Treatment of primary ciliary dyskinesia with synthetic messenger RNA

Abstract

To provide polyribonucleotides capable of encoding selected proteins, and to provide compositions comprising the same.SOLUTION: Provided is a composition comprising a nucleic acid construct encoding a polypeptide at least 95% identical to a coiled-coil domain-containing 39 (CCDC39) polypeptide, the composition comprising a cationic lipid; a fusogenic lipid; cholesterol; and a polyethylene glycol (PEG) lipid, and the nucleic acid construct comprising 1-methylpseudouridine. The composition is for delivering to an epithelial cell a polypeptide at least 95% identical to a coiled-coil domain-containing 39 (CCDC39) polypeptide.SELECTED DRAWING: Figure 3

Description

Technical Field

[0001] Cross - reference to related applications This application claims priority under 35 U.S.C.§120 to U.S. Provisional Patent Application No. 62 / 342,784, filed May 27, 2016, which is hereby incorporated by reference in its entirety herein. and is a continuation of

Background Art

[0002] Messenger RNA (mRNA) is a polymer composed of a number of linked nucleotides, each consisting of a sugar, a phosphate, and a base. Each mRNA polymer stores genetic information along the nucleotide chain. Messenger RNA polymers transfer genetic information from DNA in the cell nucleus to the cytoplasm where proteins are made. Each triplet of nucleotides in mRNA is called a codon, and each codon defines the identity of an amino acid in the translated protein.

[0003] Cells can also take up and translate exogenous RNA, but many factors affect efficient uptake and translation. For example, the immune system recognizes many exogenous RNAs as foreign and induces responses aimed at inactivating those RNAs.

Summary of the Invention

Means for Solving the Problems

[0004] The present disclosure provides a polynucleotide capable of encoding a selected protein and a composition containing the same. In some cases, the present disclosure provides a method for treating a subject having or at risk of having primary ciliary dyskinesia, the method comprising A composition comprising a nucleic acid construct encoding an axonemal dynein intermediate chain 1 protein or a variant thereof is administered to a subject, thereby treating the subject having or at risk of having primary ciliary dyskinesia. The nucleic acid construct comprises codons that provide for heterologous or enhanced expression of axonemal dynein intermediate chain 1 protein or a variant thereof in the cells of the subject. The nucleic acid construct can be, for example, a complementary deoxyribonucleic acid (DNA) template. The nucleic acid construct can encode at least about 1.5-fold, at least about 5-fold, or another suitable multiple higher level of axonemal dynein intermediate chain 1 protein or a variant thereof compared to the level in cells exposed to a composition comprising a nucleic acid construct that does not contain codons encoding axonemal dynein intermediate chain 1 protein or a variant thereof. In some cases, the codons of the construct are at least 70% homologous to mammalian axonemal dynein intermediate chain 1 mRNA, such as human. In some cases, the construct comprises 5' and / or 3' untranslated regions (UTRs) adjacent to the codon sequence encoding axonemal dynein intermediate chain 1, and the (one or more) untranslated regions enhance protein expression in the cells of the subject. The 3' non-coding region can comprise a 3'-cap-independent translational enhancer (3'-CITE). In some cases, the 3' non-coding region can also comprise at least one intervening sequence region between the codon sequence and the 3' non-coding region or 5' non-coding region, or a 3' stem-loop region derived from the nucleotide sequence of a histone protein. In some cases, the codon sequence comprises an open reading frame (ORF). The 3' non-coding region adjacent to the codon sequence (e.g., ORF)

[0005] ​​​​​​​​​​​​​​​​, may include a polyadenosine tail, and the number of adenosines in the polyadenosine tail improves the translation efficiency of axonemal dynein intermediate chain 1 mRNA and extends the half-life. In some cases, the length of the polyadenosine tail is at most 200 adenosines. The polyadenosine tail may contain a certain percentage of chemically modified nucleotides. In some cases, less than 20% of the nucleotides in the polyadenosine tail are chemically modified. In some cases, less than 30% of the nucleotides encoding axonemal dynein intermediate chain 1 in the construct are chemically modified. When the nucleotide contains a chemically modified nucleotide, the chemically modified nucleotide is selected from the group consisting of pseudouridine, 1-methylpseudouridine, 2-thiouridine, 5-iodouridine , 5-methyluridine, 5-methylcytidine, and 5-iodocytidine. In some cases, the chemically modified nucleotide is 1-methylpseudouridine. In some cases, the modified nucleotide is pseudouridine. In another case, the modified nucleotide is a combination of 1-methylpseudouridine and pseudouridine. In addition to the composition containing a polynucleotide for treating a subject having or at risk of having primary ciliary dyskinesia, in some cases, the present disclosure provides armadillo repeat-containing 4 (ARMC4), chromosome 21 open reading frame 59 (C21orf59), coiled-coil domain-containing 103 (CCDC103), coiled-coil domain-containing 114 (CCDC114), coiled-coil domain-containing 39 ( CCDC39), coiled-coil domain-containing 40 (CCDC40), coiled-coil domain-containing 65 (CCDC65), cyclin O (CCNO), dynein (axoneme) assembly and the like. Nucleotides less than 30% of the nucleotides encoding axonemal dynein intermediate chain 1 in the construct are chemically modified. When the nucleotide contains a chemically modified nucleotide, the chemically modified nucleotide is selected from the group consisting of pseudouridine, 1-methylpseudouridine, 2-thiouridine, 5-iodouridine , 5-methyluridine, 5-methylcytidine, and 5-iodocytidine. In some cases, the chemically modified nucleotide is 1-methylpseudouridine. In some cases, the modified nucleotide is pseudouridine. In another case, the modified nucleotide is a combination of 1-methylpseudouridine and pseudouridine. In addition to the composition containing a polynucleotide for treating a subject having or at risk of having primary ciliary dyskinesia, in some cases, the present disclosure provides armadillo repeat-containing 4 (ARMC4), chromosome 21 open reading frame 59 (C21orf59), coiled-coil domain-containing 103 (CCDC103), coiled-coil domain-containing 114 (CCDC114), coiled-coil domain-containing 39 ( CCDC39), coiled-coil domain-containing 40 (CCDC40), coiled-coil domain-containing 65 (CCDC65), cyclin O (CCNO), dynein (axoneme) assembly and the like. Nucleotides less than 30% of the nucleotides encoding axonemal dynein intermediate chain 1 in the construct are chemically modified. When the nucleotide contains a chemically modified nucleotide, the chemically modified nucleotide is selected from the group consisting of pseudouridine, 1-methylpseudouridine, 2-thiouridine, 5-iodouridine , 5-methyluridine, 5-methylcytidine, and 5-iodocytidine. In some cases, the chemically modified nucleotide is 1-methylpseudouridine. In some cases, the modified nucleotide is pseudouridine. In another case, the modified nucleotide is a combination of 1-methylpseudouridine and pseudouridine. In addition to the composition containing a polynucleotide for treating a subject having or at risk of having primary ciliary dyskinesia, in some cases, the present disclosure provides armadillo repeat-containing 4 (ARMC4), chromosome 21 open reading frame 59 (C21orf59), coiled-coil domain-containing 103 (CCDC103), coiled-coil domain-containing 114 (CCDC114), coiled-coil domain-containing 39 ( CCDC39), coiled-coil domain-containing 40 (CCDC40), coiled-coil domain-containing 65 (CCDC65), cyclin O (CCNO), dynein (axoneme) assembly and the like. Nucleotides less than 30% of the nucleotides encoding axonemal dynein intermediate chain 1 in the construct are chemically modified. When the nucleotide contains a chemically modified nucleotide, the chemically modified nucleotide is selected from the group consisting of pseudouridine, 1-methylpseudouridine, 2-thiouridine, 5-iodouridine , 5-methyluridine, 5-methylcytidine, and 5-iodocytidine. In some cases, the chemically modified nucleotide is 1-methylpseudouridine. In some cases, the modified nucleotide is pseudouridine. In another case, the modified nucleotide is a combination of 1-methylpseudouridine and pseudouridine. In addition to the composition containing a polynucleotide for treating a subject having or at risk of having primary ciliary dyskinesia, in some cases, the present disclosure provides armadillo repeat-containing 4 (ARMC4), chromosome 21 open reading frame 59 (C21orf59), coiled-coil domain-containing 103 (CCDC103), coiled-coil domain-containing 114 (CCDC114), coiled-coil domain-containing 39 ( CCDC39), coiled-coil domain-containing 40 (CCDC40), coiled-coil domain-containing 65 (CCDC65), cyclin O (CCNO), dynein (axoneme) assembly and the like. Nucleotides less than 30% of the nucleotides encoding axonemal dynein intermediate chain 1 in the construct are chemically modified. When the nucleotide contains a chemically modified nucleotide, the chemically modified nucleotide is selected from the group consisting of pseudouridine, 1-methylpseudouridine, 2-thiouridine, 5-iodouridine , 5-methyluridine, 5-methylcytidine, and 5-iodocytidine. In some cases, the chemically modified nucleotide is 1-methylpseudouridine. In some cases, the modified nucleotide is pseudouridine. In another case, the modified nucleotide is a combination of 1-methylpseudouridine and pseudouridine. In addition to the composition containing a polynucleotide for treating a subject having or at risk of having primary ciliary dyskinesia, in some cases, the present disclosure provides armadillo repeat-containing 4 (ARMC4), chromosome 21 open reading frame 59 (C21orf59), coiled-coil domain-containing 103 (CCDC103), coiled-coil domain-containing 114 (CCDC114), coiled-coil domain-containing 39 ( CCDC39), coiled-coil domain-containing 40 (CCDC40), coiled-coil domain-containing 65 (CCDC65), cyclin O (CCNO), dynein (axoneme) assembly and the like. Nucleotides less than 30% of the nucleotides encoding axonemal dynein intermediate chain 1 in the construct are chemically modified. When the nucleotide contains a chemically modified nucleotide, the chemically modified nucleotide is selected from the group consisting of pseudouridine, 1-methylpseudouridine, 2-thiouridine, 5-iodouridine CCDC39), coiled-coil domain-containing 40 (CCDC40), coiled-coil domain-containing 65 (CCDC65), cyclin O (CCNO), dynein (axoneme) assembly Dynein axoneme assembly factor 1 (DNAAF1), dynein (axoneme) assembly factor 2 (DNAAF2), dynein (axoneme) assembly factor 3 (DNAAF3), dynein (axoneme) assembly factor 5 (DNAAF5), axonemal dynein heavy chain 11 (DNAH11), axonemal dynein heavy chain 5 (DN AH5), axonemal dynein heavy chain 6 (DNAH6), axonemal dynein heavy chain 8 (DNAH8), axon emal dynein intermediate chain 2 (DNAI2), axonemal dynein light chain 1 (DNAL1), dynein regulatory complex subunit 1 (DRC1), dyslexia susceptibility 1 candidate 1 (DYX1C1), growth arrest specific 8 (GAS8), axoneme central pair apparatus protein (HYDIN), leucine rich repeat containing 6 (LRRC6), NME / NM23 family member 8 (NME8), oral -facial-digital syndrome 1 (OFD1), retinitis pigmentosa GTPase regulator (RPGR), radial spoke head 1 homolog (Chlamydomonas) ( RSPH1), radial spoke head 4 homolog A (Chlamydomonas) (RSPH4A ), radial spoke head 9 homolog (Chlamydomonas) (RSPH9), sperm associated antigen 1 (SPAG1) and zinc finger MYND type containing 10 (ZMYND10), and providing a composition comprising at least one additional nucleic acid construct encoding a protein selected from the group consisting of.

