on protein translation.
  • it is desirable to reduce the uridine content of the reference nucleic acid sequence in certain embodiments the uridine content, and in particular the local uridine content, of some subsequences of the reference nucleic acid sequence can be increased.
  • uridine content optimization can be combined with ramp design, since using the rarest codons for most amino acids will, with a few exceptions, reduce the U content.
  • the uridine-modified sequence is designed to induce a lower Toll-Like Receptor (TLR) response when compared to the reference nucleic acid sequence.
  • TLR Toll-Like Receptor
  • ds Double-stranded
  • ss Single-stranded
  • Single-stranded (ss)RNA activates TLR7. See Diebold et al. (2004) Science 303:1529-1531.
  • RNA oligonucleotides for example RNA with phosphorothioate internucleotide linkages, are ligands of human TLR8. See Heil et al. (2004) Science 303:1526-1529. DNA containing unmethylated CpG motifs, characteristic of bacterial and viral DNA, activate TLR9. See Hemmi et al. (2000) Nature, 408: 740-745.
  • TLR response is defined as the recognition of single-stranded RNA by a TLR7 receptor, and in some embodiments encompasses the degradation of the RNA and/or physiological responses caused by the recognition of the single-stranded RNA by the receptor.
  • Methods to determine and quantitate the binding of an RNA to a TLR7 are known in the art.
  • methods to determine whether an RNA has triggered a TLR7-mediated physiological response are well known in the art.
  • a TLR response can be mediated by TLR3, TLR8, or TLR9 instead of TLR7.
  • Human rRNA for example, has ten times more pseudouridine (P) and 25 times more 2′-O-methylated nucleosides than bacterial rRNA.
  • Bacterial mRNA contains no nucleoside modifications, whereas mammalian mRNAs have modified nucleosides such as 5-methylcytidine (m5C), N6-methyladenosine (m6A), inosine and many 2′-O-methylated nucleosides in addition to N7-methylguanosine (m7G).
  • modified nucleosides such as 5-methylcytidine (m5C), N6-methyladenosine (m6A), inosine and many 2′-O-methylated nucleosides in addition to N7-methylguanosine (m7G).
  • one or more of the optimization methods disclosed herein comprises reducing the uridine content (locally and/or globally) and/or reducing or modifying uridine clustering to reduce or to suppress a TLR7-mediated response.
  • the TLR response (e.g., a response mediated by TLR7) caused by the uridine-modified sequence is at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% lower than the TLR response caused by the reference nucleic acid sequence.
  • the TLR response caused by the reference nucleic acid sequence is at least about 1-fold, at least about 1.1-fold, at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at least about 1.9-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold higher than the TLR response caused by the uridine-modified sequence.
  • the uridine content (average global uridine content) (absolute or relative) of the uridine-modified sequence is higher than the uridine content (absolute or relative) of the reference nucleic acid sequence. Accordingly, in some embodiments, the uridine-modified sequence contains at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% more uridine that the reference nucleic acid sequence.
  • the uridine content (average global uridine content) (absolute or relative) of the uridine-modified sequence is lower than the uridine content (absolute or relative) of the reference nucleic acid sequence. Accordingly, in some embodiments, the uridine-modified sequence contains at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% less uridine that the reference nucleic acid sequence.
  • the uridine content (average global uridine content) (absolute or relative) of the uridine-modified sequence is less than 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of the total nucleobases in the uridine-modified sequence.
  • the uridine content of the uridine-modified sequence is between about 10% and about 20%. In some particular embodiments, the uridine content of the uridine-modified sequence is between about 12% and about 16%.
  • the uridine content of the reference nucleic acid sequence can be measured using a sliding window.
  • the length of the sliding window is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleobases.
  • the sliding window is over 40 nucleobases in length.
  • the sliding window is 20 nucleobases in length. Based on the uridine content measured with a sliding window, it is possible to generate a histogram representing the uridine content throughout the length of the reference nucleic acid sequence and sequence optimized nucleic acids.
  • a reference nucleic acid sequence can be modified to reduce or eliminate peaks in the histogram that are above or below a certain percentage value. In some embodiments, the reference nucleic acid sequence can be modified to eliminate peaks in the sliding-window representation which are above 65%, 60%, 55%, 50%, 45%, 40%, 35%, or 30% uridine. In another embodiment, the reference nucleic acid sequence can be modified so no peaks are over 30% uridine in the sequence optimized nucleic acid, as measured using a 20 nucleobase sliding window.
  • the reference nucleic acid sequence can be modified so no more or no less than a predetermined number of peaks in the sequence optimized nucleic sequence, as measured using a 20 nucleobase sliding window, are above or below a certain threshold value.
  • the reference nucleic acid sequence can be modified so no peaks or no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 peaks in the sequence optimized nucleic acid are above 10%, 15%, 20%, 25% or 30% uridine.
  • the sequence optimized nucleic acid contains between 0 peaks and 2 peaks with uridine contents 30% of higher.
  • a reference nucleic acid sequence can be sequence optimized to reduce the incidence of consecutive uridines. For example, two consecutive leucines could be encoded by the sequence CUUUUG, which would include a four uridine cluster. Such subsequence could be substituted with CUGCUC, which would effectively remove the uridine cluster. Accordingly, a reference nucleic sequence can be sequence optimized by reducing or eliminating uridine pairs (UU), uridine triplets (UUU) or uridine quadruplets (UUUU). Higher order combinations of U are not considered combinations of lower order combinations. Thus, for example, UUUU is strictly considered a quadruplet, not two consecutive U pairs; or UUUUUU is considered a sextuplet, not three consecutive U pairs, or two consecutive U triplets, etc.
  • all uridine pairs (UU) and/or uridine triplets (UUU) and/or uridine quadruplets (UUUU) can be removed from the reference nucleic acid sequence.
  • uridine pairs (UU) and/or uridine triplets (UUU) and/or uridine quadruplets (UUUU) can be reduced below a certain threshold, e.g., no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 occurrences in the sequence optimized nucleic acid.
  • the sequence optimized nucleic acid contains less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 uridine pairs.
  • the sequence optimized nucleic acid contains no uridine pairs and/or triplets.
  • Phenylalanine codons i.e., UUC or UUU
  • UUC or UUU comprise a uridine pair or triplet and therefore sequence optimization to reduce uridine content can at most reduce the phenylalanine U triplet to a phenylalanine U pair.
  • the occurrence of uridine pairs (UU) and/or uridine triplets (UUU) refers only to non-phenylalanine U pairs or triplets.
  • non-phenylalanine uridine pairs (UU) and/or uridine triplets (UUU) can be reduced below a certain threshold, e.g., no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 occurrences in the sequence optimized nucleic acid.
  • the sequence optimized nucleic acid contains less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 non-phenylalanine uridine pairs and/or triplets.
  • the sequence optimized nucleic acid contains no non-phenylalanine uridine pairs and/or triplets.
  • the reduction in uridine combinations (e.g., pairs, triplets, quadruplets) in the sequence optimized nucleic acid can be expressed as a percentage reduction with respect to the uridine combinations present in the reference nucleic acid sequence.
  • a sequence optimized nucleic acid can contain about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% of the total number of uridine pairs present in the reference nucleic acid sequence.
  • a sequence optimized nucleic acid can contain about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% of the total number of uridine triplets present in the reference nucleic acid sequence.
  • a sequence optimized nucleic acid can contain about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% of the total number of uridine quadruplets present in the reference nucleic acid sequence.
  • a sequence optimized nucleic acid can contain about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% of the total number of non-phenylalanine uridine pairs present in the reference nucleic acid sequence.
  • a sequence optimized nucleic acid can contain about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% of the total number of non-phenylalanine uridine triplets present in the reference nucleic acid sequence.
  • the uridine content in the sequence optimized sequence can be expressed with respect to the theoretical minimum uridine content in the sequence.
  • the term “theoretical minimum uridine content” is defined as the uridine content of a nucleic acid sequence as a percentage of the sequence's length after all the codons in the sequence have been replaced with synonymous codon with the lowest uridine content.
  • the uridine content of the sequence optimized nucleic acid is identical to the theoretical minimum uridine content of the reference sequence (e.g., a wild type sequence).
  • the uridine content of the sequence optimized nucleic acid is about 100%, about 105%, about 110%, about 115%, about 120%, about 125%, about 130%, about 135%, about 140%, about 145%, about 150%, about 155%, about 160%, about 165%, about 170%, about 175%, about 180%, about 185%, about 190%, about 195%, about 200%, about 210%, about 220%, about 230%, about 240% or about 250% of the theoretical minimum uridine content of the reference sequence (e.g., a wild type sequence).
  • the reference sequence e.g., a wild type sequence
  • the uridine content of the sequence optimized nucleic acid is identical to the theoretical minimum uridine content of the reference sequence (e.g., a wild type sequence).
  • the reference nucleic acid sequence can comprise uridine clusters which due to their number, size, location, distribution or combinations thereof have negative effects on translation.
  • uridine cluster refers to a subsequence in a reference nucleic acid sequence or sequence optimized nucleic sequence with contains a uridine content (usually described as a percentage) which is above a certain threshold.
  • a subsequence comprises more than about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65% uridine content, such subsequence would be considered a uridine cluster.
  • uridine clusters can be, for example, eliciting a TLR7 response.
  • the reference nucleic acid sequence comprises at least one uridine cluster, wherein said uridine cluster is a subsequence of the reference nucleic acid sequence wherein the percentage of total uridine nucleobases in said subsequence is above a predetermined threshold.
  • the length of the subsequence is at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, or at least about 100 nucleobases.
  • the subsequence is longer than 100 nucleobases.
  • the threshold is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24% or 25% uridine content. In some embodiments, the threshold is above 25%.
  • an amino acid sequence comprising A, D, G, S and R could be encoded by the nucleic acid sequence GCU, GAU, GGU, AGU, CGU. Although such sequence does not contain any uridine pairs, triplets, or quadruplets, one third of the nucleobases would be uridines. Such a uridine cluster could be removed by using alternative codons, for example, by using GCC, GAC, GGC, AGC, and CGC, which would contain no uridines.
  • the reference nucleic acid sequence comprises at least one uridine cluster, wherein said uridine cluster is a subsequence of the reference nucleic acid sequence wherein the percentage of uridine nucleobases of said subsequence as measured using a sliding window that is above a predetermined threshold.
  • the length of the sliding window is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleobases.
  • the sliding window is over 40 nucleobases in length.
  • the threshold is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24% or 25% uridine content. In some embodiments, the threshold is above 25%.
  • the reference nucleic acid sequence comprises at least two uridine clusters.
  • the uridine-modified sequence contains fewer uridine-rich clusters than the reference nucleic acid sequence.
  • the uridine-modified sequence contains more uridine-rich clusters than the reference nucleic acid sequence.
  • the uridine-modified sequence contains uridine-rich clusters with are shorter in length than corresponding uridine-rich clusters in the reference nucleic acid sequence.
  • the uridine-modified sequence contains uridine-rich clusters which are longer in length than the corresponding uridine-rich cluster in the reference nucleic acid sequence.
  • a reference nucleic acid sequence can be sequence optimized using methods comprising altering the Guanine/Cytosine (G/C) content (absolute or relative) of the reference nucleic acid sequence.
  • G/C Guanine/Cytosine
  • Such optimization can comprise altering (e.g., increasing or decreasing) the global G/C content (absolute or relative) of the reference nucleic acid sequence; introducing local changes in G/C content in the reference nucleic acid sequence (e.g., increase or decrease G/C in selected regions or subsequences in the reference nucleic acid sequence); altering the frequency, size, and distribution of G/C clusters in the reference nucleic acid sequence, or combinations thereof.
  • the sequence optimized nucleic acid encoding PBGD comprises an overall increase in G/C content (absolute or relative) relative to the G/C content (absolute or relative) of the reference nucleic acid sequence.
  • the overall increase in G/C content (absolute or relative) is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% relative to the G/C content (absolute or relative) of the reference nucleic acid sequence.
  • the sequence optimized nucleic acid encoding PBGD comprises an overall decrease in G/C content (absolute or relative) relative to the G/C content of the reference nucleic acid sequence.
  • the overall decrease in G/C content (absolute or relative) is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% relative to the G/C content (absolute or relative) of the reference nucleic acid sequence.
  • the sequence optimized nucleic acid encoding PBGD comprises a local increase in Guanine/Cytosine (G/C) content (absolute or relative) in a subsequence (i.e., a G/C modified subsequence) relative to the G/C content (absolute or relative) of the corresponding subsequence in the reference nucleic acid sequence.
  • G/C Guanine/Cytosine
  • the local increase in G/C content is by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% relative to the G/C content (absolute or relative) of the corresponding subsequence in the reference nucleic acid sequence.
  • the sequence optimized nucleic acid encoding PBGD comprises a local decrease in Guanine/Cytosine (G/C) content (absolute or relative) in a subsequence (i.e., a G/C modified subsequence) relative to the G/C content (absolute or relative) of the corresponding subsequence in the reference nucleic acid sequence.
  • G/C Guanine/Cytosine
  • the local decrease in G/C content is by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% relative to the G/C content (absolute or relative) of the corresponding subsequence in the reference nucleic acid sequence.
  • the G/C content (absolute or relative) is increased or decreased in a subsequence which is at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleobases in length.
  • the G/C content (absolute or relative) is increased or decreased in a subsequence which is at least about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890
  • the G/C content (absolute or relative) is increased or decreased in a subsequence which is at least about 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800, 7900, 8000, 8100, 8200, 8300, 8400, 8500, 8
  • G and C content can be conducted by replacing synonymous codons with low G/C content with synonymous codons having higher G/C content, or vice versa.
  • L has 6 synonymous codons: two of them have 2 G/C (CUC, CUG), 3 have a single G/C (UUG, CUU, CUA), and one has no G/C (UUA). So if the reference nucleic acid had a CUC codon in a certain position, G/C content at that position could be reduced by replacing CUC with any of the codons having a single G/C or the codon with no G/C.
  • a nucleic acid sequence encoding PBGD disclosed herein can be sequence optimized using methods comprising the use of modifications in the frequency of use of one or more codons relative to other synonymous codons in the sequence optimized nucleic acid with respect to the frequency of use in the non-codon optimized sequence.
  • codon frequency refers to codon usage bias, i.e., the differences in the frequency of occurrence of synonymous codons in coding DNA/RNA. It is generally acknowledged that codon preferences reflect a balance between mutational biases and natural selection for translational optimization. Optimal codons help to achieve faster translation rates and high accuracy. As a result of these factors, translational selection is expected to be stronger in highly expressed genes. In the field of bioinformatics and computational biology, many statistical methods have been proposed and used to analyze codon usage bias. See, e.g., Comeron & Aguadé (1998) J. Mol. Evol. 47: 268-74.
  • Multivariate statistical methods such as correspondence analysis and principal component analysis, are widely used to analyze variations in codon usage among genes (Suzuki et al. (2008) DNA Res. 15 (6): 357-65; Sandhu et al., In Silico Biol. 2008; 8(2):187-92).
  • nucleic acid sequence encoding a PBGD polypeptide disclosed herein can be codon optimized using methods comprising substituting at least one codon in the reference nucleic acid sequence with an alternative codon having a higher or lower codon frequency in the synonymous codon set; wherein the resulting sequence optimized nucleic acid has at least one optimized property with respect to the reference nucleic acid sequence.
  • At least one codon in the reference nucleic acid sequence encoding PBGD is substituted with an alternative codon having a codon frequency higher than the codon frequency of the substituted codon in the synonymous codon set, and at least one codon in the reference nucleic acid sequence is substituted with an alternative codon having a codon frequency lower than the codon frequency of the substituted codon in the synonymous codon set.
  • At least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, or at least about 75% of the codons in the reference nucleic acid sequence encoding PBGD are substituted with alternative codons, each alternative codon having a codon frequency higher than the codon frequency of the substituted codon in the synonymous codon set.
  • At least one alternative codon having a higher codon frequency has the highest codon frequency in the synonymous codon set. In other embodiments, all alternative codons having a higher codon frequency have the highest codon frequency in the synonymous codon set.
  • At least one alternative codon having a lower codon frequency has the lowest codon frequency in the synonymous codon set. In some embodiments, all alternative codons having a higher codon frequency have the highest codon frequency in the synonymous codon set.
  • At least one alternative codon has the second highest, the third highest, the fourth highest, the fifth highest or the sixth highest frequency in the synonymous codon set. In some specific embodiments, at least one alternative codon has the second lowest, the third lowest, the fourth lowest, the fifth lowest, or the sixth lowest frequency in the synonymous codon set.
  • Optimization based on codon frequency can be applied globally, as described above, or locally to the reference nucleic acid sequence encoding a PBGD polypeptide.
  • regions of the reference nucleic acid sequence when applied locally, can modified based on codon frequency, substituting all or a certain percentage of codons in a certain subsequence with codons that have higher or lower frequencies in their respective synonymous codon sets.