[0006] The present disclosure provides a composition comprising a nucleic acid construct encoding axonemal dynein intermediate chain 1, the nucleic acid construct comprising codons that provide for heterologous or enhanced expression of axonemal dynein intermediate chain 1 protein or a variant thereof in cells of a subject having or at risk of having primary ciliary dyskinesia. The compositions described herein comprise at least 4 modified polylys ines. The ratio of the number of moles of amine groups of the cationic polymer to the number of moles of phosphate groups of the bonucleotide may be included. In some cases, the composition is formulated into nanoparticles or nanocapsules. In another case, the composition is formulated into a cationic lipid, a cationic polymer or a nanoemulsion The composition may be formulated for administration to a subject. The nucleic acid construct in the composition provides for heterologous or enhanced expression of the axonemal dynein intermediate chain 1 protein or a variant thereof in cells of a subject having or at risk of having primary ciliary dyskinesia and may contain codons. In some cases, less than 30% of the ribonucleotides encoding axonemal dynein intermediate chain 1 are chemically modified nucleotides. In some cases, the codon of the construct is at least 70% homologous to the axonemal dynein intermediate chain 1 mRNA of a mammal such as a human In some cases, the construct includes a 5' or 3' non-coding region adjacent to the codon sequence encoding axonemal dynein intermediate chain 1, and this non-coding region enhances the expression of the protein in the cells of the subject In another case, the construct includes a 3' non-coding region adjacent to the codon sequence encoding axonemal dynein intermediate chain 1, and this 3' non-coding region includes a 3'-cap-independent translation enhancer (3'-CITE). The 3' non-coding region may include a 3' stem-loop region derived from the nucleotide sequence of a histone protein The 3' non-coding region may include a 3' triple helix structure derived from the nucleotide sequence of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). The 3' non-coding region adjacent to the codon sequence may include a polyadenosine tail, and the number of adenosines in the polyadenosine tail is the axonemal dynein intermediate chain 1 ta In some cases, the composition is formulated for administration to a subject. The nucleic acid construct in the composition provides for heterologous or enhanced expression of the axonemal dynein intermediate chain 1 protein or a variant thereof in cells of a subject having or at risk of having primary ciliary dyskinesia and may contain codons. In some cases, less than 30% of the ribonucleotides encoding axonemal dynein intermediate chain 1 are chemically modified nucleotides. In some cases, the codon of the construct is at least 70% homologous to the axonemal dynein intermediate chain 1 mRNA of a mammal such as a human In some cases, the construct includes a 5' or 3' non-coding region adjacent to the codon sequence encoding axonemal dynein intermediate chain 1, and this non-coding region enhances the expression of the protein in the cells of the subject In another case, the construct includes a 3' non-coding region adjacent to the codon sequence encoding axonemal dynein intermediate chain 1, and this 3' non-coding region includes a 3'-cap-independent translation enhancer (3'-CITE). The 3' non-coding region may include a 3' stem-loop region derived from the nucleotide sequence of a histone protein The 3' non-coding region may include a 3' triple helix structure derived from the nucleotide sequence of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). The 3' non-coding region adjacent to the codon sequence may include a polyadenosine tail, and the number of adenosines in the polyadenosine tail is the axonemal dynein intermediate chain 1 ta In some cases, the composition is formulated for administration to a subject. The nucleic acid construct in the composition Improve the translation efficiency of proteins. In some cases, the number of adenosines in the polyadenosine tail Improve the half-life of the axonemal dynein intermediate chain 1 protein. In some cases, the polyadeno The length of the sine tail is at most 200 adenosines. In some cases, a certain percentage of the poly Adenosine tails contain chemically modified nucleotides. In some cases, the poly(A) tail Less than 20% of the adenosines are modified. In some cases, the construct has a certain percentage of Contains chemically modified nucleotides. In some cases, less than 30% of the nucleotides encoding axonemal dynein intermediate chain 1 Are chemically modified. When chemically modified nucleotides are present They can be selected from the group consisting of pseudouridine, 1-methylpseudouridine, 5-methoxyuridine Lysine, 2-thiouridine, 5-iodouridine, 5-methyluridine, 5-methylcytidine Din, 2'-amino-2'-deoxycytidine, 2'-fluoro-2'-deoxycytidine And 5-iodocytidine. In some cases, the chemically modified Nucleotides are pseudouridine or 1-methylpseudouridine. In some In cases, the composition further comprises at least one additional nucleic acid construct. This at least One additional nucleic acid construct is armadillo repeat-containing 4 (ARMC4), chromosome 21 Open reading frame 59 (C21orf59), coiled-coil domain-containing 103 (CCDC103), coiled-coil domain-containing 114 (CCDC114), co iled-coil domain-containing 39 (CCDC39), coiled-coil domain-containing 40 (C CDC40), coiled-coil domain-containing 65 (CCDC65), cyclin O (CC NO), dynein (axoneme) assembly factor 1 (DNAAF1), dynein (axoneme) assembly Blepharoplast factor 2 (DNAAF2), dynein (axoneme) assembly factor 3 (DNAAF3), dy nein (axoneme) assembly factor 5 (DNAAF5), axonemal dynein heavy chain 11 (DNAH1 1), axonemal dynein heavy chain 5 (DNAH5), axonemal dynein heavy chain 6 (DNAH6), axonemal dy nein heavy chain 8 (DNAH8), axonemal dynein intermediate chain 2 (DNAI2), axonemal dynein light chain 1 (DNAL1), dynein regulatory complex subunit 1 (DRC1), dyslexia susceptibility 1 candidate complement 1 (DYX1C1), growth arrest specific 8 (GAS8), axonemal central pair apparatus protein( HYDIN), leucine rich repeat containing 6 (LRRC6), NME / NM23 family member 8 (NME8), orofaciodigital syndrome 1 (OFD1), retinitis pigmentosa GTP ase regulator (RPGR), radial spoke head 1 homolog (Chlamydomonas)( RSPH1), radial spoke head 4 homolog A (Chlamydomonas)(RSPH4A ), radial spoke head 9 homolog (Chlamydomonas)(RSPH9), sperm associated antigen 1 (SPAG1) and zinc finger MYND type containing 10 (ZMYND10). The present disclosure also provides nucleic acid constructs, vectors or isolated nucleic acids formulated for administration to a subject. In some cases, the formulation comprises a therapeutically effective amount of a nucleic acid construct encoding axonemal dynein intermediate chain 1. The nucleic acid construct can be a cDNA construct encoding axonemal dynein intermediate chain 1 protein or a variant thereof, or any one of the additional nucleic acid constructs described above. In some cases, the present disclosure comprises a nucleic acid construct encoding axonemal dynein intermediate chain 1.

[0007] The present disclosure also provides nucleic acid constructs, vectors or isolated nucleic acids formulated for administration to a subject. In some cases, the formulation comprises a therapeutically effective amount of a nucleic acid construct encoding axonemal dynein intermediate chain 1. The nucleic acid construct can be a cDNA construct encoding axonemal dynein intermediate chain 1 protein or a variant thereof, or any one of the additional nucleic acid constructs described above. In some cases, the present disclosure comprises a nucleic acid construct encoding axonemal dynein intermediate chain 1. The present disclosure also provides nucleic acid constructs, vectors or isolated nucleic acids formulated for administration to a subject. In some cases, the formulation comprises a therapeutically effective amount of a nucleic acid construct encoding axonemal dynein intermediate chain 1. The nucleic acid construct can be a cDNA construct encoding axonemal dynein intermediate chain 1 protein or a variant thereof, or any one of the additional nucleic acid constructs described above. In some cases, the present disclosure comprises a nucleic acid construct encoding axonemal dynein intermediate chain 1. The present disclosure also provides nucleic acid constructs, vectors or isolated nucleic acids formulated for administration to a subject. In some cases, the formulation comprises a therapeutically effective amount of a nucleic acid construct encoding axonemal dynein intermediate chain 1. The nucleic acid construct can be a cDNA construct encoding axonemal dynein intermediate chain 1 protein or a variant thereof, or any one of the additional nucleic acid constructs described above. In some cases, the present disclosure comprises a nucleic acid construct encoding axonemal dynein intermediate chain 1. The nucleic acid construct can be a cDNA construct encoding axonemal dynein intermediate chain 1 protein or a variant thereof, or any one of the additional nucleic acid constructs described above. In some cases, the present disclosure comprises a nucleic acid construct encoding axonemal dynein intermediate chain 1. To provide a composition, the nucleic acid construct includes any one of SEQ ID NOs: 14 to 16. In part In some cases, the present disclosure provides a composition comprising a nucleic acid construct encoding axonemal dynein heavy chain 5 and the nucleic acid construct includes any one of SEQ ID NOs: 17 to 18.

[0008] Further aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, which illustrates and describes only exemplary embodiments of the present disclosure. As will be understood, the present disclosure is capable of other and different embodiments, and some of the details thereof may be modified in various obvious respects all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature and not as restrictive. should not be

[0009] Incorporation by reference All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference.

[0010] The novel features of the invention are set forth with particularity in the appended claims. Referring to the following detailed description which illustrates exemplary embodiments in which the principles of the invention are utilized, and the accompanying drawings, a better understanding of the features and advantages of the invention will be obtained.

Brief Description of the Drawings

[0011]

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BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Although various embodiments of the present invention are shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many variations, modifications, and substitutions may be envisioned by those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be used. The term "subject" as used herein generally refers to a human. In some cases,

[0013] the subject may be an animal such as a mouse, rat, guinea pig, dog, cat, horse, rabbit, and various other animals. The subject may be of any age. For example, the subject may be an infant or a child. It can be an infant, a toddler, a child, a pre-adolescent individual, an adolescent individual, an adult or an elderly individual.

[0014] As used herein, the term "disease" generally refers to a disorder (e.g., primary ciliary dyskinesia) or an abnormal physiological condition that affects some or all of a subject, such as an abnormality that causes defects in the inner lining of various airways (lower and upper airways, sinuses, eustachian tubes, middle ear), various lung cells, the activity of cilia in the fallopian tubes, or the flagella of sperm cells.

[0015] As used herein, the term "polynucleotide" or "nucleic acid" refers to a polymer form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides, that include purine and pyrimidine bases, purine and pyrimidine analogs, chemically or biochemically modified, natural or non-natural, or derivatized nucleotide bases. Polynucleotides include sequences of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or DNA copies (cDNA) of ribonucleic acid, all of which can be recombinantly produced, artificially synthesized, or isolated and purified from natural sources. Polynucleotides and nucleic acids can exist as single-stranded or double-stranded. The backbone of a polynucleotide can contain sugar and phosphate groups, as typically found in RNA or DNA, or analogs or substituted sugars or phosphate groups. Polynucleotides can include natural or non-natural nucleotides, such as methylated nucleotides and nucleotide analogs.

[0016] As used herein, the term "polynucleotide" generally refers to a polynucleotide polymer containing ribonucleic acid. This term also refers to polynucleotide polymers containing chemically modified ribonucleotides. Polynucleotides can be formed with the d-ribose sugars found in nature.

[0017] As used herein, the term "polypeptide" generally refers to a polymer chain of amino acid residues linked together via amide bonds (peptide bonds). Polypeptides can be chains of at least three amino acids, proteins, recombinant proteins, antigens, epitopes, enzymes, receptors or structural analogs, or combinations thereof. When used herein, the abbreviations for the L-enantiomer amino acids forming polypeptides are as follows: alanine (A, Ala); arginine (R, Arg); asparagine (N, Asn); aspartic acid (D, Asp); cysteine (C, Cys); glutamic acid (E, Glu); glutamine (Q, Gln); glycine (G, Gly); histidine (H, His); isoleucine (I, Ile); leucine (L, Leu); lysine (K, Lys); methionine (M, Met); phenylalanine (F, Phe); proline (P, Pro); serine (S, Ser); threonine (T, Thr); tryptophan (W, Trp); tyrosine (Y, Tyr); valine (V, Val). X or Xaa can represent any amino acid.

[0018] As used herein, the term "engineered" generally refers to being genetically designed and engineered to provide polynucleotides within cells, engineered polynucleotides, vectors and nuclei. Refers to an acid construct. The engineered polynucleotide can be partially or fully synthesized in vitro and can also be cloned. The engineered polynucleotide can contain one or more bases or sugar analogs, such as ribonucleotides that are not naturally present in messenger RNA. The engineered polyribonucleotide can be present as a nucleotide analog in transfer RNA (tRNA), ribosomal RNA (rRNA), guide RNA (gRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), SmY RNA, splice leader RNA (SL RNA), CRISPR RNA, long non-coding RNA (lncRNA), microRNA (miRNA), or another suitable RNA. The engineered polynucleotide can contain one or more bases or sugar analogs, such as ribonucleotides that are not naturally present in messenger RNA. The engineered polynucleotide can contain one or more bases or sugar analogs, such as ribonucleotides that are not naturally present in messenger RNA. The engineered polyribonucleotide can be present as a nucleotide analog in transfer RNA (tRNA), ribosomal RNA (rRNA), guide RNA (gRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), SmY RNA, splice leader RNA (SL RNA), CRISPR RNA, long non-coding RNA (lncRNA), microRNA (miRNA), or another suitable RNA. The engineered polyribonucleotide can be present as a nucleotide analog in transfer RNA (tRNA), ribosomal RNA (rRNA), guide RNA (gRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), SmY RNA, splice leader RNA (SL RNA), CRISPR RNA, long non-coding RNA (lncRNA), microRNA (miRNA), or another suitable RNA. The engineered polyribonucleotide can be present as a nucleotide analog in transfer RNA (tRNA), ribosomal RNA (rRNA), guide RNA (gRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), SmY RNA, splice leader RNA (SL RNA), CRISPR RNA, long non-coding RNA (lncRNA), microRNA (miRNA), or another suitable RNA. The engineered polyribonucleotide can be present as a nucleotide analog in transfer RNA (tRNA), ribosomal RNA (rRNA), guide RNA (gRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), SmY RNA, splice leader RNA (SL RNA), CRISPR RNA, long non-coding RNA (lncRNA), microRNA (miRNA), or another suitable RNA. The engineered polyribonucleotide can be present as a nucleotide analog in transfer RNA (tRNA), ribosomal RNA (rRNA), guide RNA (gRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), SmY RNA, splice leader RNA (SL RNA), CRISPR RNA, long non-coding RNA (lncRNA), microRNA (miRNA), or another suitable RNA.

[0019] Summary The present disclosure provides compositions and methods for treating conditions related to cilia maintenance and function using nucleic acids encoding a protein or protein fragment(s). Many eukaryotic cells often carry appendages called cilia or flagella, and their inner core contains a cytoskeletal structure called the axoneme. The axoneme can function as a backbone of the cytoskeletal structure, support the structure, and in some cases, bend the structure. Usually, the internal structure of the axoneme is common to both cilia and flagella. Cilia are often found in the airways, the genital system, and the inner layers of other organs and tissues. Flagella are tail-like structures that can propel cells, such as sperm cells, forward, similar to cilia. The present disclosure provides compositions and methods for treating conditions related to cilia maintenance and function using nucleic acids encoding a protein or protein fragment(s). Many eukaryotic cells often carry appendages called cilia or flagella, and their inner core contains a cytoskeletal structure called the axoneme. The axoneme can function as a backbone of the cytoskeletal structure, support the structure, and in some cases, bend the structure. The axoneme can function as a backbone of the cytoskeletal structure, support the structure, and in some cases, bend the structure. Usually, the internal structure of the axoneme is common to both cilia and flagella. Cilia are often found in the airways, the genital system, and the inner layers of other organs and tissues. Flagella are tail-like structures that can propel cells, such as sperm cells, forward, similar to cilia. Flagella are tail-like structures that can propel cells, such as sperm cells, forward, similar to cilia.

[0020] If cilia do not function properly in the airways, bacteria can remain in the airways and cause infection. In the airway, cilia move back and forth in a coordinated manner to move mucus toward the throat. This mucus movement helps to eliminate fluids, bacteria, and particles from the lungs. Many infants suffering from ciliary and flagellar dysfunction experience breathing problems at birth, suggesting that cilia play an important role in removing fetal fluid from the lungs. Starting in infancy, subjects suffering from ciliary dysfunction can develop frequent respiratory

[0021] infections. Primary ciliary dyskinesia is a condition characterized by chronic airway infections, abnormally positioned internal organs, and the inability to have children (infertility). The signs and symptoms of this condition are caused by abnormal cilia and flagella. Subjects suffering from primary ciliary dyskinesia often have nasal congestion and chronic cough throughout the year. Chronic airway infections can lead to a condition

[0022] called bronchiectasis, which can damage the airways leading from the trachea to the lungs and cause life-threatening respiratory disorders. In some cases, the nucleic acid constructs, vectors, or compositions of the present disclosure include one or more nucleotide sequences encoding axonemal dynein intermediate chain 1 protein or variants thereof, which sequences provide for heterologous or enhanced expression of axonemal dynein intermediate chain 1 protein or variants thereof in the cells of a subject. In some cases, the nucleic acid TIFF2025090635000003.tif69170

[0023] Primary ciliary dyskinesia, related conditions and their treatment The methods, constructs and compositions of the present disclosure provide methods for treating primary ciliary dyskinesia (PCD), also known as primary ciliary dyskinesia syndrome or Kartagener syndrome. PCD is typically a rare, ciliopathic autosomal recessive disorder that causes defects in the activity of cilia in the inner lining of the airways (lower and upper airways, sinuses, eustachian tubes, middle ear) and in the fallopian tubes, as well as in the flagella of sperm cells. It is thought to be occurring.

[0024] Some individuals with primary ciliary dyskinesia have organs that occupy abnormal positions within the chest and abdomen. These abnormalities occur early in embryogenesis when the left - right differences in the body are established. Approximately 50% of people with primary ciliary dyskinesia have situs inversus (complete situs inversus) of the internal organs. For example, in these individuals, the heart is on the right side of the body instead of the left. When a person with primary ciliary dyskinesia has complete situs inversus, they are often said to have Kartagener syndrome.

[0025] Approximately 12% of people with primary ciliary dyskinesia have a condition known as situs ambiguus or visceral heterotaxy, characterized by abnormalities of the heart, liver, intestine or spleen. These organs can be structurally abnormal or in inappropriate positions. Additionally, affected individuals may not have a spleen (asplenia) or may have multiple spleens (polysplenia). Situs ambiguus results from problems establishing the left - right sides of the body during embryogenesis. The severity of situs ambiguus varies widely among affected individuals. ​​​​​​

[0026] Primary ciliary dyskinesia can also lead to infertility. To move sperm cells towards the female egg cell, intense movement of the flagellum may be required. Since the sperm of subjects suffering from primary ciliary dyskinesia do not move properly, men with primary ciliary dyskinesia usually cannot become fathers. Infertility occurs in some affected women and is usually associated with abnormal cilia in the fallopian tubes. To move sperm cells towards the female egg cell, intense movement of the flagellum may be required. Since the sperm of subjects suffering from primary ciliary dyskinesia do not move properly, men with primary ciliary dyskinesia usually cannot become fathers. Infertility occurs in some affected women and is usually associated with abnormal cilia in the fallopian tubes. Since the sperm of subjects suffering from primary ciliary dyskinesia do not move properly, men with primary ciliary dyskinesia usually cannot become fathers. Infertility occurs in some affected women and is usually associated with abnormal cilia in the fallopian tubes. Since the sperm of subjects suffering from primary ciliary dyskinesia do not move properly, men with primary ciliary dyskinesia usually cannot become fathers. Infertility occurs in some affected women and is usually associated with abnormal cilia in the fallopian tubes. Infertility occurs in some affected women and is usually associated with abnormal cilia in the fallopian tubes.

[0027] Another feature of primary ciliary dyskinesia is recurrent ear infections (otitis media), especially in infants. Otitis media can lead to permanent hearing loss if not treated. Ear infections are likely to be associated with abnormal cilia in the inner ear. Another feature of primary ciliary dyskinesia is recurrent ear infections (otitis media), especially in infants. Otitis media can lead to permanent hearing loss if not treated. Ear infections are likely to be associated with abnormal cilia in the inner ear. Another feature of primary ciliary dyskinesia is recurrent ear infections (otitis media), especially in infants. Otitis media can lead to permanent hearing loss if not treated. Ear infections are likely to be associated with abnormal cilia in the inner ear.

[0028] Rarely, individuals with primary ciliary dyskinesia have fluid accumulation in the brain (hydrocephalus), probably due to abnormal cilia in the brain. Rarely, individuals with primary ciliary dyskinesia have fluid accumulation in the brain (hydrocephalus), probably due to abnormal cilia in the brain.

[0029] The polynucleotides of the present disclosure can be used, for example, to treat subjects having or at risk of having primary ciliary dyskinesia or any other condition associated with a defect or dysfunction of a gene whose function is linked to the maintenance and function of cilia. The polynucleotides of the present disclosure can be used, for example, to treat subjects having or at risk of having primary ciliary dyskinesia or any other condition associated with a defect or dysfunction of a gene whose function is linked to the maintenance and function of cilia. The polynucleotides of the present disclosure can be used, for example, to treat subjects having or at risk of having primary ciliary dyskinesia or any other condition associated with a defect or dysfunction of a gene whose function is linked to the maintenance and function of cilia. Non-limiting examples of genes associated with primary ciliary dyskinesia include armadillo repeat containing 4 (ARMC4), chromosome 21 open reading frame 59 (C21orf59), coiled-coil domain containing 103 (CCDC103), coiled-coil domain containing 114 (CCDC114), coiled-coil domain containing 39 (CCDC39), coiled coiled-coil domain containing 103 (CCDC103), coiled-coil domain containing 114 (CCDC114), coiled-coil domain containing 39 (CCDC39), coiled coiled-coil domain containing 114 (CCDC114), coiled-coil domain containing 39 (CCDC39), coiled coil domain containing 40 (CCDC40), coiled-coil domain containing 65 (CCDC65), C65), Cyclin O (CCNO), Dynein (axoneme) assembly factor 1 (DNAAF 1), Dynein (axoneme) assembly factor 2 (DNAAF2), Dynein (axoneme) assembly factor 3 (DNAAF3), Dynein (axoneme) assembly factor 5 (DNAAF5), axoneme dynein heavy chain 11 (DNAH11), axoneme dynein heavy chain 5 (DNAH5), axoneme dynein heavy chain 6 (DNAH6), axoneme dynein heavy chain 8 (DNAH8), axoneme dynein intermediate chain 2 (D NAI2), axoneme dynein light chain 1 (DNAL1), dynein regulatory complex subunit 1 ( DRC1), dyslexia susceptibility 1 candidate 1 (DYX1C1), growth arrest specific 8 (GAS8) , axoneme central pair apparatus protein (HYDIN), leucine rich repeat containing 6 (LRRC 6), NME / NM23 family member 8 (NME8), oral-facial-digital syndrome 1 (OF D1), retinitis pigmentosa GTPase regulator (RPGR), radial spoke head 1 homolog (Chlamydomonas) (RSPH1), radial spoke head 4 homolog A (Ch lamydomonas) (RSPH4A), radial spoke head 9 homolog (Chlamydomonas )(RSPH9), sperm associated antigen 1 (SPAG1) and zinc finger MYND type containing 10 (ZMYND10) are included.

[0030] In some cases, the composition comprises a nucleic acid construct encoding axoneme dynein intermediate chain 1 (DNAI1), which, when translated within the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The DNAI1 gene can provide instructions for making a protein that is part of a group (complex) of proteins called dynein. This complex functions within cilia. The coordinated functioning of cilia of cilia of cilia The back-and-forth movement can move the cell or the liquid surrounding the cell, and dynein is responsible for the movement of the cilia to generate the necessary force. Within the core (axoneme) of the cilium, the dynein complex is part of a structure known as the inner dynein arm (IDA) and the outer dynein arm (ODA) depending on their positions . The coordinated movement of the dynein arms bends the entire axoneme back and forth. The IDA and ODA have various combinations of protein components (subunits) classified as heavy, intermediate, or light chains by weight . The DNAI1 gene provides instructions for making intermediate chain 1 found in the ODA . Other subunits can be generated from various genes administered to a subject in the same or separate compositions. Alternatively, other subunits can be generated by a single nucleic acid construct encoding a functional component of the inner or outer dynein arm .

[0031] At least 21 mutations in the DNAI1 gene cause primary ciliary dyskinesia, a condition characterized by airway infections, abnormal organ placement, and the inability to have children (infertility). DNAI1 gene mutations result in the absence or abnormal intermediate chain 1 . Without a normal version of this subunit, the ODA cannot form properly and can be shortened or absent. As a result, the cilia are unable to generate the force necessary to bend back and forth. Ciliary defects lead to the characteristics of primary ciliary dyskinesia . In some cases, the present disclosure provides a nucleic acid engineered to replace or complement the function of the endogenous DNAI1 protein, including the IVS1+2_3insT(219+ 3insT) mutation. In some cases, the present disclosure provides the second most ​​​ Replace or supplement the function of the endogenous DNAI1 protein containing the common A538T mutation Or provide a nucleic acid engineered to supplement it.

[0032] In some cases, the composition comprises a nucleic acid construct encoding axonemal dynein intermediate chain 2 (DNAI2), which, when translated in the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. DNAI2 The gene is part of the dynein complex of respiratory cilia and sperm flagella. Mutations in this gene are associated with primary ciliary dyskinesia type 9, a disorder characterized by abnormal motile cilia, chronic inflammation and respiratory infections leading to bronchiectasis, as well as Abnormalities in the sperm tail. In some cases, the composition comprises a nucleic acid construct encoding armadillo repeat-containing 4 (ARMC4), which, when translated in the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the ARMC4 Gene contains 10 armadillo repeat motifs (ARM) and 1 HEAT repeat and has been shown to localize to the ciliary axoneme and the base of the cilia of respiratory cells. Mutations in the ARMC4 gene can cause partial dynein outer arm (ODA) deficiency of respiratory cilia.

[0033] In some cases, the composition comprises a nucleic acid construct encoding chromosome 21 open reading frame 59 (C21 orf59), which, when translated in the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. This construct Is a subject who has or is at risk of having primary ciliary dyskinesia. Po The ID=42 And has been shown to localize to the ciliary axoneme and the base of the cilia of respiratory cells. Mutations in the ARMC4 gene can cause partial dynein outer arm (ODA) deficiency of respiratory cilia.

[0034] In some cases, the composition comprises a nucleic acid construct encoding chromosome 21 open reading frame 59 (C21 orf59), which, when translated in the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. This construct Is a subject who has or is at risk of having primary ciliary dyskinesia. Po It produces a peptide. The protein encoded by the C21orf59 gene can play an important role in dynein arm assembly and motile cilia function. Mutations in this gene can lead to primary ciliary dyskinesia. In some cases, the composition contains a nucleic acid construct encoding coiled-coil domain-containing 103 (CCDC103), which, when translated within the cells of a subject, produces a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the CCDC103 gene can function as a dynein binding factor required for ciliary motility. In some cases, the composition contains a nucleic acid construct encoding coiled-coil domain-containing 114 (CCDC114), which, when translated within the cells of a subject, produces a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the CCDC114 gene can function as a component of the dynein outer arm docking complex in ciliated cells. Mutations in this gene can cause primary ciliary dyskinesia type 20.

[0035] In some cases, the composition contains a nucleic acid construct encoding coiled-coil domain-containing 103 (CCDC103), which, when translated within the cells of a subject, produces a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the CCDC103 gene can function as a dynein binding factor required for ciliary motility. In some cases, the composition contains a nucleic acid construct encoding coiled-coil domain-containing 114 (CCDC114), which, when translated within the cells of a subject, produces a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the CCDC114 gene can function as a component of the dynein outer arm docking complex in ciliated cells. Mutations in this gene can cause primary ciliary dyskinesia type 20. In some cases, the composition contains a nucleic acid construct encoding coiled-coil domain-containing 39 (CCDC39), which, when translated within the cells of a subject, produces a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the CCDC39 gene regulates ciliary beating and is a dynein regulator.

[0036] In some cases, the composition contains a nucleic acid construct encoding coiled-coil domain-containing 114 (CCDC114), which, when translated within the cells of a subject, produces a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the CCDC114 gene can function as a component of the dynein outer arm docking complex in ciliated cells. Mutations in this gene can cause primary ciliary dyskinesia type 20. In some cases, the composition contains a nucleic acid construct encoding coiled-coil domain-containing 39 (CCDC39), which, when translated within the cells of a subject, produces a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the CCDC39 gene regulates ciliary beating and is a dynein regulator. In some cases, the composition contains a nucleic acid construct encoding coiled-coil domain-containing 39 (CCDC39), which, when translated within the cells of a subject, produces a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the CCDC39 gene regulates ciliary beating and is a dynein regulator.

[0037] In some cases, the composition contains a nucleic acid construct encoding coiled-coil domain-containing 39 (CCDC39), which, when translated within the cells of a subject, produces a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the CCDC39 gene regulates ciliary beating and is a dynein regulator. In some cases, the composition contains a nucleic acid construct encoding coiled-coil domain-containing 39 (CCDC39), which, when translated within the cells of a subject, produces a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the CCDC39 gene regulates ciliary beating and is a dynein regulator. The protein encoded by the CCDC39 gene regulates ciliary beating and is a dynein regulator. It can function as an assembly of the node and the dynein inner arm complex. Deletion of this gene is the cause of primary ciliary dyskinesia type 14 (CCDC39).

[0038] In some cases, the composition includes a nucleic acid construct encoding coiled-coil domain-containing 40 (CCDC40), which, when translated within the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the CCDC40 gene can function with CCDC39 to form a molecular ruler that determines the repeat length of 96 nanometers (nm) and the arrangement (by similarity) of components in cilia and flagella. CCDC40 is not required for the assembly of the dynein outer arm complex but may be required for the axonemal recruitment of CCDC39. In some cases, CCD40 and CCD39 can be generated from various genes administered to a subject in the same or separate compositions. Alternatively, CCD40 and CCD39 can be generated by a single nucleic acid construct encoding a functional component of the dynein inner arm or the dynein outer arm. Deletion of the CCD40 gene is the cause of primary ciliary dyskinesia type 14 (CILD14). In some cases, the composition includes a nucleic acid construct encoding coiled-coil domain-containing 65 (CCDC65), which, when translated within the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the CCDC65 gene functions as a sperm cell protein. In some cases, CCD40 and CCD39 are administered to a subject in the same or separate compositions. Alternatively, CCD40 and CCD39 can be generated from various genes administered to a subject in the same or separate compositions. Alternatively, CCD40 and CCD39 can be generated by a single nucleic acid construct encoding a functional component of the dynein inner arm or the dynein outer arm. Deletion of the CCD40 gene is the cause of primary ciliary dyskinesia type 14 (CILD14). In some cases, the composition includes a nucleic acid construct encoding coiled-coil domain-containing 65 (CCDC65), which, when translated within the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the CCDC65 gene functions as a sperm cell protein.

[0039] In some cases, the composition includes a nucleic acid construct encoding coiled-coil domain-containing 65 (CCDC65), which, when translated within the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the CCDC65 gene functions as a sperm cell protein. In some cases, the composition includes a nucleic acid construct encoding coiled-coil domain-containing 65 (CCDC65), which, when translated within the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the CCDC65 gene functions as a sperm cell protein. It can be done. CCDC65 is highly expressed in adult testes, spermatocytes and spermatids. It has been shown to be expressed. This protein plays an important role in the assembly of the nexin-dynein regulatory complex. Mutations in this gene are associated with primary ciliary dyskinesia type 27.

[0040] In some cases, the composition comprises a nucleic acid construct encoding cyclin O (CCNO), which, when translated within the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia.

[0041] In some cases, the composition comprises a nucleic acid construct encoding dynein (axoneme) assembly factor 1 (DNAAF1), which, when translated within the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the DNAAF1 gene is thought to be cilia-specific and may be required for the stability of the ciliary structure. Mutations in this gene are associated with primary ciliary dyskinesia type 13.

[0042] In some cases, the composition comprises a nucleic acid construct encoding dynein (axoneme) assembly factor 2 (DNAAF2), which, when translated within the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the DNAAF2 gene may be involved in the pre-assembly of the dynein arm complex that activates cilia. Mutations in this gene are associated with primary ciliary dyskinesia type 10 (CILD10).