  • At least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100% of the codons in a subsequence of the reference nucleic acid sequence are substituted with alternative codons, each alternative codon having a codon frequency higher than the codon frequency of the substituted codon in the synonymous codon set.
  • At least one codon in a subsequence of the reference nucleic acid sequence encoding a PBGD polypeptide is substituted with an alternative codon having a codon frequency higher than the codon frequency of the substituted codon in the synonymous codon set, and at least one codon in a subsequence of the reference nucleic acid sequence is substituted with an alternative codon having a codon frequency lower than the codon frequency of the substituted codon in the synonymous codon set.
  • At least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, or at least about 75% of the codons in a subsequence of the reference nucleic acid sequence encoding a PBGD polypeptide are substituted with alternative codons, each alternative codon having a codon frequency higher than the codon frequency of the substituted codon in the synonymous codon set.
  • At least one alternative codon substituted in a subsequence of the reference nucleic acid sequence encoding a PBGD polypeptide and having a higher codon frequency has the highest codon frequency in the synonymous codon set.
  • all alternative codons substituted in a subsequence of the reference nucleic acid sequence and having a lower codon frequency have the lowest codon frequency in the synonymous codon set.
  • At least one alternative codon substituted in a subsequence of the reference nucleic acid sequence encoding a PBGD polypeptide and having a lower codon frequency has the lowest codon frequency in the synonymous codon set. In some embodiments, all alternative codons substituted in a subsequence of the reference nucleic acid sequence and having a higher codon frequency have the highest codon frequency in the synonymous codon set.
  • a sequence optimized nucleic acid encoding a PBGD polypeptide can comprise a subsequence having an overall codon frequency higher or lower than the overall codon frequency in the corresponding subsequence of the reference nucleic acid sequence at a specific location, for example, at the 5′ end or 3′ end of the sequence optimized nucleic acid, or within a predetermined distance from those region (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 codons from the 5′ end or 3′ end of the sequence optimized nucleic acid).
  • a sequence optimized nucleic acid encoding a PBGD polypeptide can comprise more than one subsequence having an overall codon frequency higher or lower than the overall codon frequency in the corresponding subsequence of the reference nucleic acid sequence.
  • subsequences with overall higher or lower overall codon frequencies can be organized in innumerable patterns, depending on whether the overall codon frequency is higher or lower, the length of the subsequence, the distance between subsequences, the location of the subsequences, etc.
  • Structural motifs Motifs encoded by an arrangement of nucleotides that tends to form a certain secondary structure.
  • motifs that fit into the category of disadvantageous motifs.
  • Some examples include, for example, restriction enzyme motifs, which tend to be relatively short, exact sequences such as the restriction site motifs forXbaI (TCTAGA), EcoRI (GAATTC), EcoRII (CCWGG, wherein W means A or T, per the IUPAC ambiguity codes), or HindIII (AAGCTT); enzyme sites, which tend to be longer and based on consensus not exact sequence, such in the T7 RNA polymerase (GnnnnWnCRnCTCnCnWnD, wherein n means any nucleotide, R means A or G, W means A or T, D means A or G or T but not C); structural motifs, such as GGGG repeats (Kim et al. (1991) Nature 351(6324):331-2); or other motifs such as CUG-triplet repeats (Querido et al. (2014) J. Cell Sci. 124:1703-1714).
  • nucleic acid sequence encoding a PBGD polypeptide disclosed herein can be sequence optimized using methods comprising substituting at least one destabilizing motif in a reference nucleic acid sequence, and removing such disadvantageous motif or replacing it with an advantageous motif.
  • the optimization process comprises identifying advantageous and/or disadvantageous motifs in the reference nucleic sequence, wherein such motifs are, e.g., specific subsequences that can cause a loss of stability in the reference nucleic acid sequence prior or during the optimization process. For example, substitution of specific bases during optimization can generate a subsequence (motif) recognized by a restriction enzyme. Accordingly, during the optimization process the appearance of disadvantageous motifs can be monitored by comparing the sequence optimized sequence with a library of motifs known to be disadvantageous. Then, the identification of disadvantageous motifs could be used as a post-hoc filter, i.e., to determine whether a certain modification which potentially could be introduced in the reference nucleic acid sequence should be actually implemented or not.
  • motifs are, e.g., specific subsequences that can cause a loss of stability in the reference nucleic acid sequence prior or during the optimization process. For example, substitution of specific bases during optimization can generate a subsequence (motif) recognized by a restriction
  • the identification of disadvantageous motifs can be used prior to the application of the sequence optimization methods disclosed herein, i.e., the identification of motifs in the reference nucleic acid sequence encoding a PBGD polypeptide and their replacement with alternative nucleic acid sequences can be used as a preprocessing step, for example, before uridine reduction.
  • the identification of disadvantageous motifs and their removal is used as an additional sequence optimization technique integrated in a multiparametric nucleic acid optimization method comprising two or more of the sequence optimization methods disclosed herein.
  • a disadvantageous motif identified during the optimization process would be removed, for example, by substituting the lowest possible number of nucleobases in order to preserve as closely as possible the original design principle(s) (e.g., low U, high frequency, etc.).
  • sequence optimization of a reference nucleic acid sequence encoding a PBGD polypeptide can be conducted using a limited codon set, e.g., a codon set wherein less than the native number of codons is used to encode the 20 natural amino acids, a subset of the 20 natural amino acids, or an expanded set of amino acids including, for example, non-natural amino acids.
  • a limited codon set e.g., a codon set wherein less than the native number of codons is used to encode the 20 natural amino acids, a subset of the 20 natural amino acids, or an expanded set of amino acids including, for example, non-natural amino acids.
  • the genetic code is highly similar among all organisms and can be expressed in a simple table with 64 entries which would encode the 20 standard amino acids involved in protein translation plus start and stop codons.
  • the genetic code is degenerate, i.e., in general, more than one codon specifies each amino acid.
  • the amino acid leucine is specified by the UUA, UUG, CUU, CUC, CUA, or CUG codons
  • the amino acid serine is specified by UCA, UCG, UCC, UCU, AGU, or AGC codons (difference in the first, second, or third position).
  • Native genetic codes comprise 62 codons encoding naturally occurring amino acids.
  • optimized codon sets comprising less than 62 codons to encode 20 amino acids can comprise 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, or 20 codons.
  • the limited codon set comprises less than 20 codons.
  • an optimized codon set comprises as many codons as different types of amino acids are present in the protein encoded by the reference nucleic acid sequence.
  • the optimized codon set comprises 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or even 1 codon.
  • At least one amino acid selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Tyr, and Val i.e., amino acids which are naturally encoded by more than one codon, is encoded with less codons than the naturally occurring number of synonymous codons.
  • Ala can be encoded in the sequence optimized nucleic acid by 3, 2 or 1 codons; Cys can be encoded in the sequence optimized nucleic acid by 1 codon; Asp can be encoded in the sequence optimized nucleic acid by 1 codon; Glu can be encoded in the sequence optimized nucleic acid by 1 codon; Phe can be encoded in the sequence optimized nucleic acid by 1 codon; Gly can be encoded in the sequence optimized nucleic acid by 3 codons, 2 codons or 1 codon; His can be encoded in the sequence optimized nucleic acid by 1 codon; Ile can be encoded in the sequence optimized nucleic acid by 2 codons or 1 codon; Lys can be encoded in the sequence optimized nucleic acid by 1 codon; Leu can be encoded in the sequence optimized nucleic acid by 5 codons, 4 codons, 3 codons, 2 codons or 1 codon; Asn can be encoded in the sequence optimized nucleic acid by 1 codon;
  • the sequence optimized nucleic acid is a DNA and the limited codon set consists of 20 codons, wherein each codon encodes one of 20 amino acids.
  • the sequence optimized nucleic acid is a DNA and the limited codon set comprises at least one codon selected from the group consisting of GCT, GCC, GCA, and GCG; at least a codon selected from the group consisting of CGT, CGC, CGA, CGG, AGA, and AGG; at least a codon selected from AAT or ACC; at least a codon selected from GAT or GAC; at least a codon selected from TGT or TGC; at least a codon selected from CAA or CAG; at least a codon selected from GAA or GAG; at least a codon selected from the group consisting of GGT, GGC, GGA, and GGG; at least a codon selected from CAT or CAC; at least a codon selected from the group consisting of ATT, ATC, and
  • the sequence optimized nucleic acid is an RNA (e.g., an mRNA) and the limited codon set consists of 20 codons, wherein each codon encodes one of 20 amino acids.
  • the sequence optimized nucleic acid is an RNA and the limited codon set comprises at least one codon selected from the group consisting of GCU, GCC, GCA, and GCG; at least a codon selected from the group consisting of CGU, CGC, CGA, CGG, AGA, and AGG; at least a codon selected from AAU or ACC; at least a codon selected from GAU or GAC; at least a codon selected from UGU or UGC; at least a codon selected from CAA or CAG; at least a codon selected from GAA or GAG; at least a codon selected from the group consisting of GGU, GGC, GGA, and GGG; at least a codon selected from CAU or CAC; at least a codon selected from the group consisting of GGU, GGC
  • the limited codon set has been optimized for in vivo expression of a sequence optimized nucleic acid (e.g., a synthetic mRNA) following administration to a certain tissue or cell.
  • a sequence optimized nucleic acid e.g., a synthetic mRNA
  • the optimized codon set (e.g., a 20 codon set encoding 20 amino acids) complies at least with one of the following properties:
  • the optimized codon set has a higher average G/C content than the original or native codon set;
  • the optimized codon set has a lower average U content than the original or native codon set
  • the optimized codon set is composed of codons with the highest frequency
  • the optimized codon set is composed of codons with the lowest frequency
  • At least one codon in the optimized codon set has the second highest, the third highest, the fourth highest, the fifth highest or the sixth highest frequency in the synonymous codon set. In some specific embodiments, at least one codon in the optimized codon has the second lowest, the third lowest, the fourth lowest, the fifth lowest, or the sixth lowest frequency in the synonymous codon set.
  • the term “native codon set” refers to the codon set used natively by the source organism to encode the reference nucleic acid sequence.
  • the term “original codon set” refers to the codon set used to encode the reference nucleic acid sequence before the beginning of sequence optimization, or to a codon set used to encode an optimized variant of the reference nucleic acid sequence at the beginning of a new optimization iteration when sequence optimization is applied iteratively or recursively.
  • 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of codons in the codon set are those with the highest frequency.
  • 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of codons in the codon set are those with the lowest frequency.
  • 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of codons in the codon set are those with the highest uridine content. In some embodiments, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of codons in the codon set are those with the lowest uridine content.
  • the average G/C content (absolute or relative) of the codon set is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% higher than the average G/C content (absolute or relative) of the original codon set.
  • the average G/C content (absolute or relative) of the codon set is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% lower than the average G/C content (absolute or relative) of the original codon set.
  • the uracil content (absolute or relative) of the codon set is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% higher than the average uracil content (absolute or relative) of the original codon set.
  • the uracil content (absolute or relative) of the codon set is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% lower than the average uracil content (absolute or relative) of the original codon set.
  • the polynucleotide e.g., a RNA, e.g., an mRNA
  • a sequence optimized nucleic acid disclosed herein encoding a PBGD polypeptide can be tested to determine whether at least one nucleic acid sequence property (e.g., stability when exposed to nucleases) or expression property has been improved with respect to the non-sequence optimized nucleic acid.
  • expression property refers to a property of a nucleic acid sequence either in vivo (e.g., translation efficacy of a synthetic mRNA after administration to a subject in need thereof) or in vitro (e.g., translation efficacy of a synthetic mRNA tested in an in vitro model system).
  • Expression properties include but are not limited to the amount of protein produced by an mRNA encoding a PBGD polypeptide after administration, and the amount of soluble or otherwise functional protein produced.
  • sequence optimized nucleic acids disclosed herein can be evaluated according to the viability of the cells expressing a protein encoded by a sequence optimized nucleic acid sequence (e.g., a RNA, e.g., an mRNA) encoding a PBGD polypeptide disclosed herein.
  • a sequence optimized nucleic acid sequence e.g., a RNA, e.g., an mRNA
  • a plurality of sequence optimized nucleic acids disclosed herein e.g., a RNA, e.g., an mRNA
  • a property of interest for example an expression property in an in vitro model system, or in vivo in a target tissue or cell.
  • the desired property of the polynucleotide is an intrinsic property of the nucleic acid sequence.
  • the nucleotide sequence e.g., a RNA, e.g., an mRNA
  • the nucleotide sequence can be sequence optimized for in vivo or in vitro stability.
  • the nucleotide sequence can be sequence optimized for expression in a particular target tissue or cell.
  • the nucleic acid sequence is sequence optimized to increase its plasma half-life by preventing its degradation by endo and exonucleases.
  • the nucleic acid sequence is sequence optimized to increase its resistance to hydrolysis in solution, for example, to lengthen the time that the sequence optimized nucleic acid or a pharmaceutical composition comprising the sequence optimized nucleic acid can be stored under aqueous conditions with minimal degradation.
  • sequence optimized nucleic acid can be optimized to increase its resistance to hydrolysis in dry storage conditions, for example, to lengthen the time that the sequence optimized nucleic acid can be stored after lyophilization with minimal degradation.
  • the desired property of the polynucleotide is the level of expression of a PBGD polypeptide encoded by a sequence optimized sequence disclosed herein.
  • Protein expression levels can be measured using one or more expression systems.
  • expression can be measured in cell culture systems, e.g., CHO cells or HEK293 cells.
  • expression can be measured using in vitro expression systems prepared from extracts of living cells, e.g., rabbit reticulocyte lysates, or in vitro expression systems prepared by assembly of purified individual components.
  • the protein expression is measured in an in vivo system, e.g., mouse, rabbit, monkey, etc.
  • protein expression in solution form can be desirable.
  • a reference sequence can be sequence optimized to yield a sequence optimized nucleic acid sequence having optimized levels of expressed proteins in soluble form.
  • Levels of protein expression and other properties such as solubility, levels of aggregation, and the presence of truncation products (i.e., fragments due to proteolysis, hydrolysis, or defective translation) can be measured according to methods known in the art, for example, using electrophoresis (e.g., native or SDS-PAGE) or chromatographic methods (e.g., HPLC, size exclusion chromatography, etc.).
  • heterologous therapeutic proteins encoded by a nucleic acid sequence can have deleterious effects in the target tissue or cell, reducing protein yield, or reducing the quality of the expressed product (e.g., due to the presence of protein fragments or precipitation of the expressed protein in inclusion bodies), or causing toxicity.
  • sequence optimization of a nucleic acid sequence disclosed herein can be used to increase the viability of target cells expressing the protein encoded by the sequence optimized nucleic acid.
  • Heterologous protein expression can also be deleterious to cells transfected with a nucleic acid sequence for autologous or heterologous transplantation. Accordingly, in some embodiments of the present disclosure the sequence optimization of a nucleic acid sequence disclosed herein can be used to increase the viability of target cells expressing the protein encoded by the sequence optimized nucleic acid sequence. Changes in cell or tissue viability, toxicity, and other physiological reaction can be measured according to methods known in the art.
  • the administration of a sequence optimized nucleic acid encoding PBGD polypeptide or a functional fragment thereof can trigger an immune response, which could be caused by (i) the therapeutic agent (e.g., an mRNA encoding a PBGD polypeptide), or (ii) the expression product of such therapeutic agent (e.g., the PBGD polypeptide encoded by the mRNA), or (iv) a combination thereof.
  • the therapeutic agent e.g., an mRNA encoding a PBGD polypeptide
  • the expression product of such therapeutic agent e.g., the PBGD polypeptide encoded by the mRNA
  • nucleic acid sequence e.g., an mRNA
  • sequence optimization of nucleic acid sequence can be used to decrease an immune or inflammatory response triggered by the administration of a nucleic acid encoding a PBGD polypeptide or by the expression product of PBGD encoded by such nucleic acid.
  • an inflammatory response can be measured by detecting increased levels of one or more inflammatory cytokines using methods known in the art, e.g., ELISA.
  • inflammatory cytokine refers to cytokines that are elevated in an inflammatory response.
  • inflammatory cytokines include interleukin-6 (IL-6), CXCL1 (chemokine (C—X—C motif) ligand 1; also known as GRO ⁇ , interferon- ⁇ (IFN ⁇ ), tumor necrosis factor ⁇ (TNF ⁇ ), interferon ⁇ -induced protein 10 (IP-10), or granulocyte-colony stimulating factor (G-CSF).
  • inflammatory cytokines includes also other cytokines associated with inflammatory responses known in the art, e.g., interleukin-1 (IL-1), interleukin-8 (IL-8), interleukin-12 (IL-12), interleukin-13 (Il-13), interferon ⁇ (IFN- ⁇ ), etc.