[0043] In some cases, the composition comprises a nucleic acid construct encoding dynein (axoneme) assembly factor 3 (DNAAF3), which construct gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the DNAAF3 gene may be required for the assembly of the inner and outer arms of axonemal dynein and can play a role in the assembly of the dynein complex for transport into cilia. Mutations in this gene are associated with primary ciliary dyskinesia type 2 (CILD2). In some cases, the composition comprises a nucleic acid construct encoding dynein (axoneme) assembly factor 5 (DNAAF5), which construct gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia when translated within the cells of the subject. The protein encoded by the DNAAF5 gene is thought to be required for preassembly or stability of the axonemal dynein arms and is found only in organisms having motile cilia and flagella. Mutations in this gene are associated with primary ciliary dyskinesia type 18. In some cases, the composition comprises a nucleic acid construct encoding axonemal dynein heavy chain 11 (DNAH11), which construct gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia when translated within the cells of the subject. The protein encoded by the DNAH11 gene can generate a ciliary dynein outer arm protein. DNAH11 is a microtubule-dependent motor involved in the movement of respiratory cilia In some cases, the composition comprises a nucleic acid construct encoding dynein (axoneme) assembly factor 3 (DNAAF3), which construct gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the DNAAF3 gene may be required for the assembly of the inner and outer arms of axonemal dynein and can play a role in the assembly of the dynein complex for transport into cilia. Mutations in this gene are associated with primary ciliary dyskinesia type 2 (CILD2). In some cases, the composition comprises a nucleic acid construct encoding dynein (axoneme) assembly factor 5 (DNAAF5), which construct gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia when translated within the cells of the subject. The protein encoded by the DNAAF5 gene is thought to be required for preassembly or stability of the axonemal dynein arms and is found only in organisms having motile cilia and flagella. Mutations in this gene are associated with primary ciliary dyskinesia type 18. In some cases, the composition comprises a nucleic acid construct encoding axonemal dynein heavy chain 11 (DNAH11), which construct gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia when translated within the cells of the subject. The protein encoded by the DNAH11 gene can generate a ciliary dynein outer arm protein. DNAH11 is a microtubule-dependent motor involved in the movement of respiratory cilia

[0044] In some cases, the composition comprises a nucleic acid construct encoding dynein (axoneme) assembly factor 3 (DNAAF3), which construct gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the DNAAF3 gene may be required for the assembly of the inner and outer arms of axonemal dynein and can play a role in the assembly of the dynein complex for transport into cilia. Mutations in this gene are associated with primary ciliary dyskinesia type 2 (CILD2). In some cases, the composition comprises a nucleic acid construct encoding dynein (axoneme) assembly factor 5 (DNAAF5), which construct gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia when translated within the cells of the subject. The protein encoded by the DNAAF5 gene is thought to be required for preassembly or stability of the axonemal dynein arms and is found only in organisms having motile cilia and flagella. Mutations in this gene are associated with primary ciliary dyskinesia type 18. In some cases, the composition comprises a nucleic acid construct encoding axonemal dynein heavy chain 11 (DNAH11), which construct gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia when translated within the cells of the subject. The protein encoded by the DNAH11 gene can generate a ciliary dynein outer arm protein. DNAH11 is a microtubule-dependent motor involved in the movement of respiratory cilia In some cases, the composition comprises a nucleic acid construct encoding dynein (axoneme) assembly factor 3 (DNAAF3), which construct gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the DNAAF3 gene may be required for the assembly of the inner and outer arms of axonemal dynein and can play a role in the assembly of the dynein complex for transport into cilia. Mutations in this gene are associated with primary ciliary dyskinesia type 2 (CILD2). In some cases, the composition comprises a nucleic acid construct encoding dynein (axoneme) assembly factor 5 (DNAAF5), which construct gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia when translated within the cells of the subject. The protein encoded by the DNAAF5 gene is thought to be required for preassembly or stability of the axonemal dynein arms and is found only in organisms having motile cilia and flagella. Mutations in this gene are associated with primary ciliary dyskinesia type 18. In some cases, the composition comprises a nucleic acid construct encoding axonemal dynein heavy chain 11 (DNAH11), which construct gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia when translated within the cells of the subject. The protein encoded by the DNAH11 gene can generate a ciliary dynein outer arm protein. DNAH11 is a microtubule-dependent motor involved in the movement of respiratory cilia In some cases, the composition comprises a nucleic acid construct encoding dynein (axoneme) assembly factor 3 (DNAAF3), which construct gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the DNAAF3 gene may be required for the assembly of the inner and outer arms of axonemal dynein and can play a role in the assembly of the dynein complex for transport into cilia. Mutations in this gene are associated with primary ciliary dyskinesia type 2 (CILD2).

[0045] In some cases, the composition comprises a nucleic acid construct encoding axonemal dynein heavy chain 11 (DNAH11), which construct gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia when translated within the cells of the subject. The protein encoded by the DNAH11 gene can generate a ciliary dynein outer arm protein. DNAH11 is a microtubule-dependent motor involved in the movement of respiratory cilia In some cases, the composition comprises a nucleic acid construct encoding axonemal dynein heavy chain 11 (DNAH11), which construct gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia when translated within the cells of the subject. The protein encoded by the DNAH11 gene can generate a ciliary dynein outer arm protein. DNAH11 is a microtubule-dependent motor involved in the movement of respiratory cilia In some cases, the composition comprises a nucleic acid construct encoding axonemal dynein heavy chain 11 (DNAH11), which construct gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia when translated within the cells of the subject. The protein encoded by the DNAH11 gene can generate a ciliary dynein outer arm protein. DNAH11 is a microtubule-dependent motor involved in the movement of respiratory cilia In some cases, the composition comprises a nucleic acid construct encoding axonemal dynein heavy chain 11 (DNAH11), which construct gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia when translated within the cells of the subject. The protein encoded by the DNAH11 gene can generate a ciliary dynein outer arm protein. DNAH11 is a microtubule-dependent motor involved in the movement of respiratory cilia In some cases, the composition comprises a nucleic acid construct encoding axonemal dynein heavy chain 11 (DNAH11), which construct gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia when translated within the cells of the subject. The protein encoded by the DNAH11 gene can generate a ciliary dynein outer arm protein. DNAH11 is a microtubule-dependent motor involved in the movement of respiratory cilia It is thought to be P-ase. Mutations in this gene are associated with primary ciliary dyskinesia type 7 (C ILD7) and situs inversus syndrome.

[0046] In some cases, the composition comprises a nucleic acid construct encoding axonemal dynein heavy chain 5 (DNAH5), which, when translated within the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The DNAH5 gene can provide instructions for making a protein that is part of a group (complex) of proteins called dynein. The coordinated back-and-forth movement of cilia can move the fluid within or around the cell. Dynein can generate the force necessary for the cilia to move. More than 80 mutations in DNAH5 are associated with primary ciliary dyskinesia. Mutations in this gene are associated with primary ciliary dyskinesia and situs inversus syndrome. In some cases, the composition comprises a nucleic acid construct encoding axonemal dynein heavy chain 5 (DNAH5), which, when translated within the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The DNAH5 gene can provide instructions for making a protein that is part of a group (complex) of proteins called dynein. The coordinated back-and-forth movement of cilia can move the fluid within or around the cell. Dynein can generate the force necessary for the cilia to move. More than 80 mutations in DNAH5 are associated with primary ciliary dyskinesia. Mutations in this gene are associated with primary ciliary dyskinesia and situs inversus syndrome. The DNAH5 gene can provide instructions for making a protein that is part of a group (complex) of proteins called dynein. The coordinated back-and-forth movement of cilia can move the fluid within or around the cell. Dynein can generate the force necessary for the cilia to move. More than 80 mutations in DNAH5 are associated with primary ciliary dyskinesia. Mutations in this gene are associated with primary ciliary dyskinesia and situs inversus syndrome. The DNAH5 gene can provide instructions for making a protein that is part of a group (complex) of proteins called dynein. The coordinated back-and-forth movement of cilia can move the fluid within or around the cell. Dynein can generate the force necessary for the cilia to move. More than 80 mutations in DNAH5 are associated with primary ciliary dyskinesia. Mutations in this gene are associated with primary ciliary dyskinesia and situs inversus syndrome.

[0047] In some cases, the composition comprises a nucleic acid construct encoding axonemal dynein heavy chain 6 (DNAH6), which, when translated within the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. In some cases, the composition comprises a nucleic acid construct encoding axonemal dynein heavy chain 6 (DNAH6), which, when translated within the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. In some cases, the composition comprises a nucleic acid construct encoding axonemal dynein heavy chain 6 (DNAH6), which, when translated within the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia.

[0048] In some cases, the composition comprises a nucleic acid construct encoding axonemal dynein heavy chain 8 (DNAH8), which, when translated within the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. In some cases, the composition comprises a nucleic acid construct encoding axonemal dynein heavy chain 8 (DNAH8), which, when translated within the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the DNAH8 gene can function as a force-generating protein for respiratory cilia. DNAH8 can generate force towards the minus end of microtubules. The protein encoded by the DNAH8 gene can function as a force-generating protein for respiratory cilia. DNAH8 can generate force towards the minus end of microtubules. Dynein has ATPase activity; the power stroke that generates force is thought to occur upon release of ADP. DNAH8 may be involved in sperm motility and sperm flagellar assembly. DNAH8 is also known as ATPase and hdhc9.

[0049] In some cases, the composition comprises a nucleic acid construct encoding axonemal dynein light chain 1 (DNAL1), which, when translated within the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the DNAL1 gene can function as a force-generating protein for respiratory cilia. DNAL1 can function as a component of the dynein outer arm complex. This complex acts as a molecular motor that provides the force to move cilia in an ATP-dependent manner. Mutations in this gene are associated with primary ciliary dyskinesia type 16 (CILD16). In some cases, the composition comprises a nucleic acid construct encoding axonemal dynein light chain 1 (DNAL1), which, when translated within the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the DNAL1 gene can function as a force-generating protein for respiratory cilia. DNAL1 can function as a component of the dynein outer arm complex. This complex acts as a molecular motor that provides the force to move cilia in an ATP-dependent manner. Mutations in this gene are associated with primary ciliary dyskinesia type 16 (CILD16). In some cases, the composition comprises a nucleic acid construct encoding axonemal dynein light chain 1 (DNAL1), which, when translated within the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the DNAL1 gene can function as a force-generating protein for respiratory cilia. DNAL1 can function as a component of the dynein outer arm complex. This complex acts as a molecular motor that provides the force to move cilia in an ATP-dependent manner. Mutations in this gene are associated with primary ciliary dyskinesia type 16 (CILD16). In some cases, the composition comprises a nucleic acid construct encoding axonemal dynein light chain 1 (DNAL1), which, when translated within the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the DNAL1 gene can function as a force-generating protein for respiratory cilia. DNAL1 can function as a component of the dynein outer arm complex. This complex acts as a molecular motor that provides the force to move cilia in an ATP-dependent manner. Mutations in this gene are associated with primary ciliary dyskinesia type 16 (CILD16). In some cases, the composition comprises a nucleic acid construct encoding axonemal dynein light chain 1 (DNAL1), which, when translated within the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the DNAL1 gene can function as a force-generating protein for respiratory cilia. DNAL1 can function as a component of the dynein outer arm complex. This complex acts as a molecular motor that provides the force to move cilia in an ATP-dependent manner. Mutations in this gene are associated with primary ciliary dyskinesia type 16 (CILD16). In some cases, the composition comprises a nucleic acid construct encoding axonemal dynein light chain 1 (DNAL1), which, when translated within the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the DNAL1 gene can function as a force-generating protein for respiratory cilia. DNAL1 can function as a component of the dynein outer arm complex. This complex acts as a molecular motor that provides the force to move cilia in an ATP-dependent manner. Mutations in this gene are associated with primary ciliary dyskinesia type 16 (CILD16). In some cases, the composition comprises a nucleic acid construct encoding axonemal dynein light chain 1 (DNAL1), which, when translated within the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the DNAL1 gene can function as a force-generating protein for respiratory cilia. DNAL1 can function as a component of the dynein outer arm complex. This complex acts as a molecular motor that provides the force to move cilia in an ATP-dependent manner. Mutations in this gene are associated with primary ciliary dyskinesia type 16 (CILD16).

[0050] In some cases, the composition comprises a nucleic acid construct encoding dynein regulatory complex subunit 1 (DRC1), which, when translated within the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the DRC1 gene can function as a force-generating protein for respiratory cilia. DRC1 can encode the central component of the nexine-dynein complex (N-DRC) that regulates the assembly of ciliary dynein. Mutations in this gene are associated with primary ciliary dyskinesia type 21 (CILD21). In some cases, the composition comprises a nucleic acid construct encoding dynein regulatory complex subunit 1 (DRC1), which, when translated within the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the DRC1 gene can function as a force-generating protein for respiratory cilia. DRC1 can encode the central component of the nexine-dynein complex (N-DRC) that regulates the assembly of ciliary dynein. Mutations in this gene are associated with primary ciliary dyskinesia type 21 (CILD21). In some cases, the composition comprises a nucleic acid construct encoding dynein regulatory complex subunit 1 (DRC1), which, when translated within the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the DRC1 gene can function as a force-generating protein for respiratory cilia. DRC1 can encode the central component of the nexine-dynein complex (N-DRC) that regulates the assembly of ciliary dynein. Mutations in this gene are associated with primary ciliary dyskinesia type 21 (CILD21). In some cases, the composition comprises a nucleic acid construct encoding dynein regulatory complex subunit 1 (DRC1), which, when translated within the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the DRC1 gene can function as a force-generating protein for respiratory cilia. DRC1 can encode the central component of the nexine-dynein complex (N-DRC) that regulates the assembly of ciliary dynein. Mutations in this gene are associated with primary ciliary dyskinesia type 21 (CILD21). In some cases, the composition comprises a nucleic acid construct encoding dynein regulatory complex subunit 1 (DRC1), which, when translated within the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the DRC1 gene can function as a force-generating protein for respiratory cilia. DRC1 can encode the central component of the nexine-dynein complex (N-DRC) that regulates the assembly of ciliary dynein. Mutations in this gene are associated with primary ciliary dyskinesia type 21 (CILD21). In some cases, the composition comprises a nucleic acid construct encoding dynein regulatory complex subunit 1 (DRC1), which, when translated within the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the DRC1 gene can function as a force-generating protein for respiratory cilia. DRC1 can encode the central component of the nexine-dynein complex (N-DRC) that regulates the assembly of ciliary dynein. Mutations in this gene are associated with primary ciliary dyskinesia type 21 (CILD21). In some cases, the composition comprises a nucleic acid construct encoding dynein regulatory complex subunit 1 (DRC1), which, when translated within the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the DRC1 gene can function as a force-generating protein for respiratory cilia. DRC1 can encode the central component of the nexine-dynein complex (N-DRC) that regulates the assembly of ciliary dynein. Mutations in this gene are associated with primary ciliary dyskinesia type 21 (CILD21).