  • IL-1 interleukin-1
  • IL-8 interleukin-8
  • IL-12 interleukin-12
  • Il-13 interleukin-13
  • IFN- ⁇ interferon ⁇
  • the polynucleotide of the invention (e.g., a RNA, e.g., an mRNA) comprises a chemically modified nucleobase, e.g., 5-methoxyuracil.
  • the mRNA is a uracil-modified sequence comprising an ORF encoding a PBGD polypeptide, wherein the mRNA comprises a chemically modified nucleobase, e.g., 5-methoxyuracil.
  • the resulting modified nucleoside or nucleotide is referred to as 5-methoxyuridine.
  • uracil in the polynucleotide is at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least 90%, at least 95%, at least 99%, or about 100% 5-methoxyuracil.
  • uracil in the polynucleotide is at least 95% 5-methoxyuracil.
  • uracil in the polynucleotide is 100% 5-methoxyuracil.
  • uracil in the polynucleotide is at least 95% 5-methoxyuracil
  • overall uracil content can be adjusted such that the polynucleotide of the invention (e.g., a RNA, e.g., an mRNA) provides suitable protein expression levels while inducing little to no immune response.
  • the uracil content of the ORF is between about 105% and about 145%, about 105% and about 140%, about 110% and about 140%, about 110% and about 145%, about 115% and about 135%, about 105% and about 135%, about 110% and about 135%, about 115% and about 145%, or about 115% and about 140% of the theoretical minimum uracil content in the corresponding wild-type ORF (% U TM ). In other embodiments, the uracil content of the ORF is between about 117% and about 134% or between 118% and 132% of the % U TM .
  • the uracil content of the ORF encoding a PBGD polypeptide is about 115%, about 120%, about 125%, about 130%, about 135%, about 140%, about 145%, or about 150% of the % U TM .
  • uracil can refer to 5-methoxyuracil and/or naturally occurring uracil.
  • the uracil content in the ORF of the mRNA encoding a PBGD polypeptide of the invention is less than about 50%, about 40%, about 30%, or about 20% of the total nucleobase content in the ORF. In some embodiments, the uracil content in the ORF is between about 15% and about 25% of the total nucleobase content in the ORF. In other embodiments, the uracil content in the ORF is between about 20% and about 30% of the total nucleobase content in the ORF. In one embodiment, the uracil content in the ORF of the mRNA encoding a PBGD polypeptide is less than about 20% of the total nucleobase content in the open reading frame. In this context, the term “uracil” can refer to 5-methoxyuracil and/or naturally occurring uracil.
  • the ORF of the mRNA encoding a PBGD polypeptide having 5-methoxyuracil and adjusted uracil content has increased Cytosine (C), Guanine (G), or Guanine/Cytosine (G/C) content (absolute or relative).
  • the overall increase in C, G, or G/C content (absolute or relative) of the ORF is at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 10%, at least about 15%, at least about 20%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 100% relative to the G/C content (absolute or relative) of the wild-type ORF.
  • the G, the C, or the G/C content in the ORF is less than about 100%, less than about 90%, less than about 85%, or less than about 80% of the theoretical maximum G, C, or G/C content of the corresponding wild type nucleotide sequence encoding the PBGD polypeptide (% G TMX ; % C TMX , or % G/C TMX ). In other embodiments, the G, the C, or the G/C content in the ORF is between about 70% and about 80%, between about 71% and about 79%, between about 71% and about 78%, or between about 71% and about 77% of the % G TMX , % C TMX , or % G/C TMX .
  • the increases in G and/or C content (absolute or relative) described herein can be conducted by replacing synonymous codons with low G, C, or G/C content with synonymous codons having higher G, C, or G/C content.
  • the increase in G and/or C content (absolute or relative) is conducted by replacing a codon ending with U with a synonymous codon ending with G or C.
  • the ORF of the mRNA encoding a PBGD polypeptide of the invention comprises 5-methoxyuracil and has an adjusted uracil content containing less uracil pairs (UU) and/or uracil triplets (UUU) and/or uracil quadruplets (UUUU) than the corresponding wild-type nucleotide sequence encoding the PBGD polypeptide.
  • the ORF of the mRNA encoding a PBGD polypeptide of the invention contains no uracil pairs and/or uracil triplets and/or uracil quadruplets.
  • uracil pairs and/or uracil triplets and/or uracil quadruplets are reduced below a certain threshold, e.g., no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 occurrences in the ORF of the mRNA encoding the PBGD polypeptide.
  • the ORF of the mRNA encoding the PBGD polypeptide of the invention contains less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 non-phenylalanine uracil pairs and/or triplets.
  • the ORF of the mRNA encoding the PBGD polypeptide contains no non-phenylalanine uracil pairs and/or triplets.
  • the ORF of the mRNA encoding a PBGD polypeptide of the invention comprises 5-methoxyuracil and has an adjusted uracil content containing less uracil-rich clusters than the corresponding wild-type nucleotide sequence encoding the PBGD polypeptide.
  • the ORF of the mRNA encoding the PBGD polypeptide of the invention contains uracil-rich clusters that are shorter in length than corresponding uracil-rich clusters in the corresponding wild-type nucleotide sequence encoding the PBGD polypeptide.
  • alternative lower frequency codons are employed. At least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100% of the codons in the PBGD polypeptide-encoding ORF of the 5-methoxyuracil-comprising mRNA are substituted with alternative codons, each alternative codon having a codon frequency lower than the codon frequency of the substituted codon in the synonymous codon set.
  • the ORF also has adjusted uracil content, as described above.
  • at least one codon in the ORF of the mRNA encoding the PBGD polypeptide is substituted with an alternative codon having a codon frequency lower than the codon frequency of the substituted codon in the synonymous codon set.
  • the adjusted uracil content, PBGD polypeptide-encoding ORF of the 5-methoxyuracil-comprising mRNA exhibits expression levels of PBGD when administered to a mammalian cell that are higher than expression levels of PBGD from the corresponding wild-type mRNA.
  • the expression levels of PBGD when administered to a mammalian cell are increased relative to a corresponding mRNA containing at least 95% 5-methoxyuracil and having a uracil content of about 160%, about 170%, about 180%, about 190%, or about 200% of the theoretical minimum.
  • the expression levels of PBGD when administered to a mammalian cell are increased relative to a corresponding mRNA, wherein at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of uracils are 1-methylpseudouracil or pseudouracils.
  • the mammalian cell is a mouse cell, a rat cell, or a rabbit cell.
  • the mammalian cell is a monkey cell or a human cell.
  • the human cell is a HeLa cell, a BJ fibroblast cell, or a peripheral blood mononuclear cell (PBMC).
  • PBMC peripheral blood mononuclear cell
  • PBGD is expressed when the mRNA is administered to a mammalian cell in vivo.
  • the mRNA is administered to mice, rabbits, rats, monkeys, or humans.
  • mice are null mice.
  • the mRNA is administered to mice in an amount of about 0.01 mg/kg, about 0.05 mg/kg, about 0.1 mg/kg, or about 0.15 mg/kg.
  • the mRNA is administered intravenously or intramuscularly.
  • the PBGD polypeptide is expressed when the mRNA is administered to a mammalian cell in vitro.
  • the expression is increased by at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 50-fold, at least about 500-fold, at least about 1500-fold, or at least about 3000-fold. In other embodiments, the expression is increased by at least about 10%, about 20%, about 30%, about 40%, about 50%, 60%, about 70%, about 80%, about 90%, or about 100%.
  • adjusted uracil content, PBGD polypeptide-encoding ORF of the 5-methoxyuracil-comprising mRNA exhibits increased stability.
  • the mRNA exhibits increased stability in a cell relative to the stability of a corresponding wild-type mRNA under the same conditions.
  • the mRNA exhibits increased stability including resistance to nucleases, thermal stability, and/or increased stabilization of secondary structure.
  • increased stability exhibited by the mRNA is measured by determining the half-life of the mRNA (e.g., in a plasma, serum, cell, or tissue sample) and/or determining the area under the curve (AUC) of the protein expression by the mRNA over time (e.g., in vitro or in vivo).
  • An mRNA is identified as having increased stability if the half-life and/or the AUC is greater than the half-life and/or the AUC of a corresponding wild-type mRNA under the same conditions.
  • the mRNA of the present invention induces a detectably lower immune response (e.g., innate or acquired) relative to the immune response induced by a corresponding wild-type mRNA under the same conditions.
  • the mRNA of the present disclosure induces a detectably lower immune response (e.g., innate or acquired) relative to the immune response induced by an mRNA that encodes for a PBGD polypeptide but does not comprise 5-methoxyuracil under the same conditions, or relative to the immune response induced by an mRNA that encodes for a PBGD polypeptide and that comprises 5-methoxyuracil but that does not have adjusted uracil content under the same conditions.
  • the innate immune response can be manifested by increased expression of pro-inflammatory cytokines, activation of intracellular PRRs (RIG-I, MDA5, etc.), cell death, and/or termination or reduction in protein translation.
  • a reduction in the innate immune response can be measured by expression or activity level of Type 1 interferons (e.g., IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , and IFN- ⁇ ) or the expression of interferon-regulated genes such as the toll-like receptors (e.g., TLR7 and TLR8), and/or by decreased cell death following one or more administrations of the mRNA of the invention into a cell.
  • Type 1 interferons e.g., IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , and IFN- ⁇
  • interferon-regulated genes e.g., TLR7 and TLR8
  • the expression of Type-1 interferons by a mammalian cell in response to the mRNA of the present disclosure is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or greater than 99.9% relative to a corresponding wild-type mRNA, to an mRNA that encodes a PBGD polypeptide but does not comprise 5-methoxyuracil, or to an mRNA that encodes a PBGD polypeptide and that comprises 5-methoxyuracil but that does not have adjusted uracil content.
  • the interferon is IFN- ⁇ .
  • cell death frequency caused by administration of mRNA of the present disclosure to a mammalian cell is 10%, 25%, 50%, 75%, 85%, 90%, 95%, or over 95% less than the cell death frequency observed with a corresponding wild-type mRNA, an mRNA that encodes for a PBGD polypeptide but does not comprise 5-methoxyuracil, or an mRNA that encodes for a PBGD polypeptide and that comprises 5-methoxyuracil but that does not have adjusted uracil content.
  • the mammalian cell is a BJ fibroblast cell. In other embodiments, the mammalian cell is a splenocyte.
  • the mammalian cell is that of a mouse or a rat. In other embodiments, the mammalian cell is that of a human. In one embodiment, the mRNA of the present disclosure does not substantially induce an innate immune response of a mammalian cell into which the mRNA is introduced.
  • the polynucleotide is an mRNA that comprises an ORF that encodes a PBGD polypeptide, wherein uracil in the mRNA is at least about 95% 5-methoxyuracil, wherein the uracil content of the ORF is between about 115% and about 135% of the theoretical minimum uracil content in the corresponding wild-type ORF, and wherein the uracil content in the ORF encoding the PBGD polypeptide is less than about 30% of the total nucleobase content in the ORF.
  • the ORF that encodes the PBGD polypeptide is further modified to increase G/C content of the ORF (absolute or relative) by at least about 40%, as compared to the corresponding wild-type ORF.
  • the ORF encoding the PBGD polypeptide contains less than 20 non-phenylalanine uracil pairs and/or triplets.
  • at least one codon in the ORF of the mRNA encoding the PBGD polypeptide is further substituted with an alternative codon having a codon frequency lower than the codon frequency of the substituted codon in the synonymous codon set.
  • the expression of the PBGD polypeptide encoded by an mRNA comprising an ORF wherein uracil in the mRNA is at least about 95% 5-methoxyuracil, and wherein the uracil content of the ORF is between about 115% and about 135% of the theoretical minimum uracil content in the corresponding wild-type ORF, is increased by at least about 10-fold when compared to expression of the PBGD polypeptide from the corresponding wild-type mRNA.
  • the mRNA comprises an open ORF wherein uracil in the mRNA is at least about 95% 5-methoxyuracil, and wherein the uracil content of the ORF is between about 115% and about 135% of the theoretical minimum uracil content in the corresponding wild-type ORF, and wherein the mRNA does not substantially induce an innate immune response of a mammalian cell into which the mRNA is introduced.
  • the invention includes modified polynucleotides comprising a polynucleotide described herein (e.g., a polynucleotide, e.g., an mRNA, comprising a nucleotide sequence encoding a PBGD polypeptide).
  • the modified polynucleotides can be chemically modified and/or structurally modified.
  • the polynucleotides of the present invention are chemically and/or structurally modified the polynucleotides can be referred to as “modified polynucleotides.”
  • nucleosides and nucleotides of a polynucleotide e.g., RNA polynucleotides, such as mRNA polynucleotides
  • a polynucleotide e.g., RNA polynucleotides, such as mRNA polynucleotides
  • a “nucleoside” refers to a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as “nucleobase”).
  • a “nucleotide” refers to a nucleoside including a phosphate group.
  • Modified nucleotides can be synthesized by any useful method, such as, for example, chemically, enzymatically, or recombinantly, to include one or more modified or non-natural nucleosides.
  • Polynucleotides can comprise a region or regions of linked nucleosides. Such regions can have variable backbone linkages. The linkages can be standard phosphodiester linkages, in which case the polynucleotides would comprise regions of nucleotides.
  • modified polynucleotides disclosed herein can comprise various distinct modifications.
  • the modified polynucleotides contain one, two, or more (optionally different) nucleoside or nucleotide modifications.
  • a modified polynucleotide, introduced to a cell can exhibit one or more desirable properties, e.g., improved protein expression, reduced immunogenicity, or reduced degradation in the cell, as compared to an unmodified polynucleotide.
  • a polynucleotide of the present invention e.g., a polynucleotide comprising a nucleotide sequence encoding a PBGD polypeptide
  • a “structural” modification is one in which two or more linked nucleosides are inserted, deleted, duplicated, inverted or randomized in a polynucleotide without significant chemical modification to the nucleotides themselves. Because chemical bonds will necessarily be broken and reformed to effect a structural modification, structural modifications are of a chemical nature and hence are chemical modifications. However, structural modifications will result in a different sequence of nucleotides.
  • the polynucleotide “ATCG” can be chemically modified to “AT-5meC-G”.
  • the same polynucleotide can be structurally modified from “ATCG” to “ATCCCG”.
  • the dinucleotide “CC” has been inserted, resulting in a structural modification to the polynucleotide.
  • the polynucleotides of the present invention are chemically modified.
  • the terms “chemical modification” or, as appropriate, “chemically modified” refer to modification with respect to adenosine (A), guanosine (G), uridine (U), thymidine (T) or cytidine (C) ribo- or deoxyribonucleosides in one or more of their position, pattern, percent or population, including, but not limited to, its nucleobase, sugar, backbone, or any combination thereof.
  • these terms are not intended to refer to the ribonucleotide modifications in naturally occurring 5′-terminal mRNA cap moieties.
  • the polynucleotides of the invention can have a uniform chemical modification of all or any of the same nucleoside type or a population of modifications produced by downward titration of the same starting modification in all or any of the same nucleoside type, or a measured percent of a chemical modification of all any of the same nucleoside type but with random incorporation, such as where all uridines are replaced by a uridine analog, e.g., 5-methoxyuridine.
  • the polynucleotides can have a uniform chemical modification of two, three, or four of the same nucleoside type throughout the entire polynucleotide (such as all uridines and/or all cytidines, etc. are modified in the same way).
  • Modified nucleotide base pairing encompasses not only the standard adenine-thymine, adenine-uracil, or guanine-cytosine base pairs, but also base pairs formed between nucleotides and/or modified nucleotides comprising non-standard or modified bases, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors permits hydrogen bonding between a non-standard base and a standard base or between two complementary non-standard base structures.
  • non-standard base pairing is the base pairing between the modified nucleobase inosine and adenine, cytosine or uracil. Any combination of base/sugar or linker can be incorporated into polynucleotides of the present disclosure.
  • RNA polynucleotides e.g., RNA polynucleotides, such as mRNA polynucleotides
  • nucleotides, nucleosides, and nucleobases 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine; 2-methylthio-N6-methyladenosine; 2-methylthio-N6-threonyl carbamoyladenosine; N6-glycinylcarbamoyladenosine; N6-isopentenyladenosine; N6-methyladenosine; N6-threonylcarbamoyladenosine; 1,2′-O-dimethyladenosine; 1-methyladenosine; 2′-O-methyladenosine; 2′-O-ribosyladenosine (phosphat
  • the polynucleotide e.g., RNA polynucleotide, such as mRNA polynucleotide
  • the polynucleotide includes a combination of at least two (e.g., 2, 3, 4 or more) of the aforementioned modified nucleobases.
  • the mRNA comprises at least one chemically modified nucleoside.
  • the at least one chemically modified nucleoside is selected from the group consisting of pseudouridine ( ⁇ ), 2-thiouridine (s2U), 4′-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine, 2′-O-methyl uridine, 1-methyl-pseud
  • the at least one chemically modified nucleoside is selected from the group consisting of pseudouridine, 1-methylpseudouridine, 1-ethyl-pseudouridine, 5-methylcytosine, 5-methoxyuridine, and a combination thereof.
  • the polynucleotide e.g., RNA polynucleotide, such as mRNA polynucleotide
  • the polynucleotide includes a combination of at least two (e.g., 2, 3, 4 or more) of the aforementioned modified nucleobases.
  • the chemical modification is at nucleobases in the polynucleotides (e.g., RNA polynucleotide, such as mRNA polynucleotide).
  • modified nucleobases in the polynucleotide are selected from the group consisting of 1-methyl-pseudouridine (m1 ⁇ ), 1-ethyl-pseudouridine (e1 ⁇ ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), pseudouridine (w), ⁇ -thio-guanosine and ⁇ -thio-adenosine.
  • the polynucleotide includes a combination of at least two (e.g., 2, 3, 4 or more) of the aforementioned modified nucleobases.
  • the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises pseudouridine (v) and 5-methyl-cytidine (m5C).
  • the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises 1-methyl-pseudouridine (m1 ⁇ ).
  • the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises 1-ethyl-pseudouridine (e1 ⁇ ).
  • the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises 1-methyl-pseudouridine (m1 ⁇ ) and 5-methyl-cytidine (m5C). In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises 1-ethyl-pseudouridine (e1 ⁇ ) and 5-methyl-cytidine (m5C).
  • the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises 2-thiouridine (s2U). In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises 2-thiouridine and 5-methyl-cytidine (m5C). In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises methoxy-uridine (mo5U).
  • the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises 5-methoxy-uridine (mo5U) and 5-methyl-cytidine (m5C).
  • the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises 2′-O-methyl uridine.
  • the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises 2′-O-methyl uridine and 5-methyl-cytidine (m5C).
  • the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises N6-methyl-adenosine (m6A). In some embodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises N6-methyl-adenosine (m6A) and 5-methyl-cytidine (m5C).
  • m6A N6-methyl-adenosine
  • m5C 5-methyl-cytidine
  • the polynucleotide e.g., mRNA polynucleotide, such as mRNA polynucleotide
  • mRNA polynucleotide is uniformly modified (e.g., fully modified, modified throughout the entire sequence) for a particular modification.
  • a polynucleotide can be uniformly modified with 5-methyl-cytidine (m5C), meaning that all cytosine residues in the mRNA sequence are replaced with 5-methyl-cytidine (m5C).
  • m5C 5-methyl-cytidine
  • a polynucleotide can be uniformly modified for any type of nucleoside residue present in the sequence by replacement with a modified residue such as any of those set forth above.
  • the chemically modified nucleosides in the open reading frame are selected from the group consisting of uridine, adenine, cytosine, guanine, and any combination thereof.
  • the modified nucleobase is a modified cytosine.
  • nucleobases and nucleosides having a modified cytosine include N4-acetyl-cytidine (ac4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, 2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine.
  • a modified nucleobase is a modified uridine.
  • Example nucleobases and nucleosides having a modified uridine include 5-cyano uridine or 4′-thio uridine.
  • a modified nucleobase is a modified adenine.
  • Example nucleobases and nucleosides having a modified adenine include 7-deaza-adenine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenine (m6A), and 2,6-Diaminopurine.
  • a modified nucleobase is a modified guanine.
  • Example nucleobases and nucleosides having a modified guanine include inosine (I), 1-methyl-inosine (m1I), wyosine (imG), methylwyosine (mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), 7-methyl-guanosine (m7G), 1-methyl-guanosine (m1G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine.
  • the nucleobase modified nucleotides in the polynucleotide are 5-methoxyuridine.
  • the polynucleotide e.g., RNA polynucleotide, such as mRNA polynucleotide
  • the polynucleotide includes a combination of at least two (e.g., 2, 3, 4 or more) of modified nucleobases.
  • At least 95% of a type of nucleobases (e.g., uracil) in a polynucleotide of the invention are modified nucleobases.
  • at least 95% of uracil in a polynucleotide of the present invention is 5-methoxyuracil.
  • the polynucleotide e.g., RNA polynucleotide, such as mRNA polynucleotide
  • the polynucleotide comprises 5-methoxyuridine (5mo5U) and 5-methyl-cytidine (m5C).
  • the polynucleotide e.g., RNA polynucleotide, such as mRNA polynucleotide
  • RNA polynucleotide such as mRNA polynucleotide
  • mRNA polynucleotide is uniformly modified (e.g., fully modified, modified throughout the entire sequence) for a particular modification.
  • a polynucleotide can be uniformly modified with 5-methoxyuridine, meaning that substantially all uridine residues in the mRNA sequence are replaced with 5-methoxyuridine.
  • a polynucleotide can be uniformly modified for any type of nucleoside residue present in the sequence by replacement with a modified residue such as any of those set forth above.
  • the modified nucleobase is a modified cytosine.
  • a modified nucleobase is a modified uracil.
  • Example nucleobases and nucleosides having a modified uracil include 5-methoxyuracil.
  • a modified nucleobase is a modified adenine.
  • a modified nucleobase is a modified guanine.
  • the nucleobases, sugar, backbone, or any combination thereof in the open reading frame encoding a PBGD polypeptide are chemically modified by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100%.
  • the uridine nucleosides in the open reading frame encoding a PBGD polypeptide are chemically modified by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100%.
  • the adenosine nucleosides in the open reading frame encoding a PBGD polypeptide are chemically modified by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100%.
  • the cytidine nucleosides in the open reading frame encoding a PBGD polypeptide are chemically modified by at least at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100%.
  • the guanosine nucleosides in the open reading frame encoding a PBGD polypeptide are chemically modified by at least at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100%.
  • the polynucleotides can include any useful linker between the nucleosides.
  • linkers including backbone modifications, that are useful in the composition of the present disclosure include, but are not limited to the following: 3′-alkylene phosphonates, 3′-amino phosphoramidate, alkene containing backbones, aminoalkylphosphoramidates, aminoalkylphosphotriesters, boranophosphates, —CH 2 —O—N(CH 3 )—CH 2 —, —CH 2 —N(CH 3 )—N(CH 3 )—CH 2 —, —CH 2 —NH—CH 2 —, chiral phosphonates, chiral phosphorothioates, formacetyl and thioformacetyl backbones, methylene (methylimino), methylene formacetyl and thioformacetyl backbones, methyleneimino and methylenehydrazino backbones,
  • modified nucleosides and nucleotides which can be incorporated into a polynucleotide (e.g., RNA or mRNA, as described herein), can be modified on the sugar of the ribonucleic acid.
  • a polynucleotide e.g., RNA or mRNA, as described herein
  • the 2′ hydroxyl group (OH) can be modified or replaced with a number of different substituents.
  • substitutions at the 2′-position include, but are not limited to, H, halo, optionally substituted C 1-6 alkyl; optionally substituted C 1-6 alkoxy; optionally substituted C 6-10 aryloxy; optionally substituted C 3-8 cycloalkyl; optionally substituted C 3-8 cycloalkoxy; optionally substituted C 6-10 aryloxy; optionally substituted C 6-10 aryl-C 1-6 alkoxy, optionally substituted C 1-12 (heterocyclyl)oxy; a sugar (e.g., ribose, pentose, or any described herein); a polyethyleneglycol (PEG), —O(CH 2 CH 2 O) n CH 2 CH 2 OR, where R is H or optionally substituted alkyl, and n is an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from
  • RNA includes the sugar group ribose, which is a 5-membered ring having an oxygen.
  • modified nucleotides include replacement of the oxygen in ribose (e.g., with S, Se, or alkylene, such as methylene or ethylene); addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ring contraction of ribose (e.g., to form a 4-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., to form a 6- or 7-membered ring having an additional carbon or heteroatom, such as for anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino that also has a phosphoramidate backbone); multicyclic forms (e.
  • the sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose.
  • a polynucleotide molecule can include nucleotides containing, e.g., arabinose, as the sugar.
  • Such sugar modifications are taught International Patent Publication Nos. WO2013052523 and WO2014093924, the contents of each of which are incorporated herein by reference in their entireties.
  • polynucleotides of the invention can include a combination of modifications to the sugar, the nucleobase, and/or the intemucleoside linkage. These combinations can include any one or more modifications described herein.
  • modified nucleotides can be used to form the polynucleotides of the invention.
  • the modified nucleotides can be completely substituted for the natural nucleotides of the polynucleotides of the invention.
  • the natural nucleotide uridine can be substituted with a modified nucleoside described herein.
  • the natural nucleotide uridine can be partially substituted or replaced (e.g., about 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99.9%) with at least one of the modified nucleoside disclosed herein.
  • Any combination of base/sugar or linker can be incorporated into the polynucleotides of the invention and such modifications are taught in International Patent Publications WO2013052523 and WO2014093924, and U.S. Publ. Nos. US 20130115272 and US20150307542, the contents of each of which are incorporated herein by reference in its entirety.
  • Untranslated regions are nucleic acid sections of a polynucleotide before a start codon (5′UTR) and after a stop codon (3′UTR) that are not translated.
  • a polynucleotide e.g., a ribonucleic acid (RNA), e.g., a messenger RNA (mRNA)
  • RNA e.g., a messenger RNA (mRNA)
  • mRNA messenger RNA
  • ORF open reading frame
  • a UTR can be homologous or heterologous to the coding region in a polynucleotide.
  • the UTR is homologous to the ORF encoding the PBGD polypeptide.
  • the UTR is heterologous to the ORF encoding the PBGD polypeptide.
  • the polynucleotide comprises two or more 5′UTRs or functional fragments thereof, each of which has the same or different nucleotide sequences.
  • the polynucleotide comprises two or more 3′UTRs or functional fragments thereof, each of which has the same or different nucleotide sequences.
  • the 5′UTR or functional fragment thereof, 3′ UTR or functional fragment thereof, or any combination thereof is sequence optimized.
  • the 5′UTR or functional fragment thereof, 3′ UTR or functional fragment thereof, or any combination thereof comprises at least one chemically modified nucleobase, e.g., 1-methylpseudouridine or 5-methoxyuracil.
  • UTRs can have features that provide a regulatory role, e.g., increased or decreased stability, localization and/or translation efficiency.
  • a polynucleotide comprising a UTR can be administered to a cell, tissue, or organism, and one or more regulatory features can be measured using routine methods.
  • a functional fragment of a 5′UTR or 3′UTR comprises one or more regulatory features of a full length 5′ or 3′ UTR, respectively.
  • Natural 5′UTRs bear features that play roles in translation initiation. They harbor signatures like Kozak sequences that are commonly known to be involved in the process by which the ribosome initiates translation of many genes. Kozak sequences have the consensus CCR(A/G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another ‘G’. 5′UTRs also have been known to form secondary structures that are involved in elongation factor binding.
  • liver-expressed mRNA such as albumin, serum amyloid A, Apolipoprotein A/B/E, transferrin, alpha fetoprotein, erythropoietin, or Factor VIII, can enhance expression of polynucleotides in hepatic cell lines or liver.
  • 5′UTR from other tissue-specific mRNA to improve expression in that tissue is possible for muscle (e.g., MyoD, Myosin, Myoglobin, Myogenin, Herculin), for endothelial cells (e.g., Tie-1, CD36), for myeloid cells (e.g., C/EBP, AML1, G-CSF, GM-CSF, CD11b, MSR, Fr-1, i-NOS), for leukocytes (e.g., CD45, CD18), for adipose tissue (e.g., CD36, GLUT4, ACRP30, adiponectin) and for lung epithelial cells (e.g., SP-A/B/C/D).
  • muscle e.g., MyoD, Myosin, Myoglobin, Myogenin, Herculin
  • endothelial cells e.g., Tie-1, CD36
  • myeloid cells e.g., C/E
  • UTRs are selected from a family of transcripts whose proteins share a common function, structure, feature or property.
  • an encoded polypeptide can belong to a family of proteins (i.e., that share at least one function, structure, feature, localization, origin, or expression pattern), which are expressed in a particular cell, tissue or at some time during development.
  • the UTRs from any of the genes or mRNA can be swapped for any other UTR of the same or different family of proteins to create a new polynucleotide.
  • the 5′UTR and the 3′UTR can be heterologous. In some embodiments, the 5′UTR can be derived from a different species than the 3′UTR. In some embodiments, the 3′UTR can be derived from a different species than the 5′UTR.
  • Exemplary UTRs of the application include, but are not limited to, one or more 5′UTR and/or 3′UTR derived from the nucleic acid sequence of: a globin, such as an ⁇ - or ⁇ -globin (e.g., aXenopus, mouse, rabbit, or human globin); a strong Kozak translational initiation signal; a CYBA (e.g., human cytochrome b-245 ⁇ polypeptide); an albumin (e.g., human albumin7); a HSD17B4 (hydroxysteroid (17-3) dehydrogenase); a virus (e.g., a tobacco etch virus (TEV), a Venezuelan equine encephalitis virus (VEEV), a Dengue virus, a cytomegalovirus (CMV) (e.g., CMV immediate early 1 (IE1)), a hepatitis virus (e.g., hepatitis B virus), a Sindbis virus, or a
  • the 5′UTR is selected from the group consisting of a ⁇ -globin 5′UTR; a 5′UTR containing a strong Kozak translational initiation signal; a cytochrome b-245 ⁇ polypeptide (CYBA) 5′UTR; a hydroxysteroid (17- ⁇ ) dehydrogenase (HSD17B4) 5′UTR; a Tobacco etch virus (TEV) 5′UTR; a Vietnamese etch virus (TEV) 5′UTR; a decielen equine encephalitis virus (TEEV) 5′UTR; a 5′ proximal open reading frame of rubella virus (RV) RNA encoding nonstructural proteins; a Dengue virus (DEN) 5′UTR; a heat shock protein 70 (Hsp70) 5′UTR; a eIF4G 5′UTR; a GLUT1 5′UTR; functional fragments thereof and any combination thereof.
  • CYBA cytochrome b-2
  • the 3′UTR is selected from the group consisting of a ⁇ -globin 3′UTR; a CYBA 3′UTR; an albumin 3′UTR; a growth hormone (GH) 3′UTR; a VEEV 3′UTR; a hepatitis B virus (HBV) 3′UTR; ⁇ -globin 3′UTR; a DEN 3′UTR; a PAV barley yellow dwarf virus (BYDV-PAV) 3′UTR; an elongation factor 1 ul (EEF1A 1 ) 3′UTR; a manganese superoxide dismutase (MnSOD) 3′UTR; a ⁇ subunit of mitochondrial H(+)-ATP synthase ( ⁇ -mRNA) 3′UTR; a GLUT1 3′UTR; a MEF2A 3′UTR; a ⁇ -F1-ATPase 3′UTR; functional fragments thereof and combinations thereof.
  • GH growth hormone
  • Wild-type UTRs derived from any gene or mRNA can be incorporated into the polynucleotides of the invention.
  • a UTR can be altered relative to a wild type or native UTR to produce a variant UTR, e.g., by changing the orientation or location of the UTR relative to the ORF; or by inclusion of additional nucleotides, deletion of nucleotides, swapping or transposition of nucleotides.
  • variants of 5′ or 3′ UTRs can be utilized, for example, mutants of wild type UTRs, or variants wherein one or more nucleotides are added to or removed from a terminus of the UTR.
  • one or more synthetic UTRs can be used in combination with one or more non-synthetic UTRs. See, e.g., Mandal and Rossi, Nat. Protoc. 2013 8(3):568-82, and sequences available at www.addgene.org/Derrick_Rossi/, the contents of each are incorporated herein by reference in their entirety.
  • UTRs or portions thereof can be placed in the same orientation as in the transcript from which they were selected or can be altered in orientation or location. Hence, a 5′ and/or 3′ UTR can be inverted, shortened, lengthened, or combined with one or more other 5′ UTRs or 3′ UTRs.
  • the polynucleotide comprises multiple UTRs, e.g., a double, a triple or a quadruple 5′UTR or 3′UTR.
  • a double UTR comprises two copies of the same UTR either in series or substantially in series.
  • a double beta-globin 3′UTR can be used (see US2010/0129877, the contents of which are incorporated herein by reference in its entirety).
  • the polynucleotides of the invention comprise a 5′UTR and/or a 3′UTR selected from any of the UTRs disclosed herein.
  • the 5′UTR comprises:
  • 5′UTR-001 Upstream UTR
  • 5′UTR-002 Upstream UTR
  • 5′UTR-003 Upstream UTR
  • 5′UTR-004 Upstream UTR
  • the 3′UTR comprises:
  • 142-3p 3′UTR (UTR including miR142-3p binding site) (SEQ ID NO. 57) (UGAUAAUAGUCCAUAAAGUAGGAAACACUACAGCUGGAGCCUCGGUGGCC AUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACC CGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC); 142-3p 3′UTR (UTR including miR142-3p binding site) (SEQ ID NO.