[0051] In some cases, the composition comprises a nucleic acid construct encoding dyslexia susceptibility 1 candidate 1 (DYX1C1), which, when translated within the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the DYX1C1 gene can function as a respiratory cilia force generation protein. DYX1C1 can encode a protein containing a tetratricopeptide repeat domain. The encoded protein can interact with estrogen receptors as well as heat shock proteins, Hsp70 and Hsp90. Mutations in this gene are also associated with reading and writing deficits, and chromosomal translocations involving this gene are associated with susceptibility to developmental dyslexia. In some cases, the composition comprises a nucleic acid construct encoding dyslexia susceptibility 1 candidate 1 (DYX1C1), which, when translated within the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the DYX1C1 gene can function as a respiratory cilia force generation protein. DYX1C1 can encode a protein containing a tetratricopeptide repeat domain. The encoded protein can interact with estrogen receptors as well as heat shock proteins, Hsp70 and Hsp90. Mutations in this gene are also associated with reading and writing deficits, and chromosomal translocations involving this gene are associated with susceptibility to developmental dyslexia. In some cases, the composition comprises a nucleic acid construct encoding dyslexia susceptibility 1 candidate 1 (DYX1C1), which, when translated within the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the DYX1C1 gene can function as a respiratory cilia force generation protein. DYX1C1 can encode a protein containing a tetratricopeptide repeat domain. The encoded protein can interact with estrogen receptors as well as heat shock proteins, Hsp70 and Hsp90. Mutations in this gene are also associated with reading and writing deficits, and chromosomal translocations involving this gene are associated with susceptibility to developmental dyslexia. In some cases, the composition comprises a nucleic acid construct encoding dyslexia susceptibility 1 candidate 1 (DYX1C1), which, when translated within the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the DYX1C1 gene can function as a respiratory cilia force generation protein. DYX1C1 can encode a protein containing a tetratricopeptide repeat domain. The encoded protein can interact with estrogen receptors as well as heat shock proteins, Hsp70 and Hsp90. Mutations in this gene are also associated with reading and writing deficits, and chromosomal translocations involving this gene are associated with susceptibility to developmental dyslexia. In some cases, the composition comprises a nucleic acid construct encoding dyslexia susceptibility 1 candidate 1 (DYX1C1), which, when translated within the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the DYX1C1 gene can function as a respiratory cilia force generation protein. DYX1C1 can encode a protein containing a tetratricopeptide repeat domain. The encoded protein can interact with estrogen receptors as well as heat shock proteins, Hsp70 and Hsp90. Mutations in this gene are also associated with reading and writing deficits, and chromosomal translocations involving this gene are associated with susceptibility to developmental dyslexia. In some cases, the composition comprises a nucleic acid construct encoding dyslexia susceptibility 1 candidate 1 (DYX1C1), which, when translated within the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the DYX1C1 gene can function as a respiratory cilia force generation protein. DYX1C1 can encode a protein containing a tetratricopeptide repeat domain. The encoded protein can interact with estrogen receptors as well as heat shock proteins, Hsp70 and Hsp90. Mutations in this gene are also associated with reading and writing deficits, and chromosomal translocations involving this gene are associated with susceptibility to developmental dyslexia. In some cases, the composition comprises a nucleic acid construct encoding dyslexia susceptibility 1 candidate 1 (DYX1C1), which, when translated within the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the DYX1C1 gene can function as a respiratory cilia force generation protein. DYX1C1 can encode a protein containing a tetratricopeptide repeat domain. The encoded protein can interact with estrogen receptors as well as heat shock proteins, Hsp70 and Hsp90. Mutations in this gene are also associated with reading and writing deficits, and chromosomal translocations involving this gene are associated with susceptibility to developmental dyslexia. In some cases, the composition comprises a nucleic acid construct encoding dyslexia susceptibility 1 candidate 1 (DYX1C1), which, when translated within the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the DYX1C1 gene can function as a respiratory cilia force generation protein. DYX1C1 can encode a protein containing a tetratricopeptide repeat domain. The encoded protein can interact with estrogen receptors as well as heat shock proteins, Hsp70 and Hsp90. Mutations in this gene are also associated with reading and writing deficits, and chromosomal translocations involving this gene are associated with susceptibility to developmental dyslexia. In some cases, the composition comprises a nucleic acid construct encoding dyslexia susceptibility 1 candidate 1 (DYX1C1), which, when translated within the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the DYX1C1 gene can function as a respiratory cilia force generation protein. DYX1C1 can encode a protein containing a tetratricopeptide repeat domain. The encoded protein can interact with estrogen receptors as well as heat shock proteins, Hsp70 and Hsp90. Mutations in this gene are also associated with reading and writing deficits, and chromosomal translocations involving this gene are associated with susceptibility to developmental dyslexia.

[0052] In some cases, the composition comprises a nucleic acid construct encoding growth arrest specific 8 (GAS8), which, when translated within the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. In some cases, the composition comprises a nucleic acid construct encoding growth arrest specific 8 (GAS8), which, when translated within the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. In some cases, the composition comprises a nucleic acid construct encoding growth arrest specific 8 (GAS8), which, when translated within the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia.

[0053] In some cases, the composition comprises a nucleic acid construct encoding axonemal central pair apparatus protein (HYDIN), which, when translated within the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. In some cases, the composition comprises a nucleic acid construct encoding axonemal central pair apparatus protein (HYDIN), which, when translated within the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. In some cases, the composition comprises a nucleic acid construct encoding axonemal central pair apparatus protein (HYDIN), which, when translated within the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the HYDIN gene can function in ciliary motility. Mutations in this gene are associated with primary ciliary dyskinesia type 5 (CILD5). The protein encoded by the HYDIN gene can function in ciliary motility. Mutations in this gene are associated with primary ciliary dyskinesia type 5 (CILD5).

[0054] In some cases, the composition comprises a nucleic acid construct encoding leucine rich repeat containing 6 (LRRC6). comprises a nucleic acid construct which, when translated within a cell of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. L The protein encoded by the RRC6 gene contains several leucine-rich repeat domains and is thought to be involved in ciliary motility. Mutations in this gene are associated with primary ciliary dyskinesia type 19 (CILD19).

[0055] In some cases, the composition comprises a nucleic acid construct encoding NME / NM23 family member 8 (NME8) which, when translated within a cell of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the NME8 gene can function as a respiratory ciliary force generation protein. The NME8 protein contains an N-terminal thioredoxin domain and three C-terminal nucleoside diphosphate kinase (NDK) domains. Mutations in this gene are associated with primary ciliary dyskinesia type 6 (CILD6).

[0056] In some cases, the composition comprises a nucleic acid construct encoding orofaciodigital syndrome 1 (OFD1) which, when translated within a cell of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The function of the protein produced by the OFD1 gene is not well understood but may play an important role in the early development of many parts of the body including the brain, face, limbs and kidneys. Approximately 100 mutations in the OFD1 gene are associated with type I orofaciodigital syndrome, the most common form of this disorder. ​​​​​​​It has been found in people. Mutations in this gene are associated with primary ciliary dyskinesia and Joubert syndrome.

[0057] In some cases, the composition comprises a nucleic acid construct encoding a retinitis pigmentosa GTPase regulator (RPGR), which, when translated in the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the RPGR gene may be important for normal vision and ciliary function. Mutations in this gene are associated with primary ciliary dyskinesia, X-linked retinitis pigmentosa, progressive vision loss, chronic respiratory and sinus infections, recurrent ear infections (otitis media) and hearing loss.

[0058] In some cases, the composition comprises a nucleic acid construct encoding a radial spoke head 1 homolog (RSPH1), which, when translated in the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the RSPH1 gene may play an important role in male meiosis and the construction of the axoneme central pair and radial spokes. Mutations in this gene are associated with primary ciliary dyskinesia type 24 (CILD24).

[0059] In some cases, the composition comprises a nucleic acid construct encoding a radial spoke head 4 homolog A (RSPH4A), which, when translated in the cells of a subject, gives rise to a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. It is known. The protein encoded by the RSPH4A gene may be a component of the radial spoke head. Mutations in this gene are associated with primary ciliary dyskinesia type 11 (CILD11). 1).

[0060] In some cases, the composition comprises a nucleic acid construct encoding radial spoke head 9 homolog (RSPH9), which, when translated within the cells of a subject, yields a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the RSPH9 gene may be a component of the radial spoke head in motile cilia and flagella. Mutations in this gene are associated with primary ciliary dyskinesia type 12 (CILD12). Mutations in this gene are associated with primary ciliary dyskinesia type 12 (CILD12). 12).

[0061] In some cases, the composition comprises a nucleic acid construct encoding sperm associated antigen 1 (SPAG1), which, when translated within the cells of a subject, yields a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by the SPAG1 gene may play a role in the cytoplasmic assembly of the ciliary dynein arm. Mutations in this gene are associated with primary ciliary dyskinesia type 28 (CILD28). Mutations in this gene are associated with primary ciliary dyskinesia type 28 (CILD28). 28).

[0062] In some cases, the composition comprises a nucleic acid construct encoding zinc finger MYND type containing 10 (ZMYND10), which, when translated within the cells of a subject, yields a polypeptide that treats a subject having or at risk of having primary ciliary dyskinesia. The protein encoded by ZMYND10 is appropriate for ciliary motility. Mutations in this gene are associated with primary ciliary dyskinesia type 28 (CILD28). For axoneme assembly, the inner and outer dynein arms (IDA and ODA, respectively) are assembled into axonemal assemblies. Mutations in this gene cause primary ciliopathies. It is associated with type 22 (CILD22).

[0063] Treatment includes curing a subject (e.g., a patient with a disease and / or an experimental animal with a condition). In some cases, the condition may include treating a primary ciliary dyskinesia (PCD) or Kartagener's syndrome. In some cases, the condition is caused by a defect in the cellular structures known as cilia. In some cases, the subject is a human. The treatment may be provided to a subject prior to clinical onset of the disease. Treatment can be provided to a subject after clinical onset of the disease, for up to 5 minutes or more. minutes or later, 10 minutes or later, 30 minutes or later, 1 hour after or after, 2 hours or after, 3 hours or after, 4 hours or later, 5 hours later or later, 6 hours later or later, 12 hours later or later, 1 day or later, 1 week or later, 6 months or later or later, 12 months or later, or 2 years or later. Treatment may be initiated at least 1 minute, 10 minutes, 30 minutes, 1 hour, or 2 hours after clinical onset of the disease. hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, 12 hours or more, 1 day or more , provided to subjects for periods of 1 week or more, 1 month or more, 6 months or more, 12 months or more, or 2 years or more. Treatment may begin within 2 years, 12 months, 6 months, or 1 month after clinical onset of the disease. Within, within 1 week, within 1 day, within 12 hours, within 6 hours, within 5 hours, within 4 hours, within 3 hours within a period of within 2 hours, within 1 hour, within 30 minutes, within 10 minutes or within 1 minute, the subject body. The treatment may also include treating humans in clinical trials.

[0064] Compositions containing the engineered polynucleotides described herein are for prophylactic and / or can be administered for therapeutic treatment. In therapeutic applications, the nucleic acid construct or ve ctor provides an amount of the encoded polypeptide sufficient to cure or at least ameliorate the symptoms of the disease in a subject already suffering from a disease such as primary ciliary dyskinesia. It can be administered in an amount sufficient to provide. The nucleic acid construct, vector, engineered polynucleotide or composition can also be administered to reduce the likelihood of developing, suffering from or exacerbating a disease. The amount effective for this use depends on the severity and course of the disease or condition, the transfection efficiency of the (one or more) nucleic acid constructs, the (one or more) vectors, the (one or more) engineered polynucleotides or the (one or more) compositions, the affinity of the encoded polypeptide for the target molecule, previous treatments, the health status of the subject, body weight, response to the drug, and the judgment of the treating physician. It may vary based on. multiple) engineered polynucleotides or the transfection efficiency of the (one or more) compositions, the affinity of the encoded polypeptide for the target molecule, previous treatments, The health status of the subject, body weight, response to the drug, and the judgment of the treating physician. It may vary based on the severity and course of the disease or condition, the transfection efficiency of the (one or more) nucleic acid constructs, the (one or more) vectors, the (one or more) engineered polynucleotides or the (one or more) compositions, the affinity of the encoded polypeptide for the target molecule, previous treatments, the health status of the subject, body weight, response to the drug, and the judgment of the treating physician.

[0065] In some cases, the polynucleotides of the present disclosure are armadillo repeat-containing 4 (ARMC 4), chromosome 21 open reading frame 59 (C21orf59), coiled coiled coil domain-containing 103 (CCDC103), coiled coil domain-containing 114 (C CDC114), coiled coil domain-containing 39 (CCDC39), coiled coil Main contains 40 (CCDC40), coiled-coil domain-containing 65 (CCDC65), Cyclin O (CCNO), dynein (axoneme) assembly factor 1 (DNAAF1), dy nein (axoneme) assembly factor 2 (DNAAF2), dynein (axoneme) assembly factor 3 ( DNAAF3), dynein (axoneme) assembly factor 5 (DNAAF5), axonemal dynein heavy chain 11 (DNAH11), axonemal dynein heavy chain 5 (DNAH5), axonemal dynein heavy chain 6 (D NAH6), axonemal dynein heavy chain 8 (DNAH8), axonemal dynein intermediate chain 2 (DNAI2) , axonemal dynein light chain 1 (DNAL1), dynein regulatory complex subunit 1 (DRC1) , dyslexia susceptibility 1 candidate 1 (DYX1C1), growth arrest specific 8 (GAS8), axoneme central pairing apparatus protein (HYDIN), leucine rich repeat containing 6 (LRRC6), NM E / NM23 family member 8 (NME8), orofaciodigital syndrome 1 (OFD1), retinitis pigmentosa GTPase regulator (RPGR), radial spoke head 1 homolog ( Chlamydomonas) (RSPH1), radial spoke head 4 homolog A (Chlamydomonas s) (RSPH4A), radial spoke head 9 homolog (Chlamydomonas) (RSP H9), sperm associated antigen 1 (SPAG1) and zinc finger MYND type containing 10 (Z MYND10), etc., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% homologous to proteins associated with primary ciliary dyskinesia, can encode a peptide.

[0066] A plurality of nucleic acid constructs, vectors, engineered polynucleotides or compositions, optionally They can be administered in the order of or simultaneously with the nucleic acid construct, vector, engineered polynucleotide or composition can be packaged together or separately in a single package or in multiple packages. One or all of the nucleic acid construct, vector, engineered polynucleotide or composition can be administered in multiple doses. If not simultaneous, the timing between multiple doses may vary.

[0067] The nucleic acid construct, vector, engineered polynucleotide or composition can be administered to the subject as soon as possible after the onset of symptoms. One or more nucleic acid constructs, one or more vectors, one or more engineered polynucleotides or compositions can be administered as soon as practically possible after the onset of a detected or suspected disease or condition for the period required for the treatment of the disease, e.g., about 1 month, about 6 months, about 12 months, about 18 months, about 24 months, or any suitable period. The length of the treatment period may vary for each subject.

[0068] Altered nucleotide usage frequency in the coding region to enhance mRNA stability for transcript therapy Hydrolysis of oligonucleotides suggests that the reactivity of the phosphodiester bond linking two ribonucleotides in single-stranded (ss) RNA depends on the nature of those nucleotides. At pH 8.5, the dinucleotide cleavage sensitivity when embedded in an ssRNA dodecamer can vary by about an order of magnitude. Under nearly physiological conditions, RNA hydrolysis is usually , a 2'-oxygen nucleophile on the adjacent phosphorus target center on the opposite side of the 5'-oxyanion leaving group results in an S N type 2 attack, generating two RNA fragments with 2',3'-cyclic phosphate and 5'-hydroxyl termini. The backbone at these stages is the "in-line" conformation most readily adopted by the 2'-OH on the adjacent phosphodiester bond for an S N type 2 nucleophilic attack. Thus, more reactive cleavable phosphodiester bonds can include 5'-UpA-3' (R1 = U1, R2 = A) and 5'-CpA-3 ' (R1 = C, R2 = A). Additionally, the interferon-regulated dsRNA-activated antiviral pathway results in the production of 2'-5' oligoadenylates that bind to the ankyrin repeats to activate RNase L endoribonuclease. RNase L efficiently cleaves ssRNA at UA and UU dinucleotides. Finally, U-rich sequences are potent activators of RNA sensors including Toll-like receptors 7 and 8 and RIG-I, and a reduction in the overall uridine content represents a potentially attractive approach to reduce the immunogenicity of therapeutic mRNAs. Altered nucleotide usage frequency schemes that aim to reduce the number of more reactive 5'-U(U / A)- 3' dinucleotides within and across codons of the modified mRNA partially alleviate the constraints imposed by the inherent chemical instability of RNA.

[0069] Simultaneously, reducing the U content in the RNA transcript reduces its immunogenicity. This disclosure relates to RNA transcripts containing altered open reading frames (ORFs). In particular, a method is proposed that involves a substantial reduction of 5'-U(U / A)-3' dinucleotides within the protein-coding region that results in stabilized therapeutic mRNA.

Table 2

[0070] Nucleic acid constructs, vectors, and engineered polynucleotides ​The present disclosure provides nucleic acid molecules, such as polynucleotides, encoding one or more polypeptides of interest. The term nucleic acid includes any compound and / or substance that contains a polymer of nucleotides. Nucleotide polymers that contain greater than 50% ribose bases or ribonucleotide analogs are referred to as polynucleotides. Nucleotide polymers can use altered nucleotide usage frequencies that encode proteins such as DNAI1 or DNAH5 or functional fragments thereof. The sequence of the engineered polynucleotide can be derived from, for example, DNA, RNA, mRNA transcript, genomic DNA, mitochondrial DNA, mitochondrial RNA, or another suitable nucleic acid that contains the genetic information of the gene of interest. Nucleic acid constructs, vectors, engineered polynucleotides or compositions can be derived from nucleic acids that carry mutated genes and polymorphisms.

[0071] In addition to the four standard ribonucleotides, i.e., adenosine, guanosine, cytidine, and uridine, some cellular RNAs also contain many structurally diverse ribonucleotides. Approximately 100 structurally different nucleotides or nucleotide analogs have been identified in transfer RNA (tRNA), ribosomal RNA (rRNA), messenger RNA (mRNA), and small nuclear RNA (snRNA). In tRNA, some nucleotides can be important determinants of the specificity and efficiency of aminoacylation and codon recognition. Such structurally diverse ribonucleotides can be modified ribonucleotides or nucleotide analogs. In some cases, the polynucleotides of the present disclosure are engineered to include ribonucleotide analogs. ​

[0072] In some cases, a nucleic acid construct, vector or polynucleotide is engineered to contain four classical ribo nucleotides and can be post-transcriptionally modified after administration to a subject. For example, in some cases, the present disclosure provides a composition, vector or nucleic acid construct comprising a nucleic acid construct encoding axonemal dynein intermediate chain 1, wherein less than 30% of the nucleotides encoding axonemal dynein intermediate chain 1 are nucleotide analogs. In other cases, less than 27.5%, less than 25%, less than 22.5%, less than 20%, less than 17.5%, less than 15%, less than 12.5%, less than 10%, less than 7.5%, less than 5% or less than 2.5% of the nucleotides encoding axonemal dynein intermediate chain 1 are nucleotide

[0073] Exemplary nucleic acids that can form the polynucleotides of the present disclosure include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA) or hybrids thereof. Exemplary modified nucleotides that can form at least a portion of the polynucleotides of the present disclosure include, but are not limited to, pseudouridine (Ψ) and 1-methylpseudouridine (m 1 Ψ).

[0074] Chemical modifications can be located on one or more nucleosides or on the backbone of the nucleic acid molecule. They can be located on both the nucleoside and the backbone linkage. Modifications can be incorporated into the polynucleotide in vitro. Modified ribonucleotides and nucleic acid analogs can also be introduced post-transcriptionally by covalent modification

[0075] The nucleic acid constructs, vectors or engineered polynucleotides of the present disclosure may contain purine and pyrimidine analogs. In some cases, the polynucleotides of the present disclosure contain modified pyrimidines such as modified uridine. In some cases, uridine analogs include pseudouridine (Ψ), 1-methylpseudouridine (m 1 Ψ), 2-thiouridine (s 2 U), 5-methyluridine (m 5 U), 5-methoxyuridine (mo 5 U), 4 -thiouridine (s 4 U), 5-bromouridine (Br 5 U), 2’O-methyluridine (U2’m), 2’-amino-2’-deoxyuridine (U2’NH2), 2’-azido -2’-deoxyuridine (U2’N3) and 2’-fluoro-2’-deoxyuridine (U2’F).

[0076] In some cases, the (one or more) nucleic acid constructs, the (one or more) vectors, (one or more) engineered polynucleotides or (one or more) compositions encode at levels that are increased by at least about 1.5-fold compared to the levels in cells exposed to a composition containing a nucleic acid construct that does not contain a codon encoding the axonemal dynein intermediate chain 1 protein or a variant thereof. In some cases, the fold increase is at least about 1.1, at least about 1.2, at least about 1.3 , at least about 1.4, at least about 1.5, at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 1000, at least about 2000, at least about 3000, 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 at least about 100.

[0077] The polynucleotide can have the same nucleotide analogs or modified nucleotides, or a mixture of different nucleotide analogs or modified nucleotides. The nucleotide analogs or modified nucleotides can have structural changes that are present or not present in messenger RNA naturally. Mixtures of various analogs or modified nucleotides can be used. For example, one or more analogs within the polynucleotide can have natural modifications, and another portion can have modifications not found naturally in mRNA. Furthermore, some analogs or modified ribonucleotides can have base modifications, while other modified ribonucleotides can have sugar modifications. Similarly, all modifications can be base modifications, or all modifications can be sugar

[0078] modifications or any suitable mixture thereof. The nucleotide analogs or modified nucleotides are pyridin-4-one ribonucleosides , 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio- pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyl uridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine , 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl uridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-u - Pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl - Pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-meth yl-1-deaza-pseudouridine, Dihydrouridine, Dihydropseudouridine, 2 - Thio-dihydrouridine, 2-Thio-dihydropseudouridine, 2-Methoxyuridine , 2-Methoxy-4-thio-uridine, 4-Methoxy-pseudouridine, 4-Meth oxy-2-thio-pseudouridine, 5-Aza-cytidine, Pseudoisocytidine, 3-M ethyl-cytidine, N4-Acetylcytidine, 5-Formylcytidine, N4-Methylcytid ine, 5-Hydroxymethylcytidine, 1-Methyl-pseudoisocytidine, Pyrrolo-cyt idine, Pyrrolo-pseudoisocytidine, 2-Thio-cytidine, 2-Thio-5-methyl-c ytidine, 4-Thio-pseudoisocytidine, 4-Thio-1-methyl-pseudoisocytid ine, 4-Thio-1-methyl-1-deaza-pseudoisocytidine, 1-Methyl-1-dea za-pseudoisocytidine, Zebularine, 5-Aza-zebularine, 5-Methyl-zebular ine, 5-Aza-2-thio-zebularine, 2-Thio-zebularine, 2-Methoxy-cytidine , 2-Methoxy-5-methyl-cytidine, 4-Methoxy-pseudoisocytidine, 4-M ethoxy-1-methyl-pseudoisocytidine, 2-Aminopurine, 2,6-Diaminopur ine, 7-Deaza-adenine, 7-Deaza-8-aza-adenine, 7-Deaza-2-amin opurine, 7-Deaza-8-aza-2-aminopurine, 7-Deaza-2,6-diaminopur ine, 7-Deaza-8-aza-2,6-diaminopurine, 1-Methyladenosine, N6-M Chiladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopenten 2-Methylthio-N6-(cis-hydroxyisopentenyl)adenosine, Adenosine, N6-glycinylcarbamoyl adenosine, N6-threonylcarbamoyl adenosine Adenosine, 2-methylthio-N6-threonylcarbamoyl adenosine, N6,N6-dimethyl adenosine, 7-methyladenine, 2-methylthio-adenine, 2-methoxy-adenine Inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza Aza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine , 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thiamine O-guanosine, N2-methyl-6-thio-guanosine and N2,N2-dimethyl-6 -thio-guanosine.

[0079] In some cases, the nucleic acid construct(s), vector(s), At least about 5% of the engineered polyribonucleotide(s) or composition are not naturally occurring (e.g., modified, analogous) nucleotides such as those described herein. Some of the amino acids contain uridine, adenosine, guanine, or cytosine (analogous or engineered). In some cases, 100% of the modified nucleotides in the composition are 1-methylpseudouridine or In some cases, the nucleic acid construct(s) The product, vector(s), engineered polyribonucleoside(s), At least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 0%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 9 0%, 95% contain the non-naturally occurring uracil, adenine, guanine or cytosine. In some cases, the nucleic acid construct(s), vector(s) At most about 99% of the engineered polyribonucleotide or compositions %, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50 %, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1% are , including the non-naturally occurring uracil, adenine, guanine, or cytosine.

[0080] The nucleic acid construct(s), vector(s) or (1) of the present disclosure The engineered polyribonucleotide (or polyribonucleotides) may comprise one or more promoter sequences and The promoter sequence and / or any associated regulatory sequences may be included. The string may contain any number of modified or unmodified nucleotides, and any number of nucleic acid analogs. The promoter sequence and / or any associated regulatory sequences may be, for example, at least two amino acids. base or base pair, 3 base or base pair, 4 base or base pair, 5 base or base pair, 6 salt base or base pair, 7 base or base pair, 8 base or base pair, 9 base or base pair, 10 Base or base pair, 11 base or base pair, 12 base or base pair, 13 base or base pairs, 14 bases or base pairs, 15 bases or base pairs, 16 bases or base pairs, 17 bases or or a base pair, 18 bases or base pairs, 19 bases or base pairs, 20 bases or base pairs, 2 1 base or base pair, 22 bases or base pairs, 23 bases or base pairs, 24 bases or base pairs, 25 bases or base pairs, 26 bases or base pairs, 27 bases or base pairs, 28 bases or base pairs, 29 bases or base pairs, 30 bases or base pairs, 35 bases or base pairs, 40 bases or base pairs, 50 bases or base pairs, 75 bases or base pairs, 100 bases or base pairs, 150 bases or base pairs, 200 bases or base pairs, 300 bases or base pairs 400 bases or base pairs, 500 bases or base pairs, 600 bases or base pairs, 700 bases or base pairs, 800 bases or base pairs, 900 bases or base pairs, 1000 bases or base pairs, 2000 bases or base pairs, 3000 bases or base pairs, 4000 bases or base pairs, 5000 bases or base pairs, including at least 10000 bases or base pairs or more may be obtained. The promoter sequence and / or related regulatory sequences may have any number of modifications or non- modified nucleotides, for example, at most 10000 bases or base pairs, 5000 bases or base pairs, 4000 bases or base pairs, 3000 bases or base pairs, 2000 bases or base pairs, 1000 bases or base pairs, 900 bases or base pairs, 800 bases or base pairs, 700 bases or base pairs, 600 bases or base pairs, 500 bases or base pairs, 400 bases or base pairs, 300 bases or base pairs, 200 bases or base pairs, 100 bases or base pairs, 75 bases or base pairs, 50 bases or base pairs, 40 bases or base pairs, 35 bases or base pairs, 30 bases or base pairs, 29 bases or base pairs, 28 bases or base pairs, 27 bases or base pairs, 26 bases or or 25 bases or base pairs, 24 bases or base pairs, 23 bases or base pairs, 22 bases or base pairs, 21 bases or base pairs, 20 bases or base pairs, 19 bases or base pairs, 18 bases or base pairs, 17 bases or base pairs, 16 bases or base pairs, 15 bases or base pairs, 14 bases or base pairs, 13 bases or base pairs, 12 bases or base pairs, 11 bases or base pairs, 10 bases or base pairs, 9 bases or base pairs, 8 bases or base pairs, 7 bases or base pairs, 6 bases or base pairs, 5 bases or base pairs, 4 bases or base pairs, 3 bases or base pairs, or 2 bases or base pairs and may contain.

[0081] In some cases, fewer nucleotides than all of the nucleotides in the promoter sequence or associated regulatory region are nucleotide analogs or modified nucleotides. For example , in some cases, 99%, 95% , 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45% , 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5% or less. In some cases , all of the nucleotides in the promoter or associated regulatory region are nucleic acid analogs or modified nucleotides.

[0082] The (one or more) nucleic acid constructs, (one or more) vectors, (one or more ) engineered polyribonucleotides or compositions of the present disclosure may include an engineered 5' cap structure, or a 5' cap may be added to the polyribonucleotide intracellularly . The 5' cap structure of mRNA binds to the mRNA cap binding protein (CBP) can participate and contribute to the stability and translational ability of mRNA in cells through binding to CBP and poly(A)-binding proteins to form mature pseudo-circular mRNA species. The 5’ cap structure can also be involved in assisting nuclear transport, increasing mRNA stability, and removing 5’ proximal introns during mRNA splicing. One or more nucleic acid constructs, one or more vectors, or one or more engineered polynucleotides can be capped at the 5’ end, generating a 5’-GpppN-3’-triphosphate bond between the terminal guanosine cap residue of the mRNA molecule and the 5’-transcribed sense nucleotide. The cap structure can include a modified or unmodified 7-methylguanosine linked to the first nucleotide via a 5’-5’ triphosphate bridge. Subsequently, this 5’-guanylate cap can be methylated to generate an N7-methyl-guanylate residue (cap 0 structure). The ribose sugars of the terminal and / or pre-terminal transcribed nucleotides at the 5’ end of the mRNA can be 2’-O-methylated (cap 1 structure).