  • 3′UTR-005 ( ⁇ -globin UTR) (See SEQ ID NO. 68); 3′UTR-006 (G-CSF UTR) (See SEQ ID NO. 69); 3′UTR-007 (Col1a2; collagen, type I, alpha 2 UTR) (See SEQ ID NO. 70); 3′UTR-008 (Col6a2; collagen, type VI, alpha 2 UTR) (See SEQ ID NO. 71); 3′UTR-009 (RPN1; ribophorin I UTR) (See SEQ ID NO. 72); 3′UTR-010 (LRP1; low density lipoprotein receptor-related protein 1 UTR) (See SEQ ID NO.
  • 3′UTR-011 Nnt1; cardiotrophin-like cytokine factor 1 UTR
  • 3′UTR-012 Cold6a1; collagen, type VI, alpha 1 UTR
  • 3′UTR-013 Calr; calreticulin UTR
  • 3′UTR-014 Cold1a1; collagen, type I, alpha 1 UTR
  • 3′UTR-015 Plod1; procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1 UTR
  • 3′UTR-016 Nucb1; nucleobindin 1 UTR
  • 3′UTR-017 ⁇ -globin
  • 3′UTR-018 See SEQ ID NO. 81
  • 3′UTR miR 142-3p and miR 126-3p binding sites variant 1
  • the 5′UTR and/or 3′UTR sequence of the invention comprises a nucleotide sequence at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to a sequence selected from the group consisting of 5′UTR sequences comprising any of SEQ ID NOs: 39 to 56, 83, 189 to 191 and/or 3′UTR sequences comprises any of SEQ ID NOs: 57 to 81, 84, 149 to 151, 161 to 172, 192 to 199, and any combination thereof.
  • the polynucleotides of the invention can comprise combinations of features.
  • the ORF can be flanked by a 5′UTR that comprises a strong Kozak translational initiation signal and/or a 3′UTR comprising an oligo(dT) sequence for templated addition of a poly-A tail.
  • a 5′UTR can comprise a first polynucleotide fragment and a second polynucleotide fragment from the same and/or different UTRs (see, e.g., US2010/0293625, herein incorporated by reference in its entirety).
  • non-UTR sequences can be used as regions or subregions within the polynucleotides of the invention.
  • introns or portions of intron sequences can be incorporated into the polynucleotides of the invention. Incorporation of intronic sequences can increase protein production as well as polynucleotide expression levels.
  • the polynucleotide of the invention comprises an internal ribosome entry site (IRES) instead of or in addition to a UTR (see, e.g., Yakubov et al., Biochem. Biophys. Res. Commun. 2010 394(1):189-193, the contents of which are incorporated herein by reference in their entirety).
  • ITR internal ribosome entry site
  • the polynucleotide comprises an IRES instead of a 5′UTR sequence. In some embodiments, the polynucleotide comprises an ORF and a viral capsid sequence. In some embodiments, the polynucleotide comprises a synthetic 5′UTR in combination with a non-synthetic 3′UTR.
  • the UTR can also include at least one translation enhancer polynucleotide, translation enhancer element, or translational enhancer elements (collectively, “TEE,” which refers to nucleic acid sequences that increase the amount of polypeptide or protein produced from a polynucleotide.
  • TEE translation enhancer polynucleotide, translation enhancer element, or translational enhancer elements
  • the TEE can include those described in US2009/0226470, incorporated herein by reference in its entirety, and others known in the art.
  • the TEE can be located between the transcription promoter and the start codon.
  • the 5′UTR comprises a TEE.
  • a TEE is a conserved element in a UTR that can promote translational activity of a nucleic acid such as, but not limited to, cap-dependent or cap-independent translation.
  • the TEE comprises the TEE sequence in the 5′-leader of the Gtx homeodomain protein. See Chappell et al., PNAS 2004 101:9590-9594, incorporated herein by reference in its entirety.
  • the polynucleotide of the invention comprises one or multiple copies of a TEE.
  • the TEE in a translational enhancer polynucleotide can be organized in one or more sequence segments.
  • a sequence segment can harbor one or more of the TEEs provided herein, with each TEE being present in one or more copies.
  • multiple sequence segments are present in a translational enhancer polynucleotide, they can be homogenous or heterogeneous.
  • the multiple sequence segments in a translational enhancer polynucleotide can harbor identical or different types of the TEE provided herein, identical or different number of copies of each of the TEE, and/or identical or different organization of the TEE within each sequence segment.
  • the polynucleotide of the invention comprises a translational enhancer polynucleotide sequence.
  • TEE sequences are described in U.S. Publication 2014/0200261, the contents of which are incorporated herein by reference in their entirety.
  • MIRNA MIRNA
  • Polynucleotides of the invention can include regulatory elements, for example, microRNA (miRNA) binding sites, transcription factor binding sites, structured mRNA sequences and/or motifs, artificial binding sites engineered to act as pseudo-receptors for endogenous nucleic acid binding molecules, and combinations thereof.
  • regulatory elements for example, microRNA (miRNA) binding sites, transcription factor binding sites, structured mRNA sequences and/or motifs, artificial binding sites engineered to act as pseudo-receptors for endogenous nucleic acid binding molecules, and combinations thereof.
  • polynucleotides including such regulatory elements are referred to as including “sensor sequences”.
  • sensor sequences are described in U.S. Publication 2014/0200261, the contents of which are incorporated herein by reference in their entirety.
  • a polynucleotide e.g., a ribonucleic acid (RNA), e.g., a messenger RNA (mRNA)
  • RNA ribonucleic acid
  • mRNA messenger RNA
  • ORF open reading frame
  • miRNA binding site(s) provides for regulation of polynucleotides of the invention, and in turn, of the polypeptides encoded therefrom, based on tissue-specific and/or cell-type specific expression of naturally-occurring miRNAs.
  • compositions and formulations that comprise any of the polynucleotides described above.
  • the composition or formulation further comprises a delivery agent.
  • the composition or formulation can contain a polynucleotide comprising a sequence optimized nucleic acid sequence disclosed herein which encodes a polypeptide.
  • the composition or formulation can contain a polynucleotide (e.g., a RNA, e.g., an mRNA) comprising a polynucleotide (e.g., an ORF) having significant sequence identity to a sequence optimized nucleic acid sequence disclosed herein which encodes a polypeptide.
  • the polynucleotide further comprises a miRNA binding site, e.g., a miRNA binding site that binds
  • a miRNA e.g., a natural-occurring miRNA
  • a miRNA sequence comprises a “seed” region, i.e., a sequence in the region of positions 2-8 of the mature miRNA.
  • a miRNA seed can comprise positions 2-8 or 2-7 of the mature miRNA.
  • a miRNA seed can comprise 7 nucleotides (e.g., nucleotides 2-8 of the mature miRNA), wherein the seed-complementary site in the corresponding miRNA binding site is flanked by an adenosine (A) opposed to miRNA position 1.
  • a miRNA seed can comprise 6 nucleotides (e.g., nucleotides 2-7 of the mature miRNA), wherein the seed-complementary site in the corresponding miRNA binding site is flanked by an adenosine (A) opposed to miRNA position 1. See, for example, Grimson A, Farh K K, Johnston W K, Garrett-Engele P, Lim L P, Bartel D P; Mol Cell. 2007 Jul.
  • RNA profiling of the target cells or tissues can be conducted to determine the presence or absence of miRNA in the cells or tissues.
  • a polynucleotide e.g., a ribonucleic acid (RNA), e.g., a messenger RNA (mRNA)
  • RNA ribonucleic acid
  • mRNA messenger RNA
  • a polynucleotide of the invention comprises one or more microRNA binding sites, microRNA target sequences, microRNA complementary sequences, or microRNA seed complementary sequences.
  • sequences can correspond to, e.g., have complementarity to, any known microRNA such as those taught in US Publication US2005/0261218 and US Publication US2005/0059005, the contents of each of which are incorporated herein by reference in their entirety.
  • microRNAs derive enzymatically from regions of RNA transcripts that fold back on themselves to form short hairpin structures often termed a pre-miRNA (precursor-miRNA).
  • a pre-miRNA typically has a two-nucleotide overhang at its 3′ end, and has 3′ hydroxyl and 5′ phosphate groups.
  • This precursor-mRNA is processed in the nucleus and subsequently transported to the cytoplasm where it is further processed by DICER (a RNase III enzyme), to form a mature microRNA of approximately 22 nucleotides.
  • DICER a RNase III enzyme
  • the mature microRNA is then incorporated into a ribonuclear particle to form the RNA-induced silencing complex, RISC, which mediates gene silencing.
  • a miR referred to by number herein can refer to either of the two mature microRNAs originating from opposite arms of the same pre-miRNA (e.g., either the 3p or 5p microRNA). All miRs referred to herein are intended to include both the 3p and 5p arms/sequences, unless particularly specified by the 3p or 5p designation.
  • microRNA binding site refers to a sequence within a polynucleotide, e.g., within a DNA or within an RNA transcript, including in the 5′UTR and/or 3′UTR, that has sufficient complementarity to all or a region of a miRNA to interact with, associate with or bind to the miRNA.
  • a polynucleotide of the invention comprising an ORF encoding a polypeptide of interest and further comprises one or more miRNA binding site(s).
  • a 5′UTR and/or 3′UTR of the polynucleotide e.g., a ribonucleic acid (RNA), e.g., a messenger RNA (mRNA)
  • RNA ribonucleic acid
  • mRNA messenger RNA
  • a miRNA binding site having sufficient complementarity to a miRNA refers to a degree of complementarity sufficient to facilitate miRNA-mediated regulation of a polynucleotide, e.g., miRNA-mediated translational repression or degradation of the polynucleotide.
  • a miRNA binding site having sufficient complementarity to the miRNA refers to a degree of complementarity sufficient to facilitate miRNA-mediated degradation of the polynucleotide, e.g., miRNA-guided RNA-induced silencing complex (RISC)-mediated cleavage of mRNA.
  • miRNA-guided RNA-induced silencing complex RISC
  • the miRNA binding site can have complementarity to, for example, a 19-25 nucleotide long miRNA sequence, to a long 19-23 nucleotide miRNA sequence, or to a long 22 nucleotide miRNA sequence.
  • a miRNA binding site can be complementary to only a portion of a miRNA, e.g., to a portion less than 1, 2, 3, or 4 nucleotides of the full length of a naturally-occurring miRNA sequence, or to a portion less than 1, 2, 3, or 4 nucleotides shorter than a naturally-occurring miRNA sequence.
  • Full or complete complementarity e.g., full complementarity or complete complementarity over all or a significant portion of the length of a naturally-occurring miRNA is preferred when the desired regulation is mRNA degradation.
  • a miRNA binding site includes a sequence that has complementarity (e.g., partial or complete complementarity) with an miRNA seed sequence. In some embodiments, the miRNA binding site includes a sequence that has complete complementarity with a miRNA seed sequence. In some embodiments, a miRNA binding site includes a sequence that has complementarity (e.g., partial or complete complementarity) with an miRNA sequence. In some embodiments, the miRNA binding site includes a sequence that has complete complementarity with a miRNA sequence. In some embodiments, a miRNA binding site has complete complementarity with a miRNA sequence but for 1, 2, or 3 nucleotide substitutions, terminal additions, and/or truncations.
  • the miRNA binding site is the same length as the corresponding miRNA. In other embodiments, the miRNA binding site is one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve nucleotide(s) shorter than the corresponding miRNA at the 5′ terminus, the 3′ terminus, or both. In still other embodiments, the microRNA binding site is two nucleotides shorter than the corresponding microRNA at the 5′ terminus, the 3′ terminus, or both. The miRNA binding sites that are shorter than the corresponding miRNAs are still capable of degrading the mRNA incorporating one or more of the miRNA binding sites or preventing the mRNA from translation.
  • the miRNA binding site binds the corresponding mature miRNA that is part of an active RISC containing Dicer. In another embodiment, binding of the miRNA binding site to the corresponding miRNA in RISC degrades the mRNA containing the miRNA binding site or prevents the mRNA from being translated. In some embodiments, the miRNA binding site has sufficient complementarity to miRNA so that a RISC complex comprising the miRNA cleaves the polynucleotide comprising the miRNA binding site. In other embodiments, the miRNA binding site has imperfect complementarity so that a RISC complex comprising the miRNA induces instability in the polynucleotide comprising the miRNA binding site. In another embodiment, the miRNA binding site has imperfect complementarity so that a RISC complex comprising the miRNA represses transcription of the polynucleotide comprising the miRNA binding site.
  • the miRNA binding site has one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve mismatch(es) from the corresponding miRNA.
  • the miRNA binding site has at least about ten, at least about eleven, at least about twelve, at least about thirteen, at least about fourteen, at least about fifteen, at least about sixteen, at least about seventeen, at least about eighteen, at least about nineteen, at least about twenty, or at least about twenty-one contiguous nucleotides complementary to at least about ten, at least about eleven, at least about twelve, at least about thirteen, at least about fourteen, at least about fifteen, at least about sixteen, at least about seventeen, at least about eighteen, at least about nineteen, at least about twenty, or at least about twenty-one, respectively, contiguous nucleotides of the corresponding miRNA.
  • the polynucleotide By engineering one or more miRNA binding sites into a polynucleotide of the invention, the polynucleotide can be targeted for degradation or reduced translation, provided the miRNA in question is available. This can reduce off-target effects upon delivery of the polynucleotide. For example, if a polynucleotide of the invention is not intended to be delivered to a tissue or cell but ends up is said tissue or cell, then a miRNA abundant in the tissue or cell can inhibit the expression of the gene of interest if one or multiple binding sites of the miRNA are engineered into the 5′UTR and/or 3′UTR of the polynucleotide.
  • incorporation of one or more miRNA binding sites into an mRNA of the disclosure may reduce the hazard of off-target effects upon nucleic acid molecule delivery and/or enable tissue-specific regulation of expression of a polypeptide encoded by the mRNA.
  • incorporation of one or more miRNA binding sites into an mRNA of the disclosure can modulate immune responses upon nucleic acid delivery in vivo.
  • incorporation of one or more miRNA binding sites into an mRNA of the disclosure can modulate accelerated blood clearance (ABC) of lipid-comprising compounds and compositions described herein.
  • ABS accelerated blood clearance
  • miRNA binding sites can be removed from polynucleotide sequences in which they naturally occur in order to increase protein expression in specific tissues.
  • a binding site for a specific miRNA can be removed from a polynucleotide to improve protein expression in tissues or cells containing the miRNA.
  • Regulation of expression in multiple tissues can be accomplished through introduction or removal of one or more miRNA binding sites, e.g., one or more distinct miRNA binding sites.
  • the decision whether to remove or insert a miRNA binding site can be made based on miRNA expression patterns and/or their profilings in tissues and/or cells in development and/or disease. Identification of MiRNAs, miRNA binding sites, and their expression patterns and role in biology have been reported (e.g., Bonauer et al., Curr Drug Targets 2010 11:943-949; Anand and Cheresh Curr Opin Hematol 2011 18:171-176; Contreras and Rao Leukemia 2012 26:404-413 (2011 Dec. 20.
  • miRNAs and miRNA binding sites can correspond to any known sequence, including non-limiting examples described in U.S. Publication Nos. 2014/0200261, 2005/0261218, and 2005/0059005, each of which are incorporated herein by reference in their entirety.
  • tissues where miRNA are known to regulate mRNA, and thereby protein expression include, but are not limited to, liver (miR-122), muscle (miR-133, miR-206, miR-208), endothelial cells (miR-17-92, miR-126), myeloid cells (miR-142-3p, miR-142-5p, miR-16, miR-21, miR-223, miR-24, miR-27), adipose tissue (let-7, miR-30c), heart (miR-1d, miR-149), kidney (miR-192, miR-194, miR-204), and lung epithelial cells (let-7, miR-133, miR-126).
  • liver miR-122
  • muscle miR-133, miR-206, miR-208
  • endothelial cells miR-17-92, miR-126
  • myeloid cells miR-142-3p, miR-142-5p, miR-16, miR-21, miR-223, miR
  • miRNAs are known to be differentially expressed in immune cells (also called hematopoietic cells), such as antigen presenting cells (APCs) (e.g., dendritic cells and macrophages), macrophages, monocytes, B lymphocytes, T lymphocytes, granulocytes, natural killer cells, etc.
  • APCs antigen presenting cells
  • Immune cell specific miRNAs are involved in immunogenicity, autoimmunity, the immune-response to infection, inflammation, as well as unwanted immune response after gene therapy and tissue/organ transplantation. Immune cells specific miRNAs also regulate many aspects of development, proliferation, differentiation and apoptosis of hematopoietic cells (immune cells).