[0083] Hydrolysis and cleavage of the guanylate cap structure to remove the 5’ cap can target nucleic acid molecules such as mRNA molecules for degradation. In some cases, the cap can include further modifications, including methylation of the 2’-hydroxy groups of the first two ribose sugars at the 5’ end of the mRNA. For example, a eukaryotic cap 1 has a methylated 2’-hydroxy group on the first ribose sugar, while cap 2 has methylated 2’-hydroxy groups on the first two ribose sugars. The 5’ cap structure can also be involved in assisting nuclear transport, increasing mRNA stability, and removing 5’ proximal introns during mRNA splicing. One or more nucleic acid constructs, one or more vectors, or one or more engineered polynucleotides can be capped at the 5’ end, generating a 5’-GpppN-3’-triphosphate bond between the terminal guanosine cap residue of the mRNA molecule and the 5’-transcribed sense nucleotide. The cap structure can include a modified or unmodified 7-methylguanosine linked to the first nucleotide via a 5’-5’ triphosphate bridge. Subsequently, this 5’-guanylate cap can be methylated to generate an N7-methyl-guanylate residue (cap 0 structure). The ribose sugars of the terminal and / or pre-terminal transcribed nucleotides at the 5’ end of the mRNA can be 2’-O-methylated (cap 1 structure). Hydrolysis and cleavage of the guanylate cap structure to remove the 5’ cap can target nucleic acid molecules such as mRNA molecules for degradation. In some cases, the cap can include further modifications, including methylation of the 2’-hydroxy groups of the first two ribose sugars at the 5’ end of the mRNA. For example, a eukaryotic cap 1 has a methylated 2’-hydroxy group on the first ribose sugar, while cap 2 has methylated 2’-hydroxy groups on the first two ribose sugars. The 5’ cap structure can also be involved in assisting nuclear transport, increasing mRNA stability, and removing 5’ proximal introns during mRNA splicing. One or more nucleic acid constructs, one or more vectors, or one or more engineered polynucleotides can be capped at the 5’ end, generating a 5’-GpppN-3’-triphosphate bond between the terminal guanosine cap residue of the mRNA molecule and the 5’-transcribed sense nucleotide. The cap structure can include a modified or unmodified 7-methylguanosine linked to the first nucleotide via a 5’-5’ triphosphate bridge. Subsequently, this 5’-guanylate cap can be methylated to generate an N7-methyl-guanylate residue (cap 0 structure). The ribose sugars of the terminal and / or pre-terminal transcribed nucleotides at the 5’ end of the mRNA can be 2’-O-methylated (cap 1 structure).

[0084] Hydrolysis and cleavage of the guanylate cap structure to remove the 5’ cap can target nucleic acid molecules such as mRNA molecules for degradation. In some cases, the cap can include further modifications, including methylation of the 2’-hydroxy groups of the first two ribose sugars at the 5’ end of the mRNA. For example, a eukaryotic cap 1 has a methylated 2’-hydroxy group on the first ribose sugar, while cap 2 has methylated 2’-hydroxy groups on the first two ribose sugars. The 5’ cap structure can also be involved in assisting nuclear transport, increasing mRNA stability, and removing 5’ proximal introns during mRNA splicing. One or more nucleic acid constructs, one or more vectors, or one or more engineered polynucleotides can be capped at the 5’ end, generating a 5’-GpppN-3’-triphosphate bond between the terminal guanosine cap residue of the mRNA molecule and the 5’-transcribed sense nucleotide. The cap structure can include a modified or unmodified 7-methylguanosine linked to the first nucleotide via a 5’-5’ triphosphate bridge. P can be chemically similar to the 3’ end of an RNA molecule (the 5’ carbon of the capped ribose is bonded and the free 3’-hydroxyl at both the 5’ and 3’ ends of the capped transcript . Such a double modification can provide significant resistance to 5’ exonucleases. Non-limiting examples of 5’ cap structures that can be used with engineered polynucleotides include m m 7 G(5’)ppp(5’)N(‘cap 0), m 7 G(5’)ppp(5’)N1 mpNp(‘cap 1) and m 7 G(5’)ppp(5’)N1mpN2mp(‘ca p 2), but are not limited thereto.

[0085] The modifications to the modified mRNA of the present disclosure generate a non-hydrolyzable cap structure that prevents cap removal, thereby increasing the half-life of the mRNA while promoting efficient translation . Since hydrolysis of the cap structure requires cleavage of the 5’-ppp-5’ triphosphate bond , modified nucleotides can be used during the capping reaction. For example, vaccinia capping enzyme from New Eng land Biolabs (Ipswich, MA) can be used with guanosine α-thiophosphate nucleotides according to the manufacturer's instructions to create phosphorothioate bonds in the 5’- ppp-5’ cap. Further modified guanosine nucleotides such as α-methylphosph onic acid and selenophosphate nucleotides can be used . Further modifications include, but are not limited to, 2’-O-methylation of the ribose sugar of the 5’ end of the mRNA and / or the nucleotide immediately preceding the 5’ end on the 2’-hydroxyl group of the sugar ring . Multiple different 5’ cap structures can be used to 5’ ca p a polynucleotide and / or the 5’ end of the polynucleotide, and the 5’ end of the polynucleotide can be further modified to include a 2’-O-methyl group on the ribose sugar of the nucleotide immediately preceding the 5’ end of the polynucleotide. Caps can be generated.

[0086] Modified mRNA can be capped post-transcriptionally. According to the present disclosure, the 5' end cap can include an endogenous cap or a cap analog. According to the present disclosure, the 5' end cap can include a guanine analog. Useful guanine analogs include inosine, N1-methyl-gua nosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine , 2-amino-guanosine, LNA-guanosine and 2-azido-guanosine, but are not limited thereto.

[0087] Furthermore, the (one or more) nucleic acid constructs, (one or more) vectors or (one or more) engineered polynucleotides can include one or more internal ribosome entry sites (IRES). The IRES sequence can initiate protein synthesis in the absence of a 5' cap structure. The IRES sequence can also be the only ribosome binding site or can function as one of multiple ribosome binding sites of the mRNA. Engineered polynucleotides containing two or more functional ribosome binding sites can encode several peptides or polypeptides ( "polycistronic or multicistronic polynucleotides") to be translated by ribosomes. The engineered polynucleotides described herein can include at least one IRES sequence, two IRES sequences, three IRES sequences , four IRES sequences, five IRES sequences, six IRES sequences, seven IRES sequences , eight IRES sequences, nine IRES sequences, ten IRES sequences, or Another suitable number may be present in the engineered polyribonucleotide. Examples of IRES sequences that can be used include, but are not limited to, those derived from the tobacco etch virus (tobacc o etch virus (TEV), picornavirus es) (e.g. FMDV), plague viruses (CFFV) , polio viruses (PV), encephalomyocarditis virus (enc ephalomyocarditis viruses (EMCV), foot-and-mouth disease virus (foot-and-mouth disease viruses)(FMDV), C Hepatitis C viruses (HCV), classical porcine Relaviruses (classical swine fever viruses) (CS FV, murine leukemia virus (MLV) ), simian immune deficiency virus ruses (SIV) or cricket paralysis virus (cricket paralysis IRES sequences include those derived from the Creutzfeldt-Virus (CrPV). lontech™, GeneCopoeia™ or Sigma-Aldr The IRES sequence can be derived from a commercially available vector, such as the IRES sequence available from ich™. The IRES sequence may be, for example, at least 150 bases or base pairs, 200 bases or is 300 bases or base pairs, 400 bases or base pairs, 500 bases or Base pairs, 600 bases or base pairs, 700 bases or base pairs, 800 bases or salts base pairs, 900 bases or base pairs, 1000 bases or base pairs, 2000 bases or It may be a base pair, 3000 bases or base pairs, 4000 bases or base pairs, 5000 bases or base pairs, or 10000 bases or base pairs. The IRES sequence is often also 10000 bases or base pairs, 5000 bases or base pairs, 4000 bases or base pairs, 3000 bases or base pairs, 2000 bases or base pairs, 1000 bases or base pairs, 900 bases or base pairs, 800 bases or base pairs, 700 bases or base pairs, 600 bases or base pairs, 500 bases or base pairs, 400 bases or base pairs, 300 bases or base pairs, 200 bases or base pairs, 100 bases or base pairs, 50 bases or base pairs, or 10 bases or base pairs.

[0088] The (one or more) nucleic acid constructs, (one or more) vectors or (one or more) engineered polynucleotides of the present disclosure may include one or more untranslated regions . The untranslated region may contain any number of modified or unmodified nucleotides. The untranslated region (UTR) of a gene is transcribed into a polypeptide but not translated. In some cases, the untranslated sequence can enhance the stability and translation efficiency of the nucleic acid molecule. The regulatory features of the UTR can be incorporated into the modified mRNA molecules of the present disclosure, for example, to enhance the stability of the molecule. Certain features can also be incorporated to ensure controlled downregulation of the transcript when they are misdirected to unwanted organ sites. Some 5’UTRs play a role in translation initiation. The 5’UTR may contain a Kozak sequence that is involved in the process by which ribosomes initiate the translation of many genes. The Kozak sequence is a consensus GCC(R ​​)It can have CCAUGG, and R is a purine (adenine or guanine) located 3 bases upstream of the start codon (AUG). The 5’UTR can form a secondary structure involved in the binding of translation elongation factors. In some cases, by manipulating the features typically found in genes highly expressed in specific target organs, the stability of the engineered polynucleotide molecules of the present disclosure and protein production can be increased. For example, the introduction of the 5’UTR of mRNAs expressed in the liver, such as albumin, serum amyloid A, apolipoprotein A / B / E, transferrin, α-fetoprotein, erythropoietin or factor VIII, can be used to increase the expression of the engineered polynucleotide in the liver. Similarly, the use of the 5’UTR from muscle proteins (MyoD, myosin, myoglobin, myogenin, herculin) for endothelial cells (Tie-1, CD36), myeloid cells (C / EBP, AML1, G-CSF, GM-CSF, CD11b, MSR, Fr-1 , i-NOS), leukocytes (CD45, CD18), adipose tissue (CD36, GLUT4, aCRP30, adiponectin) and lung epithelial cells (SP-A / B / C / D) can be used to increase the expression of the engineered polynucleotide in the desired cells or tissues. Other non-UTR sequences can be incorporated into the 5’ (or 3’UTR) UTR of the polynucleotides of the present disclosure. The 5’ and / or 3’UTR can provide the stability and / or translation efficiency of the polynucleotide. For example, an intron or a part of an intron sequence can be incorporated into the adjacent region of the engineered polynucleotide.

[0089] ​ This can also enhance the translation rate of the polynucleotide by incorporating intron sequences. This can be achieved.

[0090] The 3’UTR can have a stretch of adenosines and uridines embedded within it. These AU-rich signatures are particularly widespread in genes with high turnover rates. AU-rich elements (AREs) can be classified into classes based on their sequence features and functional properties: Class I AREs contain several dispersed copies of the AUUUA motif within a U-rich region. C-Myc and MyoD contain Class I AREs. Class II AREs have two or more overlapping UUAUUUA(U / A)(U / A ) nonamers. Molecules containing this type of ARE include GM-CSF and TNF-α. Class III AREs are less clearly defined. These U-rich regions do not contain the AUUUA motif. c-Jun and myogenin are two well-studied examples of this class. Proteins that bind to AREs can destabilize the messenger but members of the ELAV family such as HuR can enhance mRNA stability. HuR can bind to AREs of all three classes. By incorporating a HuR-specific binding site into the 3’UTR of a nucleic acid molecule by manipulation, HuR binding and thus stabilization of the message in vivo can be achieved. in vivo can be achieved. but members of the ELAV family such as HuR can enhance mRNA stability. HuR can bind to AREs of all three classes. By incorporating a HuR-specific binding site into the 3’UTR of a nucleic acid molecule by manipulation, HuR binding and thus stabilization of the message in vivo can be achieved. into the 3’UTR of a nucleic acid molecule by manipulation, HuR binding and thus stabilization of the message in vivo can be achieved. in vivo can be achieved.

[0091] Manipulation of the AU-rich element (ARE) of the 3’UTR can be used to regulate the stability of the manipulated polynucleotide. Manipulation of one or more copies of the ARE can be used to regulate the stability of the manipulated polynucleotide. Manipulation of one or more copies of the ARE and regulating the stability of the polyribonucleotide. AREs increase intracellular stability and thereby improve translation and and can be identified, removed or mutated to increase production. Transfection experiments have demonstrated the transfection of ribonucleotides in relevant cell lines using engineered polyribonucleotides. Protein production can be monitored at various time points after transfection. For example, cells can be transfected with various ARE-engineered molecules. and the transfection was confirmed using ELISA kits for the relevant proteins. Proteins produced were up-regulated 6, 12, 24, 48 hours, and 7 days after incubation. You can say.

[0092] The untranslated region may contain any number of nucleotides. The untranslated region may be from about 1 to about 10 nucleotides. or base pairs, about 10 to about 20 bases or base pairs, about 20 to about 50 bases or base pairs , about 50 to about 100 bases or base pairs, about 100 to about 500 bases or base pairs, about 5 00 to about 1000 bases or base pairs, about 1000 to about 2000 bases or base pairs, about 2000 to about 3000 bases or base pairs, about 3000 to about 4000 bases or base pairs , about 4000 to about 5000 bases or base pairs, about 5000 to about 6000 bases or salts base pairs, about 6000 to about 7000 bases or base pairs, about 7000 to about 8000 bases or is about 8000 to about 9000 bases or base pairs, or about 9000 to about 1000 The untranslated region may be, for example, at least 1 base or 0 base pairs in length. is a base pair, 2 bases or base pairs, 3 bases or base pairs, 4 bases or base pairs, 5 bases a base or base pair, 6 bases or base pairs, 7 bases or base pairs, 8 bases or base pairs, 9 bases or base pairs, 10 bases or base pairs, 20 bases or base pairs, 30 bases or base pairs, 40 bases or base pairs, 50 bases or base pairs, 60 bases or base pairs, 70 bases or base pairs, 80 bases or base pairs, 90 bases or base pairs , 100 bases or base pairs, 200 bases or base pairs, 300 bases or base pairs, 400 bases or base pairs, 500 bases or base pairs, 600 bases or base pairs, 7 00 bases or base pairs, 800 bases or base pairs, 900 bases or base pairs, 10 00 bases or base pairs, 2000 bases or base pairs, 3000 bases or base pairs, 4000 bases or base pairs, 5000 bases or base pairs, 6000 bases or base pairs, 7000 bases or base pairs, 8000 bases or base pairs, 9000 bases or base pairs, or may include lengths of 10000 bases or base pairs.