  • miR-142 and miR-146 are exclusively expressed in immune cells, particularly abundant in myeloid dendritic cells. It has been demonstrated that the immune response to a polynucleotide can be shut-off by adding miR-142 binding sites to the 3′-UTR of the polynucleotide, enabling more stable gene transfer in tissues and cells. miR-142 efficiently degrades exogenous polynucleotides in antigen presenting cells and suppresses cytotoxic elimination of transduced cells (e.g., Annoni A et al., blood, 2009, 114, 5152-5161; Brown B D, et al., Nat med. 2006, 12(5), 585-591; Brown B D, et al., blood, 2007, 110(13): 4144-4152, each of which is incorporated herein by reference in its entirety).
  • An antigen-mediated immune response can refer to an immune response triggered by foreign antigens, which, when entering an organism, are processed by the antigen presenting cells and displayed on the surface of the antigen presenting cells. T cells can recognize the presented antigen and induce a cytotoxic elimination of cells that express the antigen.
  • Introducing a miR-142 binding site into the 5′UTR and/or 3′UTR of a polynucleotide of the invention can selectively repress gene expression in antigen presenting cells through miR-142 mediated degradation, limiting antigen presentation in antigen presenting cells (e.g., dendritic cells) and thereby preventing antigen-mediated immune response after the delivery of the polynucleotide.
  • the polynucleotide is then stably expressed in target tissues or cells without triggering cytotoxic elimination.
  • binding sites for miRNAs that are known to be expressed in immune cells can be engineered into a polynucleotide of the invention to suppress the expression of the polynucleotide in antigen presenting cells through miRNA mediated RNA degradation, subduing the antigen-mediated immune response. Expression of the polynucleotide is maintained in non-immune cells where the immune cell specific miRNAs are not expressed.
  • any miR-122 binding site can be removed and a miR-142 (and/or mirR-146) binding site can be engineered into the 5′UTR and/or 3′UTR of a polynucleotide of the invention.
  • a polynucleotide of the invention can include a further negative regulatory element in the 5′UTR and/or 3′UTR, either alone or in combination with miR-142 and/or miR-146 binding sites.
  • the further negative regulatory element is a Constitutive Decay Element (CDE).
  • Immune cell specific miRNAs include, but are not limited to, hsa-let-7a-2-3p, hsa-let-7a-3p, hsa-7a-5p, hsa-let-7c, hsa-let-7e-3p, hsa-let-7e-5p, hsa-let-7g-3p, hsa-let-7g-5p, hsa-let-7i-3p, hsa-let-7i-5p, miR-10a-3p, miR-10a-5p, miR-1184, hsa-let-7f-1-3p, hsa-let-7f-2-5p, hsa-let-7f-5p, miR-125b-1-3p, miR-125b-2-3p, miR-125b-5p, miR-1279, miR-130a-3p, miR-130a-5p, miR-132-3p, miR-132-5p, miR-142-3p, miR-142-5p, miR-
  • novel miRNAs can be identified in immune cell through micro-array hybridization and microtome analysis (e.g., Jima D D et al, Blood, 2010, 116:e118-e127; Vaz C et al., BMC Genomics, 2010, 11, 288, the content of each of which is incorporated herein by reference in its entirety.)
  • miRNAs that are known to be expressed in the liver include, but are not limited to, miR-107, miR-122-3p, miR-122-5p, miR-1228-3p, miR-1228-5p, miR-1249, miR-129-5p, miR-1303, miR-151a-3p, miR-151a-5p, miR-152, miR-194-3p, miR-194-5p, miR-199a-3p, miR-199a-5p, miR-199b-3p, miR-199b-5p, miR-296-5p, miR-557, miR-581, miR-939-3p, and miR-939-5p.
  • MiRNA binding sites from any liver specific miRNA can be introduced to or removed from a polynucleotide of the invention to regulate expression of the polynucleotide in the liver.
  • Liver specific miRNA binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA binding sites in a polynucleotide of the invention.
  • miRNAs that are known to be expressed in the lung include, but are not limited to, let-7a-2-3p, let-7a-3p, let-7a-5p, miR-126-3p, miR-126-5p, miR-127-3p, miR-127-5p, miR-130a-3p, miR-130a-5p, miR-130b-3p, miR-130b-5p, miR-133a, miR-133b, miR-134, miR-18a-3p, miR-18a-5p, miR-18b-3p, miR-18b-5p, miR-24-1-5p, miR-24-2-5p, miR-24-3p, miR-296-3p, miR-296-5p, miR-32-3p, miR-337-3p, miR-337-5p, miR-381-3p, and miR-381-5p.
  • miRNA binding sites from any lung specific miRNA can be introduced to or removed from a polynucleotide of the invention to regulate expression of the polynucleotide in the lung.
  • Lung specific miRNA binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA binding sites in a polynucleotide of the invention.
  • miRNAs that are known to be expressed in the heart include, but are not limited to, miR-1, miR-133a, miR-133b, miR-149-3p, miR-149-5p, miR-186-3p, miR-186-5p, miR-208a, miR-208b, miR-210, miR-296-3p, miR-320, miR-451a, miR-451b, miR-499a-3p, miR-499a-5p, miR-499b-3p, miR-499b-5p, miR-744-3p, miR-744-5p, miR-92b-3p, and miR-92b-5p.
  • mMiRNA binding sites from any heart specific microRNA can be introduced to or removed from a polynucleotide of the invention to regulate expression of the polynucleotide in the heart.
  • Heart specific miRNA binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA binding sites in a polynucleotide of the invention.
  • miRNAs that are known to be expressed in the nervous system include, but are not limited to, miR-124-5p, miR-125a-3p, miR-125a-5p, miR-125b-1-3p, miR-125b-2-3p, miR-125b-5p, miR-1271-3p, miR-1271-5p, miR-128, miR-132-5p, miR-135a-3p, miR-135a-5p, miR-135b-3p, miR-135b-5p, miR-137, miR-139-5p, miR-139-3p, miR-149-3p, miR-149-5p, miR-153, miR-181c-3p, miR-181c-5p, miR-183-3p, miR-183-5p, miR-190a, miR-190b, miR-212-3p, miR-212-5p, miR-219-1-3p, miR-219-2-3p, miR-23a-3p, miR-23a-5p, miR-30
  • miRNAs enriched in the nervous system further include those specifically expressed in neurons, including, but not limited to, miR-132-3p, miR-132-3p, miR-148b-3p, miR-148b-5p, miR-151a-3p, miR-151a-5p, miR-212-3p, miR-212-5p, miR-320b, miR-320e, miR-323a-3p, miR-323a-5p, miR-324-5p, miR-325, miR-326, miR-328, miR-922 and those specifically expressed in glial cells, including, but not limited to, miR-1250, miR-219-1-3p, miR-219-2-3p, miR-219-5p, miR-23a-3p, miR-23a-5p, miR-3065-3p, miR-3065-5p, miR-30e-3p, miR-30e-5p, miR-32-5p, miR-338-5p, and miR-657.
  • miRNA binding sites from any CNS specific miRNA can be introduced to or removed from a polynucleotide of the invention to regulate expression of the polynucleotide in the nervous system.
  • Nervous system specific miRNA binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA binding sites in a polynucleotide of the invention.
  • miRNAs that are known to be expressed in the pancreas include, but are not limited to, miR-105-3p, miR-105-5p, miR-184, miR-195-3p, miR-195-5p, miR-196a-3p, miR-196a-5p, miR-214-3p, miR-214-5p, miR-216a-3p, miR-216a-5p, miR-30a-3p, miR-33a-3p, miR-33a-5p, miR-375, miR-7-1-3p, miR-7-2-3p, miR-493-3p, miR-493-5p, and miR-944.
  • MiRNA binding sites from any pancreas specific miRNA can be introduced to or removed from a polynucleotide of the invention to regulate expression of the polynucleotide in the pancreas.
  • Pancreas specific miRNA binding sites can be engineered alone or further in combination with immune cell (e.g. APC) miRNA binding sites in a polynucleotide of the invention.
  • miRNAs that are known to be expressed in the kidney include, but are not limited to, miR-122-3p, miR-145-5p, miR-17-5p, miR-192-3p, miR-192-5p, miR-194-3p, miR-194-5p, miR-20a-3p, miR-20a-5p, miR-204-3p, miR-204-5p, miR-210, miR-216a-3p, miR-216a-5p, miR-296-3p, miR-30a-3p, miR-30a-5p, miR-30b-3p, miR-30b-5p, miR-30c-1-3p, miR-30c-2-3p, miR30c-5p, miR-324-3p, miR-335-3p, miR-335-5p, miR-363-3p, miR-363-5p, and miR-562.
  • kidney specific miRNA binding sites from any kidney specific miRNA can be introduced to or removed from a polynucleotide of the invention to regulate expression of the polynucleotide in the kidney.
  • Kidney specific miRNA binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA binding sites in a polynucleotide of the invention.
  • miRNAs that are known to be expressed in the muscle include, but are not limited to, let-7g-3p, let-7g-5p, miR-1, miR-1286, miR-133a, miR-133b, miR-140-3p, miR-143-3p, miR-143-5p, miR-145-3p, miR-145-5p, miR-188-3p, miR-188-5p, miR-206, miR-208a, miR-208b, miR-25-3p, and miR-25-5p.
  • MiRNA binding sites from any muscle specific miRNA can be introduced to or removed from a polynucleotide of the invention to regulate expression of the polynucleotide in the muscle.
  • Muscle specific miRNA binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA binding sites in a polynucleotide of the invention.
  • miRNAs are also differentially expressed in different types of cells, such as, but not limited to, endothelial cells, epithelial cells, and adipocytes.
  • miRNAs that are known to be expressed in endothelial cells include, but are not limited to, let-7b-3p, let-7b-5p, miR-100-3p, miR-100-5p, miR-101-3p, miR-101-5p, miR-126-3p, miR-126-5p, miR-1236-3p, miR-1236-5p, miR-130a-3p, miR-130a-5p, miR-17-5p, miR-17-3p, miR-18a-3p, miR-18a-5p, miR-19a-3p, miR-19a-5p, miR-19b-1-5p, miR-19b-2-5p, miR-19b-3p, miR-20a-3p, miR-20a-5p, miR-217, miR-210, miR-21-3p, miR-21-5p, miR-221-3p, miR-221-5p, miR-222-3p, miR-222-5p, miR-23a-3p, miR-23a-5p, miR-296-5p
  • miRNA binding sites from any endothelial cell specific miRNA can be introduced to or removed from a polynucleotide of the invention to regulate expression of the polynucleotide in the endothelial cells.
  • miRNAs that are known to be expressed in epithelial cells include, but are not limited to, let-7b-3p, let-7b-5p, miR-1246, miR-200a-3p, miR-200a-5p, miR-200b-3p, miR-200b-5p, miR-200c-3p, miR-200c-5p, miR-338-3p, miR-429, miR-451a, miR-451b, miR-494, miR-802 and miR-34a, miR-34b-5p, miR-34c-5p, miR-449a, miR-449b-3p, miR-449b-5p specific in respiratory ciliated epithelial cells, let-7 family, miR-133a, miR-133b, miR-126 specific in lung epithelial cells, miR-382-3p, miR-382-5p specific in renal epithelial cells, and miR-762 specific in corneal epithelial cells. miRNA binding sites from any epithelial cell
  • a large group of miRNAs are enriched in embryonic stem cells, controlling stem cell self-renewal as well as the development and/or differentiation of various cell lineages, such as neural cells, cardiac, hematopoietic cells, skin cells, osteogenic cells and muscle cells (e.g., Kuppusamy K T et al., Curr. Mol Med, 2013, 13(5), 757-764; Vidigal JA and Ventura A, Semin Cancer Biol.
  • MiRNAs abundant in embryonic stem cells include, but are not limited to, let-7a-2-3p, let-a-3p, let-7a-5p, let7d-3p, let-7d-5p, miR-103a-2-3p, miR-103a-5p, miR-106b-3p, miR-106b-5p, miR-1246, miR-1275, miR-138-1-3p, miR-138-2-3p, miR-138-5p, miR-154-3p, miR-154-5p, miR-200c-3p, miR-200c-5p, miR-290, miR-301a-3p, miR-301a-5p, miR-302a-3p, miR-302a-5p, miR-302b-3p, miR-302b-5p, miR-302c-3p, miR-302c-5p, miR-302d-3p, miR-302d-5p, miR-302e, miR-367-3p, miR-367-5p, miR-369-3p
  • the binding sites of embryonic stem cell specific miRNAs can be included in or removed from the 3′UTR of a polynucleotide of the invention to modulate the development and/or differentiation of embryonic stem cells, to inhibit the senescence of stem cells in a degenerative condition (e.g. degenerative diseases), or to stimulate the senescence and apoptosis of stem cells in a disease condition (e.g. cancer stem cells).
  • a degenerative condition e.g. degenerative diseases
  • apoptosis of stem cells e.g. cancer stem cells
  • miRNAs are selected based on expression and abundance in immune cells of the hematopoietic lineage, such as B cells, T cells, macrophages, dendritic cells, and cells that are known to express TLR7/TLR8 and/or able to secrete cytokines such as endothelial cells and platelets.
  • the miRNA set thus includes miRs that may be responsible in part for the immunogenicity of these cells, and such that a corresponding miR-site incorporation in polynucleotides of the present invention (e.g., mRNAs) could lead to destabilization of the mRNA and/or suppression of translation from these mRNAs in the specific cell type.
  • Non-limiting representative examples include miR-142, miR-144, miR-150, miR-155 and miR-223, which are specific for many of the hematopoietic cells; miR-142, miR150, miR-16 and miR-223, which are expressed in B cells; miR-223, miR-451, miR-26a, miR-16, which are expressed in progenitor hematopoietic cells; and miR-126, which is expressed in plasmacytoid dendritic cells, platelets and endothelial cells.
  • tissue expression of miRs see e.g., Teruel-Montoya, R. et al. (2014) PLoS One 9:e102259; Landgraf, P.
  • Any one miR-site incorporation in the 3′UTR and/or 5′ UTR may mediate such effects in multiple cell types of interest (e.g., miR-142 is abundant in both B cells and dendritic cells).
  • polynucleotides of the invention contain two or more (e.g., two, three, four or more) miR bindings sites from: (i) the group consisting of miR-142, miR-144, miR-150, miR-155 and miR-223 (which are expressed in many hematopoietic cells); or (ii) the group consisting of miR-142, miR150, miR-16 and miR-223 (which are expressed in B cells); or the group consisting of miR-223, miR-451, miR-26a, miR-16 (which are expressed in progenitor hematopoietic cells).
  • miR-142, miR-144, miR-150, miR-155 and miR-223 which are expressed in many hematopoietic cells
  • miR-142, miR150, miR-16 and miR-223 which are expressed in B cells
  • miR-223, miR-451, miR-26a, miR-16 which are expressed in progenitor hema
  • miR-142 and miR-126 may also be beneficial to combine various miRs such that multiple cell types of interest are targeted at the same time (e.g., miR-142 and miR-126 to target many cells of the hematopoietic lineage and endothelial cells).
  • polynucleotides of the invention comprise two or more (e.g., two, three, four or more) miRNA bindings sites, wherein: (i) at least one of the miRs targets cells of the hematopoietic lineage (e.g., miR-142, miR-144, miR-150, miR-155 or miR-223) and at least one of the miRs targets plasmacytoid dendritic cells, platelets or endothelial cells (e.g., miR-126); or (ii) at least one of the miRs targets B cells (e.g., miR-142, miR150, miR-16 or miR-223) and at least one of the miRs targets plasmacytoid dendritic cells, platelets or endothelial cells (e.g., miR-126); or (iii) at least one of the miRs targets progenitor hematopoietic cells (e.g., miR
  • polynucleotides of the present invention can comprise one or more miRNA binding sequences that bind to one or more miRs that are expressed in conventional immune cells or any cell that expresses TLR7 and/or TLR8 and secrete pro-inflammatory cytokines and/or chemokines (e.g., in immune cells of peripheral lymphoid organs and/or splenocytes and/or endothelial cells).
  • miRNA binding sequences that bind to one or more miRs that are expressed in conventional immune cells or any cell that expresses TLR7 and/or TLR8 and secrete pro-inflammatory cytokines and/or chemokines (e.g., in immune cells of peripheral lymphoid organs and/or splenocytes and/or endothelial cells).
  • incorporation into an mRNA of one or more miRs that are expressed in conventional immune cells or any cell that expresses TLR7 and/or TLR8 and secrete pro-inflammatory cytokines and/or chemokines reduces or inhibits immune cell activation (e.g., B cell activation, as measured by frequency of activated B cells) and/or cytokine production (e.g., production of IL-6, IFN- ⁇ and/or TNF ⁇ ).
  • incorporation into an mRNA of one or more miRs that are expressed in conventional immune cells or any cell that expresses TLR7 and/or TLR8 and secrete pro-inflammatory cytokines and/or chemokines can reduce or inhibit an anti-drug antibody (ADA) response against a protein of interest encoded by the mRNA.
  • ADA anti-drug antibody
  • polynucleotides of the invention can comprise one or more miR binding sequences that bind to one or more miRNAs expressed in conventional immune cells or any cell that expresses TLR7 and/or TLR8 and secrete pro-inflammatory cytokines and/or chemokines (e.g., in immune cells of peripheral lymphoid organs and/or splenocytes and/or endothelial cells).
  • incorporation into an mRNA of one or more miR binding sites reduces or inhibits accelerated blood clearance (ABC) of the lipid-comprising compound or composition for use in delivering the mRNA.
  • incorporation of one or more miR binding sites into an mRNA reduces serum levels of anti-PEG anti-IgM (e.g, reduces or inhibits the acute production of IgMs that recognize polyethylene glycol (PEG) by B cells) and/or reduces or inhibits proliferation and/or activation of plasmacytoid dendritic cells following administration of a lipid-comprising compound or composition comprising the mRNA.
  • serum levels of anti-PEG anti-IgM e.g, reduces or inhibits the acute production of IgMs that recognize polyethylene glycol (PEG) by B cells
  • PEG polyethylene glycol
  • miR sequences may correspond to any known microRNA expressed in immune cells, including but not limited to those taught in US Publication US2005/0261218 and US Publication US2005/0059005, the contents of which are incorporated herein by reference in their entirety.
  • Non-limiting examples of miRs expressed in immune cells include those expressed in spleen cells, myeloid cells, dendritic cells, plasmacytoid dendritic cells, B cells, T cells and/or macrophages.
  • miR-142-3p, miR-142-5p, miR-16, miR-21, miR-223, miR-24 and miR-27 are expressed in myeloid cells
  • miR-155 is expressed in dendritic cells
  • miR-146 is upregulated in macrophages upon TLR stimulation
  • miR-126 is expressed in plasmacytoid dendritic cells.
  • the miR(s) is expressed abundantly or preferentially in immune cells.
  • miR-142 miR-142-3p and/or miR-142-5p
  • miR-126 miR-126-3p and/or miR-126-5p
  • miR-146 miR-146-3p and/or miR-146-5p
  • miR-155 miR-155-3p and/or miR155-5p
  • polynucleotides of the present invention comprise at least one microRNA binding site for a miR selected from the group consisting of miR-142, miR-146, miR-155, miR-126, miR-16, miR-21, miR-223, miR-24 and miR-27.
  • the mRNA comprises at least two miR binding sites for microRNAs expressed in immune cells.
  • the polynucleotide of the invention comprises 1-4, one, two, three or four miR binding sites for microRNAs expressed in immune cells.
  • the polynucleotide of the invention comprises three miR binding sites.
  • miR binding sites can be for microRNAs selected from the group consisting of miR-142, miR-146, miR-155, miR-126, miR-16, miR-21, miR-223, miR-24, miR-27, and combinations thereof.
  • the polynucleotide of the invention comprises two or more (e.g., two, three, four) copies of the same miR binding site expressed in immune cells, e.g., two or more copies of a miR binding site selected from the group of miRs consisting of miR-142, miR-146, miR-155, miR-126, miR-16, miR-21, miR-223, miR-24, miR-27.
  • the polynucleotide of the invention comprises three copies of the same miR binding site.
  • use of three copies of the same miR binding site can exhibit beneficial properties as compared to use of a single miR binding site.
  • Non-limiting examples of sequences for 3′ UTRs containing three miR bindings sites are shown in SEQ ID NO: 165 (three miR-142-3p binding sites), and SEQ ID NO: 167 (three miR-142-5p binding sites).
  • the polynucleotide of the invention comprises two or more (e.g., two, three, four) copies of at least two different miR binding sites expressed in immune cells.
  • Non-limiting examples of sequences of 3′ UTRs containing two or more different miR binding sites are shown in SEQ ID NO: 164 (one miR-142-3p binding site and one miR-126-3p binding site), SEQ ID NO: 168 (two miR-142-5p binding sites and one miR-142-3p binding sites) and SEQ ID NO: 171 (two miR-155-5p binding sites and one miR-142-3p binding sites).
  • the polynucleotide of the invention comprises at least two miR binding sites for microRNAs expressed in immune cells, wherein one of the miR binding sites is for miR-142-3p.
  • the polynucleotide of the invention comprises binding sites for miR-142-3p and miR-155 (miR-155-3p or miR-155-5p), miR-142-3p and miR-146 (miR-146-3 or miR-146-5p), or miR-142-3p and miR-126 (miR-126-3p or miR-126-5p).
  • the polynucleotide of the invention comprises at least two miR binding sites for microRNAs expressed in immune cells, wherein one of the miR binding sites is for miR-126-3p.
  • the polynucleotide of the invention comprises binding sites for miR-126-3p and miR-155 (miR-155-3p or miR-155-5p), miR-126-3p and miR-146 (miR-146-3p or miR-146-5p), or miR-126-3p and miR-142 (miR-142-3p or miR-142-5p).
  • the polynucleotide of the invention comprises at least two miR binding sites for microRNAs expressed in immune cells, wherein one of the miR binding sites is for miR-142-5p.
  • the polynucleotide of the invention comprises binding sites for miR-142-5p and miR-155 (miR-155-3p or miR-155-5p), miR-142-5p and miR-146 (miR-146-3 or miR-146-5p), or miR-142-5p and miR-126 (miR-126-3p or miR-126-5p).
  • the polynucleotide of the invention comprises at least two miR binding sites for microRNAs expressed in immune cells, wherein one of the miR binding sites is for miR-155-5p.
  • the polynucleotide of the invention comprises binding sites for miR-155-5p and miR-142 (miR-142-3p or miR-142-5p), miR-155-5p and miR-146 (miR-146-3 or miR-146-5p), or miR-155-5p and miR-126 (miR-126-3p or miR-126-5p).
  • miRNA can also regulate complex biological processes such as angiogenesis (e.g., miR-132) (Anand and Cheresh Curr Opin Hematol 2011 18:171-176).
  • angiogenesis e.g., miR-132
  • miRNA binding sites that are involved in such processes can be removed or introduced, in order to tailor the expression of the polynucleotides to biologically relevant cell types or relevant biological processes.
  • the polynucleotides of the invention are defined as auxotrophic polynucleotides.
  • a polynucleotide of the invention comprises a miRNA binding site, wherein the miRNA binding site comprises one or more nucleotide sequences selected from TABLE 3, including one or more copies of any one or more of the miRNA binding site sequences.
  • a polynucleotide of the invention further comprises at least one, two, three, four, five, six, seven, eight, nine, ten, or more of the same or different miRNA binding sites selected from TABLE 3, including any combination thereof.
  • the miRNA binding site binds to miR-142 or is complementary to miR-142. In some embodiments, the miR-142 comprises SEQ ID NO:34. In some embodiments, the miRNA binding site binds to miR-142-3p or miR-142-5p. In some embodiments, the miR-142-3p binding site comprises SEQ ID NO:35. In some embodiments, the miR-142-5p binding site comprises SEQ ID NO:37. In some embodiments, the miRNA binding site comprises a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:36 or SEQ ID NO:38.
  • the miRNA binding site binds to miR-126 or is complementary to miR-126. In some embodiments, the miR-126 comprises SEQ ID NO: 156. In some embodiments, the miRNA binding site binds to miR-126-3p or miR-126-5p. In some embodiments, the miR-126-3p binding site comprises SEQ ID NO: 158. In some embodiments, the miR-126-5p binding site comprises SEQ ID NO: 160. In some embodiments, the miRNA binding site comprises a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 158 or SEQ ID NO: 160.
  • the 3′ UTR comprises two miRNA binding sites, wherein a first miRNA binding site binds to miR-142 and a second miRNA binding site binds to miR-126.
  • the 3′ UTR binding to miR-142 and miR-126 comprises, consists, or consists essentially of the sequence of SEQ ID NO: 149 or 150.
  • a miRNA binding site is inserted in the polynucleotide of the invention in any position of the polynucleotide (e.g., the 5′UTR and/or 3′UTR).
  • the 5′UTR comprises a miRNA binding site.
  • the 3′UTR comprises a miRNA binding site.
  • the 5′UTR and the 3′UTR comprise a miRNA binding site.
  • the insertion site in the polynucleotide can be anywhere in the polynucleotide as long as the insertion of the miRNA binding site in the polynucleotide does not interfere with the translation of a functional polypeptide in the absence of the corresponding miRNA; and in the presence of the miRNA, the insertion of the miRNA binding site in the polynucleotide and the binding of the miRNA binding site to the corresponding miRNA are capable of degrading the polynucleotide or preventing the translation of the polynucleotide.
  • a miRNA binding site is inserted in at least about 30 nucleotides downstream from the stop codon of an ORF in a polynucleotide of the invention comprising the ORF. In some embodiments, a miRNA binding site is inserted in at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 35 nucleotides, at least about 40 nucleotides, at least about 45 nucleotides, at least about 50 nucleotides, at least about 55 nucleotides, at least about 60 nucleotides, at least about 65 nucleotides, at least about 70 nucleotides, at least about 75 nucleotides, at least about 80 nucleotides, at least about 85 nucleotides, at least about 90 nucleotides, at least about 95 nucleotides, or at least about 100 nucle
  • a miRNA binding site is inserted in about 10 nucleotides to about 100 nucleotides, about 20 nucleotides to about 90 nucleotides, about 30 nucleotides to about 80 nucleotides, about 40 nucleotides to about 70 nucleotides, about 50 nucleotides to about 60 nucleotides, about 45 nucleotides to about 65 nucleotides downstream from the stop codon of an ORF in a polynucleotide of the invention.
  • a miRNA binding site is inserted within the 3′ UTR immediately following the stop codon of the coding region within the polynucleotide of the invention, e.g., mRNA. In some embodiments, if there are multiple copies of a stop codon in the construct, a miRNA binding site is inserted immediately following the final stop codon. in some embodiments, a miRNA binding site is inserted further downstream of the stop codon, in which case there are 3′ UTR bases between the stop codon and the miR binding site(s).
  • three non-limiting examples of possible insertion sites for a miR in a 3′ UTR are shown in SEQ ID NOs: 57, 58, and 172, which show a 3′ UTR sequence with a miR-142-3p site inserted in one of three different possible insertion sites, respectively, within the 3′ UTR.
  • one or more miRNA binding sites can be positioned within the 5′ UTR at one or more possible insertion sites.
  • three non-limiting examples of possible insertion sites for a miR in a 5′ UTR are shown in SEQ ID NOs: 189, 190, and 191, which show a 5′ UTR sequence with a miR-142-3p site inserted into one of three different possible insertion sites, respectively, within the 5′ UTR.
  • a codon optimized open reading frame encoding a polypeptide of interest comprises a stop codon and the at least one microRNA binding site is located within the 3′ UTR 1-100 nucleotides after the stop codon.
  • the codon optimized open reading frame encoding the polypeptide of interest comprises a stop codon and the at least one microRNA binding site for a miR expressed in immune cells is located within the 3′ UTR 30-50 nucleotides after the stop codon.
  • the codon optimized open reading frame encoding the polypeptide of interest comprises a stop codon and the at least one microRNA binding site for a miR expressed in immune cells is located within the 3′ UTR at least 50 nucleotides after the stop codon.
  • the codon optimized open reading frame encoding the polypeptide of interest comprises a stop codon and the at least one microRNA binding site for a miR expressed in immune cells is located within the 3′ UTR immediately after the stop codon, or within the 3′ UTR 15-20 nucleotides after the stop codon or within the 3′ UTR 70-80 nucleotides after the stop codon.
  • the 3′UTR comprises more than one miRNA bindingsite (e.g., 2-4 miRNA binding sites), wherein there can be a spacer region (e.g., of 10-100, 20-70 or 30-50 nucleotides in length) between each miRNA bindingsite.
  • the 3′ UTR comprises a spacer region between the end of the miRNA bindingsite(s) and the poly A tail nucleotides.
  • a spacer region of 10-100, 20-70 or 30-50 nucleotides in length can be situated between the end of the miRNA bindingsite(s) and the beginning of the poly A tail.
  • a codon optimized open reading frame encoding a polypeptide of interest comprises a start codon and the at least one microRNA binding site is located within the 5′ UTR 1-100 nucleotides before (upstream of) the start codon.
  • the codon optimized open reading frame encoding the polypeptide of interest comprises a start codon and the at least one microRNA binding site for a miR expressed in immune cells is located within the 5′ UTR 10-50 nucleotides before (upstream of) the start codon.
  • the codon optimized open reading frame encoding the polypeptide of interest comprises a start codon and the at least one microRNA binding site for a miR expressed in immune cells is located within the 5′ UTR at least 25 nucleotides before (upstream of) the start codon.
  • the codon optimized open reading frame encoding the polypeptide of interest comprises a start codon and the at least one microRNA binding site for a miR expressed in immune cells is located within the 5′ UTR immediately before the start codon, or within the 5′ UTR 15-20 nucleotides before the start codon or within the 5′ UTR 70-80 nucleotides before the start codon.
  • the 5′UTR comprises more than one miRNA bindingsite (e.g., 2-4 miRNA binding sites), wherein there can be a spacer region (e.g., of 10-100, 20-70 or 30-50 nucleotides in length) between each miRNA bindingsite.
  • a spacer region e.g., of 10-100, 20-70 or 30-50 nucleotides in length
  • the 3′ UTR comprises more than one stop codon, wherein at least one miRNA bindingsite is positioned downstream of the stop codons.
  • a 3′ UTR can comprise 1, 2 or 3 stop codons.
  • triple stop codons that can be used include: UGAUAAUAG, UGAUAGUAA, UAAUGAUAG, UGAUAAUAA, UGAUAGUAG, UAAUGAUGA, UAAUAGUAG, UGAUGAUGA, UAAUAAUAA and UAGUAGUAG.
  • 1, 2, 3 or 4 miRNA binding sites e.g., miR-142-3p binding sites
  • these binding sites can be positioned directly next to each other in the construct (i.e., one after the other) or, alternatively, spacer nucleotides can be positioned between each binding site.
  • the 3′ UTR comprises three stop codons with a single miR-142-3p binding site located downstream of the 3rd stop codon.
  • Non-limiting examples of sequences of 3′ UTR having three stop codons and a single miR-142-3p binding site located at different positions downstream of the final stop codon are shown in SEQ ID NOs: 57, 58, 62, and 172.
  • the polynucleotide of the invention comprises a 5′ UTR, a codon optimized open reading frame encoding a polypeptide of interest, a 3′ UTR comprising the at least one miRNA binding site for a miR expressed in immune cells, and a 3′ tailing region of linked nucleosides.
  • the 3′ UTR comprises 1-4, at least two, one, two, three or four miRNA binding sites for miRs expressed in immune cells, preferably abundantly or preferentially expressed in immune cells.
  • the at least one miRNA expressed in immune cells is a miR-142-3p microRNA binding site.
  • the miR-142-3p microRNA binding site comprises the sequence shown in SEQ ID NO: 36.
  • the 3′ UTR of the mRNA comprising the miR-142-3p microRNA binding site comprises the sequence shown in SEQ ID NO: 161.
  • the at least one miRNA expressed in immune cells is a miR-126 microRNA binding site.
  • the miR-126 binding site is a miR-126-3p binding site.
  • the miR-126-3p microRNA binding site comprises the sequence shown in SEQ ID NO: 158.
  • the 3′ UTR of the mRNA of the invention comprising the miR-126-3p microRNA binding site comprises the sequence shown in SEQ ID NO: 162.
  • Non-limiting exemplary sequences for miRs to which a microRNA binding site(s) of the disclosure can bind include the following: miR-142-3p (SEQ ID NO: 35), miR-142-5p (SEQ ID NO: 37), miR-146-3p (SEQ ID NO: 173), miR-146-5p (SEQ ID NO: 174), miR-155-3p (SEQ ID NO: 175), miR-155-5p (SEQ ID NO: 176), miR-126-3p (SEQ ID NO: 157), miR-126-5p (SEQ ID NO: 159), miR-16-3p (SEQ ID NO: 177), miR-16-5p (SEQ ID NO: 178), miR-21-3p (SEQ ID NO: 179), miR-21-5p (SEQ ID NO: 180), miR-223-3p (SEQ ID NO: 181), miR-223-5p (SEQ ID NO: 182), miR-24-3p (SEQ ID NO: 183), miR-24-5p (SEQ
  • miR sequences expressed in immune cells are known and available in the art, for example at the University of Manchester's microRNA database, miRBase. Sites that bind any of the aforementioned miRs can be designed based on Watson-Crick complementarity to the miR, typically 100% complementarity to the miR, and inserted into an mRNA construct of the disclosure as described herein.
  • a polynucleotide of the present invention (e.g., and mRNA, e.g., the 3′ UTR thereof) can comprise at least one miRNA bindingsite to thereby reduce or inhibit accelerated blood clearance, for example by reducing or inhibiting production of IgMs, e.g., against PEG, by B cells and/or reducing or inhibiting proliferation and/or activation of pDCs, and can comprise at least one miRNA bindingsite for modulating tissue expression of an encoded protein of interest.
  • miRNA gene regulation can be influenced by the sequence surrounding the miRNA such as, but not limited to, the species of the surrounding sequence, the type of sequence (e.g., heterologous, homologous, exogenous, endogenous, or artificial), regulatory elements in the surrounding sequence and/or structural elements in the surrounding sequence.
  • the miRNA can be influenced by the 5′UTR and/or 3′UTR.
  • a non-human 3′UTR can increase the regulatory effect of the miRNA sequence on the expression of a polypeptide of interest compared to a human 3′UTR of the same sequence type.
  • other regulatory elements and/or structural elements of the 5′UTR can influence miRNA mediated gene regulation.
  • a regulatory element and/or structural element is a structured IRES (Internal Ribosome Entry Site) in the 5′UTR, which is necessary for the binding of translational elongation factors to initiate protein translation. EIF4A 2 binding to this secondarily structured element in the 5′-UTR is necessary for miRNA mediated gene expression (Meijer H A et al., Science, 2013, 340, 82-85, herein incorporated by reference in its entirety).
  • the polynucleotides of the invention can further include this structured 5′UTR in order to enhance microRNA mediated gene regulation.
  • At least one miRNA binding site can be engineered into the 3′UTR of a polynucleotide of the invention.
  • at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more miRNA binding sites can be engineered into a 3′UTR of a polynucleotide of the invention.
  • 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 2, or 1 miRNA binding sites can be engineered into the 3′UTR of a polynucleotide of the invention.
  • miRNA binding sites incorporated into a polynucleotide of the invention can be the same or can be different miRNA sites.
  • a combination of different miRNA binding sites incorporated into a polynucleotide of the invention can include combinations in which more than one copy of any of the different miRNA sites are incorporated.
  • miRNA binding sites incorporated into a polynucleotide of the invention can target the same or different tissues in the body.
  • tissue-, cell-type-, or disease-specific miRNA binding sites in the 3′-UTR of a polynucleotide of the invention through the introduction of tissue-, cell-type-, or disease-specific miRNA binding sites in the 3′-UTR of a polynucleotide of the invention, the degree of expression in specific cell types (e.g., myeloid cells, endothelial cells, etc.) can be reduced.
  • tissue-, cell-type-, or disease-specific miRNA binding sites in the 3′-UTR of a polynucleotide of the invention the degree of expression in specific cell types (e.g., myeloid cells, endothelial cells, etc.) can be reduced.
  • specific cell types e.g., myeloid cells, endothelial cells, etc.
  • a miRNA binding site can be engineered near the 5′ terminus of the 3′UTR, about halfway between the 5′ terminus and 3′ terminus of the 3′UTR and/or near the 3′ terminus of the 3′UTR in a polynucleotide of the invention.
  • a miRNA binding site can be engineered near the 5′ terminus of the 3′UTR and about halfway between the 5′ terminus and 3′ terminus of the 3′UTR.
  • a miRNA binding site can be engineered near the 3′ terminus of the 3′UTR and about halfway between the 5′ terminus and 3′ terminus of the 3′UTR.
  • a miRNA binding site can be engineered near the 5′ terminus of the 3′UTR and near the 3′ terminus of the 3′UTR.
  • a 3′UTR can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 miRNA binding sites.
  • the miRNA binding sites can be complementary to a miRNA, miRNA seed sequence, and/or miRNA sequences flanking the seed sequence.
  • the expression of a polynucleotide of the invention can be controlled by incorporating at least one sensor sequence in the polynucleotide and formulating the polynucleotide for administration.
  • a polynucleotide of the invention can be targeted to a tissue or cell by incorporating a miRNA binding site and formulating the polynucleotide in a lipid nanoparticle comprising a ionizable lipid, including any of the lipids described herein.
  • a polynucleotide of the invention can be engineered for more targeted expression in specific tissues, cell types, or biological conditions based on the expression patterns of miRNAs in the different tissues, cell types, or biological conditions.
  • tissue-specific miRNA binding sites Through introduction of tissue-specific miRNA binding sites, a polynucleotide of the invention can be designed for optimal protein expression in a tissue or cell, or in the context of a biological condition.
  • a polynucleotide of the invention can be designed to incorporate miRNA binding sites that either have 100% identity to known miRNA seed sequences or have less than 100% identity to miRNA seed sequences.
  • a polynucleotide of the invention can be designed to incorporate miRNA binding sites that have at least: 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to known miRNA seed sequences.
  • the miRNA seed sequence can be partially mutated to decrease miRNA binding affinity and as such result in reduced downmodulation of the polynucleotide.
  • the degree of match or mis-match between the miRNA binding site and the miRNA seed can act as a rheostat to more finely tune the ability of the miRNA to modulate protein expression.
  • mutation in the non-seed region of a miRNA binding site can also impact the ability of a miRNA to modulate protein expression.
  • a miRNA sequence can be incorporated into the loop of a stem loop.
  • a miRNA seed sequence can be incorporated in the loop of a stem loop and a miRNA binding site can be incorporated into the 5′ or 3′ stem of the stem loop.
  • a translation enhancer element can be incorporated on the 5′end of the stem of a stem loop and a miRNA seed can be incorporated into the stem of the stem loop.
  • a TEE can be incorporated on the 5′ end of the stem of a stem loop, a miRNA seed can be incorporated into the stem of the stem loop and a miRNA binding site can be incorporated into the 3′ end of the stem or the sequence after the stem loop.
  • the miRNA seed and the miRNA binding site can be for the same and/or different miRNA sequences.
  • the incorporation of a miRNA sequence and/or a TEE sequence changes the shape of the stem loop region which can increase and/or decrease translation.
  • a miRNA sequence and/or a TEE sequence changes the shape of the stem loop region which can increase and/or decrease translation.
  • the 5′-UTR of a polynucleotide of the invention can comprise at least one miRNA sequence.
  • the miRNA sequence can be, but is not limited to, a 19 or 22 nucleotide sequence and/or a miRNA sequence without the seed.
  • the miRNA sequence in the 5′UTR can be used to stabilize a polynucleotide of the invention described herein.
  • a miRNA sequence in the 5′UTR of a polynucleotide of the invention can be used to decrease the accessibility of the site of translation initiation such as, but not limited to a start codon.
  • a site of translation initiation such as, but not limited to a start codon.
  • LNA antisense locked nucleic acid
  • EJCs exon-junction complexes
  • a polynucleotide of the invention can comprise a miRNA sequence, instead of the LNA or EJC sequence described by Matsuda et al, near the site of translation initiation in order to decrease the accessibility to the site of translation initiation.
  • the site of translation initiation can be prior to, after or within the miRNA sequence.
  • the site of translation initiation can be located within a miRNA sequence such as a seed sequence or binding site.
  • a polynucleotide of the invention can include at least one miRNA in order to dampen the antigen presentation by antigen presenting cells.
  • the miRNA can be the complete miRNA sequence, the miRNA seed sequence, the miRNA sequence without the seed, or a combination thereof.
  • a miRNA incorporated into a polynucleotide of the invention can be specific to the hematopoietic system.
  • a miRNA incorporated into a polynucleotide of the invention to dampen antigen presentation is miR-142-3p.
  • a polynucleotide of the invention can include at least one miRNA in order to dampen expression of the encoded polypeptide in a tissue or cell of interest.
  • a polynucleotide of the invention can include at least one miR-142-3p binding site, miR-142-3p seed sequence, miR-142-3p binding site without the seed, miR-142-5p binding site, miR-142-5p seed sequence, miR-142-5p binding site without the seed, miR-146 binding site, miR-146 seed sequence and/or miR-146 binding site without the seed sequence.
  • a polynucleotide of the invention can comprise at least one miRNA binding site in the 3′UTR in order to selectively degrade mRNA therapeutics in the immune cells to subdue unwanted immunogenic reactions caused by therapeutic delivery.
  • the miRNA binding site can make a polynucleotide of the invention more unstable in antigen presenting cells.
  • these miRNAs include mir-142-5p, mir-142-3p, mir-146a-5p, and mir-146-3p.
  • a polynucleotide of the invention comprises at least one miRNA sequence in a region of the polynucleotide that can interact with a RNA binding protein.
  • the polynucleotide of the invention e.g., a RNA, e.g., an mRNA
  • a RNA e.g., an mRNA
  • a sequence-optimized nucleotide sequence e.g., an ORF
  • a PBGD polypeptide e.g., the wild-type sequence, functional fragment, or variant thereof
  • a miRNA binding site e.g., a miRNA binding site that binds to miR-142
  • the polynucleotide of the invention comprises a uracil-modified sequence encoding a PBGD polypeptide disclosed herein and a miRNA binding site disclosed herein, e.g., a miRNA binding site that binds to miR-142 and/or a miRNA binding site that binds to miR-126.
  • the polynucleotide of the invention comprises a uracil-modified sequence encoding a polypeptide disclosed herein and a miRNA binding site disclosed herein, e.g., a miRNA binding site that binds to miR-142miR-126, miR-142, miR-144, miR-146, miR-150, miR-155, miR-16, miR-21, miR-223, miR-24, miR-27 or miR-26a.
  • the miRNA binding site binds to miR126-3p, miR-142-3p, miR-142-5p, or miR-155.
  • the polynucleotide of the invention comprises a uracil-modified sequence encoding a polypeptide disclosed herein and at least two different microRNA binding sites, wherein the microRNA is expressed in an immune cell of hematopoietic lineage or a cell that expresses TLR7 and/or TLR8 and secretes pro-inflammatory cytokines and/or chemokines, and wherein the polynucleotide comprises one or more modified nucleobases.
  • the uracil-modified sequence encoding a PBGD polypeptide comprises at least one chemically modified nucleobase, e.g., 5-methoxyuracil.
  • At least 95% of a type of nucleobase (e.g., uracil) in a uracil-modified sequence encoding a PBGD polypeptide of the invention are modified nucleobases.
  • at least 95% of uricil in a uracil-modified sequence encoding a PBGD polypeptide is 5-methoxyuridine.
  • the polynucleotide (e.g., a RNA, e.g., an mRNA) disclosed herein, e.g., comprising an miRNA binding site, is formulated with a delivery agent comprising, e.g., a compound having the Formula (I), e.g., any of Compounds 1-232, e.g., Compound 18; a compound having the Formula (III), (IV), (V), or (VI), e.g., any of Compounds 233-342, e.g., Compound 236; or a compound having the Formula (VIII), e.g., any of Compounds 419-428, e.g., Compound 428, or any combination thereof.
  • the delivery agent comprises Compound 18, DSPC, Cholesterol, and Compound 428, e.g., with a mole ratio of about 50:10:38.5:1.5.
  • a polynucleotide of the present invention e.g., a polynucleotide comprising a nucleotide sequence encoding a PBGD polypeptide of the invention
  • a polynucleotide of the present invention further comprises a 3′ UTR.
  • 3′-UTR is the section of mRNA that immediately follows the translation termination codon and often contains regulatory regions that post-transcriptionally influence gene expression. Regulatory regions within the 3′-UTR can influence polyadenylation, translation efficiency, localization, and stability of the mRNA.
  • the 3′-UTR useful for the invention comprises a binding site for regulatory proteins or microRNAs.
  • the 3′ UTR useful for the polynucleotides of the invention comprises a 3′UTR selected from the group consisting of SEQ ID NO: 57 to 81, 84, 149 to 151, 161 to 172, 192 to 199, or any combination thereof.
  • the 3′ UTR comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 149 to 151, or any combination thereof.
  • the 3′ UTR comprises a nucleic acid sequence of SEQ ID NO: 150.
  • the 3′ UTR comprises a nucleic acid sequence of SEQ ID NO: 151.
  • the 3′ UTR sequence useful for the invention comprises a nucleotide sequence at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to a sequence selected from the group consisting of SEQ ID NO: 57 to 81, 84, 149 to 151, 161 to 172, 192 to 199, or any combination thereof.
  • the invention also includes a polynucleotide that comprises both a 5′ Cap and a polynucleotide of the present invention (e.g., a polynucleotide comprising a nucleotide sequence encoding a PBGD polypeptide).
  • the 5′ cap structure of a natural mRNA is involved in nuclear export, increasing mRNA stability and binds the mRNA Cap Binding Protein (CBP), which is responsible for mRNA stability in the cell and translation competency through the association of CBP with poly(A) binding protein to form the mature cyclic mRNA species.
  • CBP mRNA Cap Binding Protein
  • the cap further assists the removal of 5′ proximal introns during mRNA splicing.
  • Endogenous mRNA molecules can be 5′-end capped generating a 5′-ppp-5′-triphosphate linkage between a terminal guanosine cap residue and the 5′-terminal transcribed sense nucleotide of the mRNA molecule.
  • This 5′-guanylate cap can then be methylated to generate an N7-methyl-guanylate residue.
  • the ribose sugars of the terminal and/or anteterminal transcribed nucleotides of the 5′ end of the mRNA can optionally also be 2′-O-methylated.
  • 5′-decapping through hydrolysis and cleavage of the guanylate cap structure can target a nucleic acid molecule, such as an mRNA molecule, for degradation.
  • the polynucleotides of the present invention incorporate a cap moiety.
  • polynucleotides of the present invention comprise a non-hydrolyzable cap structure preventing decapping and thus increasing mRNA half-life. Because cap structure hydrolysis requires cleavage of 5′-ppp-5′ phosphorodiester linkages, modified nucleotides can be used during the capping reaction.
  • Vaccinia Capping Enzyme from New England Biolabs (Ipswich, Mass.) can be used with ⁇ -thio-guanosine nucleotides according to the manufacturer's instructions to create a phosphorothioate linkage in the 5′-ppp-5′ cap.
  • Additional modified guanosine nucleotides can be used such as a-methyl-phosphonate and seleno-phosphate nucleotides.
  • Additional modifications include, but are not limited to, 2′-O-methylation of the ribose sugars of 5′-terminal and/or 5′-anteterminal nucleotides of the polynucleotide (as mentioned above) on the 2′-hydroxyl group of the sugar ring.
  • 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 of the invention.
  • the Anti-Reverse Cap Analog (ARCA) cap contains two guanines linked by a 5′-5′-triphosphate group, wherein one guanine contains an N7 methyl group as well as a 3′-O-methyl group (i.e., N7,3′-O-dimethyl-guanosine-5′-triphosphate-5′-guanosine (m 7 G-3′mppp-G; which can equivalently be designated 3′ O-Me-m7G(5′)ppp(5′)G).
  • the 3′-0 atom of the other, unmodified, guanine becomes linked to the 5′-terminal nucleotide of the capped polynucleotide.
  • the N7- and 3′-O-methylated guanine provides the terminal moiety of the capped polynucleotide.
  • mCAP is similar to ARCA but has a 2′-O-methyl group on guanosine (i.e., N7,2′-O-dimethyl-guanosine-5′-triphosphate-5′-guanosine, m 7 Gm-ppp-G).
  • the cap is a dinucleotide cap analog.
  • the dinucleotide cap analog can be modified at different phosphate positions with a boranophosphate group or a phosphoroselenoate group such as the dinucleotide cap analogs described in U.S. Pat. No. 8,519,110, the contents of which are herein incorporated by reference in its entirety.
  • the cap is a cap analog is a N7-(4-chlorophenoxyethyl) substituted dinucleotide form of a cap analog known in the art and/or described herein.
  • Non-limiting examples of a N7-(4-chlorophenoxyethyl) substituted dinucleotide form of a cap analog include a N7-(4-chlorophenoxyethyl)-G(5′)ppp(5′)G and a N7-(4-chlorophenoxyethyl)-m 3′-O G(5′)ppp(5′)G cap analog (See, e.g., the various cap analogs and the methods of synthesizing cap analogs described in Kore et al.
  • a cap analog of the present invention is a 4-chloro/bromophenoxyethyl analog.
  • cap analogs allow for the concomitant capping of a polynucleotide or a region thereof, in an in vitro transcription reaction, up to 20% of transcripts can remain uncapped. This, as well as the structural differences of a cap analog from an endogenous 5′-cap structures of nucleic acids produced by the endogenous, cellular transcription machinery, can lead to reduced translational competency and reduced cellular stability.
  • Polynucleotides of the invention can also be capped post-manufacture (whether IVT or chemical synthesis), using enzymes, in order to generate more authentic 5′-cap structures.
  • the phrase “more authentic” refers to a feature that closely mirrors or mimics, either structurally or functionally, an endogenous or wild type feature.
  • a “more authentic” feature is better representative of an endogenous, wild-type, natural or physiological cellular function and/or structure as compared to synthetic features or analogs, etc., of the prior art, or which outperforms the corresponding endogenous, wild-type, natural or physiological feature in one or more respects.
  • Non-limiting examples of more authentic 5′cap structures of the present invention 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).

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US20230112986A1 (en) 2023-04-13

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