[0093] The engineered polynucleotides of the present disclosure may include one or more introns. The introns may include any number of modified or unmodified nucleotides. The introns, for example, may include at least 1 base or base pair, 50 bases or base pairs, 100 bases or base pairs , 150 bases or base pairs, 200 bases or base pairs, 300 bases or base pairs, 400 bases or base pairs, 500 bases or base pairs, 600 bases or base pairs, 7 00 bases or base pairs, 800 bases or base pairs, 900 bases or base pairs, 10 00 bases or base pairs, 2000 bases or base pairs, 3000 bases or base pairs, It may contain 4000 bases or base pairs, or 5000 bases or base pairs. In some cases the intron may contain, for example, at most 10000 bases or base pairs, 5000 bases or base pairs, 4000 bases or base pairs, 3000 bases or base pairs, 2000 bases or base pairs, 1000 bases or base pairs, 900 bases or base pairs, 800 bases or base pairs, 700 bases or base pairs, 600 bases or base pairs, 500 bases or base pairs, 400 bases or base pairs, 300 bases or base pairs, 200 bases or base pairs, or 100 bases or base pairs.

[0094] In some cases, a certain proportion of nucleotides in the intron are modified. For example, in some cases, 99%, 95%, 90%, 85%, 80% 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30% 25%, 20%, 15%, 10%, 5% or less than 1% of the nucleotides in the intron are modified. In some cases all of the nucleotides in the intron are modified.

[0095] The engineered polynucleotide of the present disclosure may contain a polyA sequence. The polyA sequence (e.g., a polyA tail) may contain any number of nucleotides. The polyA sequence may be about 1 to about 10 bases or base pairs, about 10 to about 20 bases or base pairs, about 20 to about 50 bases or base pairs, about 50 to about 100 bases or base pairs, about 100 to about 500 bases or base pairs , about 500 to about 1000 bases or base pairs, about 1000 to about 2000 bases or base pairs, about 2000 to about 3000 bases or base pairs, about 3000 to about 4000 bases or Base pairs, about 4000 to about 5000 bases or base pairs, about 5000 to about 6000 bases or base pairs, about 6000 to about 7000 bases or base pairs, about 7000 to about 8000 bases or base pairs, about 8000 to about 9000 bases or base pairs, or about 9000 to about 1 0000 bases or base pairs in length. In some examples, the polyA sequence is at least about 100, 110, 120, 130, 140, 150, 160, 170, 180, 19 0 or 200 nucleotides in length. The polyA sequence can be, for example, at least 1 base or base pair, 2 bases or base pairs, 3 bases or base pairs, 4 bases or base pairs, 5 bases or base pairs, 6 bases or base pairs, 7 bases or base pairs, 8 bases or base pairs , 9 bases or base pairs, 10 bases or base pairs, 20 bases or base pairs, 30 bases or base pairs, 40 bases or base pairs, 50 bases or base pairs, 60 bases or base pairs, 70 bases or base pairs, 80 bases or base pairs, 90 bases or base pairs, 100 bases or base pairs, 200 bases or base pairs, 300 bases or base pairs, 4 00 bases or base pairs, 500 bases or base pairs, 600 bases or base pairs, 70 0 bases or base pairs, 800 bases or base pairs, 900 bases or base pairs, 100 0 bases or base pairs, 2000 bases or base pairs, 3000 bases or base pairs, 4 000 bases or base pairs, 5000 bases or base pairs, 6000 bases or base pairs , 7000 bases or base pairs, 8000 bases or base pairs, 9000 bases or base pairs, or 10000 bases or base pairs in length. The polyA sequence can be at most 100 bases or base pairs, 90 bases or base pairs, 80 bases or base pairs, 70 bases a base or base pair, 60 bases or base pairs, 50 bases or base pairs, 40 bases or base pairs, 30 bases or base pairs, 20 bases or base pairs, 10 bases or base pairs , or may include a length of 5 bases or base pairs.

[0096] In some cases, a certain proportion of the nucleotides in the polyA sequence are modified. For example, in some cases, 99%, 95%, 90%, 85%, 80 %, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30 %, 25%, 20%, 15%, 10%, 5% or less than 1% of the nucleotides in the polyA are modified. In some cases all of the nucleotides in the polyA are modified.

[0097] The linker sequence can contain any number of nucleotides. The linker can be attached to the modified nucleobase at the N-3 or C- 5 position. The linker attached to the nucleobase can be a diethylene glycol, dipropylene glycol, triethylene glycol, tripropylene glycol, tetraethylene glycol, tetraethylene glycol, a divalent alkyl, an alkenyl, alkynyl moiety, an ester, an amide or an ether moiety. The linker sequence can be about 1 to about 10 bases or base pairs, about 10 to about 20 bases or base pairs, about 20 to about 50 bases or base pairs, about 50 to about 100 bases or base pairs, about 100 to about 50 0 bases or base pairs, about 500 to about 1000 bases or base pairs, about 1000 to about 20 00 bases or base pairs, about 2000 to about 3000 bases or base pairs, about 3000 to about 4000 bases or base pairs, about 4000 to about 5000 bases or base pairs, about 5000 ~about 6000 bases or base pairs, about 6000 to about 7000 bases or base pairs, about 7 00 to about 8000 bases or base pairs, about 8000 to about 9000 bases or base pairs, or about 9000 to about 10000 bases or base pairs in length. The linker sequence can include, for example, at least 1 base or base pair, 2 bases or base pairs, 3 bases or base pairs , 4 bases or base pairs, 5 bases or base pairs, 6 bases or base pairs, 7 bases or base pairs, 8 bases or base pairs, 9 bases or base pairs, 10 bases or base pairs, 2 0 bases or base pairs, 30 bases or base pairs, 40 bases or base pairs, 50 bases or base pairs, 60 bases or base pairs, 70 bases or base pairs, 80 bases or base pairs, 90 bases or base pairs, 100 bases or base pairs, 200 bases or base pairs , 300 bases or base pairs, 400 bases or base pairs, 500 bases or base pairs, 600 bases or base pairs, 700 bases or base pairs, 800 bases or base pairs, 9 00 bases or base pairs, 1000 bases or base pairs, 2000 bases or base pairs, 3000 bases or base pairs, 4000 bases or base pairs, 5000 bases or base pairs, 6000 bases or base pairs, 7000 bases or base pairs, 8000 bases or base pairs, 9000 bases or base pairs, or at least 10000 bases or base pairs in length. At most 10000 bases or base pairs, 5000 bases or base pairs, 4000 bases or base pairs, 3000 bases or base pairs, 2000 bases or base pairs, 1000 bases or base pairs, 900 bases or base pairs, 800 bases or base pairs, 700 bases or base pairs, 600 bases or base pairs, 500 bases or salt a base pair, a linker of 400 bases or base pairs, 300 bases or base pairs, 200 bases or base pairs, or 100 bases or base pairs in length.

[0098] In some cases, a certain percentage of nucleotides in the linker sequence are modified. For example , in some cases, 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or less than 1% of the nucleotides in the linker sequence are modified. In some cases, all of the nucleotides in the linker sequence are modified.

[0099] In some cases, the (one or more) nucleic acid constructs, the (one or more) vectors or the (one or more) engineered polynucleotides may contain at least one stop codon before the 3' untranslated region (UTR ). In some cases, the (one or more) nucleic acid constructs, the (one or more) vectors or the (one or more) engineered poly ribonucleotides contain multiple stop codons. The stop codons can be selected from TGA, TAA and TAG. The stop codons may or may not be modified. In some cases, the (one or more) nucleic acid constructs, the (one or more) vectors or the (one or more) engineered polynucleotides contain the stop codon TGA and one additional stop codon. In some cases, the (one or more) nucleic acid constructs , the (one or more) vectors or the (one or more) engineered polynucleotides contain the addition of the TAA stop codon.

[0100] Encoded polypeptide In some cases, the present disclosure relates to a method of treating a subject having or at risk of having primary ciliary dyskinesia, which comprises axonemal dynein intermediate chain 1 protein (DNAI1), armadillo repeat-containing 4 (ARMC4), chromosome 21 open reading frame 59 (C21orf59), coiled-coil domain-containing 103 (CCDC103), co iled-coil domain-containing 114 (CCDC114), coiled-coil domain-containing 39 (CCDC39), coiled-coil domain-containing 40 (CCDC40), coiled-coil domain-containing 65 (CCDC65), dynein (axonemal) assembly factor 1 (DNAAF1 ), dynein (axonemal) assembly factor 2 (DNAAF2), dynein (axonemal) assembly factor 3 (DNAAF3), dynein (axonemal) assembly factor 5 (DNAAF5), axonemal dy nein heavy chain 11 (DNAH11), axonemal dynein heavy chain 5 (DNAH5), axonemal dynein heavy chain 8 (DNAH8), axonemal dynein intermediate chain 2 (DNAI2), axonemal dynein light chain 1 (DN AL1), dynein regulatory complex subunit 1 (DRC1), dyslexia susceptibility 1 candidate 1 ( DYX1C1), axoneme central pair apparatus protein (HYDIN), leucine-rich repeat-containing 6 (LRRC6), NME / NM23 family member 8 (NME8), oral-facial-digital syndrome 1 (OFD1), retinitis pigmentosa GTPase regulator (RPGR), radial spoke head 1 homolog (Chlamydomonas) (RSPH1), radial spoke head 4 homolog A (Chlamydomonas) (RSPH4A), radial spoke head 9 homolog ( Chlamydomonas) (RSPH9), sperm-associated antigen 1 (SPAG1) and zinc finger - A composition comprising a nucleic acid construct encoding MYND-type containing 10 (ZMYND10) or a variant of any of the foregoing is administered to a subject, and this nucleic acid construct contains codons that provide for heterologous expression or enhanced expression of the (one or more) said protein or its variant in the cells of the subject, thereby treating a subject having or at risk of having primary ciliary dyskinesia. The polypeptide encoded is a polymer chain consisting of amino acid residue monomers linked together via amide bonds (peptide bonds). The amino acids can be l - optical isomers, d - optical isomers, or combinations thereof. The polypeptide can be a chain of at least three amino acids, a peptidomimetic, a protein, a recombinant protein, an antibody (monoclonal or polyclonal), an antigen, an epitope, an enzyme, a receptor, a vitamin, or a structural analogue, or combinations thereof. The polyribonucleotide translated in the body of the subject can provide a sufficient supply of a specific peptide or protein within the cells, tissues, or across many cells and tissues of the subject. In some cases, the polyribonucleotide can be translated in vivo within the cytosol of (one or more) specific target cell types or target tissues. In some cases, the polyribonucleotide can be translated in vivo to provide a protein whose gene is related to a protein associated with primary ciliary dyskinesia, a functional fragment thereof, or a protein that is at least 70% homologous to human DNAI1 or human DNAH5 protein. In some cases, the polyribonucleotide can be

[0101] ​​​​​​​​​​​​​​​​can be translated in vivo in a type or (one or more) target tissues. Non-limiting examples of cells that are target cells or non-target cells include a) skin cells such as keratinocytes, melanocytes, urothelial cells; b) nerve cells such as neurons, Schwann cells, oligodendrocytes, astrocytes; c) hepatocytes such as hepatocytes; d) intestinal cells such as goblet cells, enterocytes; e) blood cells such as lymphocytes or bone marrow cells; and f) germ cells such as sperm and eggs. Non-limiting examples of tissues include connective tissue, muscle tissue, nerve tissue or epithelial tissue. In some cases, the target cells or target tissues are cancerous cells, tissues or organs.

[0102] The polynucleotide sequence can be derived from one or more species. For example, the polynucleotide sequence can be derived from human (Homo sapiens), mouse (e.g., Mus musculus), rat (e.g., Rattus norvegicus or Rattus rattus), microorganism (e.g., Chlamydomonas genus), or any other suitable organism. The polynucleotide sequence can be a chimeric combination of sequences from one or more species. or more species.

[0103] In some cases, the endogenous translation machinery can add post-translational modifications to the encoded peptide. Post-translational modifications can include the addition of a hydrophobic group that can target the polypeptide for membrane localization, the addition of a cofactor for increased enzyme activity, or the addition of a smaller chemical group. The encoded polypeptide can also be attached to other peptide or protein moieties ​​​​​​​​​ For example, ubiquitination can be achieved by the addition of ubiquitin to the protein encoded by ubiquitin. SUMOylation can result in the covalent attachment of SUMO (small molecule Small Ubiquitin-related Modifiers and can provide a covalent bond between the ISG-linked polypeptide and the polypeptide encoded by the ISG-linked polypeptide. ISG15 (Interferon-Stimulated Gene 15) This can result in covalent bonding of the carboxylate gene 15).

[0104] In some cases, the encoded polypeptide may undergo other types of structural changes. It may be post-translationally modified. For example, the encoded polypeptide may be proteolytically cleaved. and one or more proteolytic fragments can modulate the activity of an intracellular pathway. The encoded polypeptide may be folded within the cell. The polypeptide to be folded is folded in the presence of cofactors and molecular chaperones. A folded polypeptide can have secondary and tertiary structure. The folded polypeptide can then associate with other folded peptides to form quaternary structures. Folded peptides can be used to create functional macromolecules such as antibody molecules that have a tetrameric quaternary structure. They can form two-subunit complexes that define antibody classes or isotypes. A variety of polypeptides can be expressed from polyribonucleotides.

[0105] The encoded polypeptide may be modified to alter the chemical properties of the encoded amino acids. For example, the encoded polypeptide may be modified post-translationally by the substitution of arginine for citrulline. It can undergo post-translational citrullination or deimination, which is a conversion of The encoded polypeptide can undergo post-translational deamidation, which is a conversion of glutamine to glutamic acid or asparagine to aspartic acid. The encoded polypeptide can undergo elimination, i.e., β-elimination of phosphothreonine and phosphoserine, or conversion of alkenes by dehydration of threonine and serine and decarboxylation of cysteine. The encoded peptide can also undergo carbamylation, which is a conversion of lysine to homocitrulline. The encoded peptide can undergo racemization, e.g., racemization of proline by prolyl isomerase or racemization of serine by protein-serine epimerase. In some cases, the encoded peptide can undergo phosphorylation of serine, threonine, and tyrosine.

[0106] The activity of multiple biomolecules can be modulated by a molecule encoded by a polynucleotide. Non-limiting examples of molecules whose activity can be modulated by the encoded polynucleotide include amino acids, peptides, peptidomimetics, proteins, recombinant proteins, antibodies (monoclonal or polyclonal), antibody fragments, antigens, epitopes, carbohydrates, lipids, fatty acids, enzymes, natural products, nucleic acids (including DNA, RNA, nucleosides, nucleotides, structural analogs, or combinations thereof), nutrients, receptors, and vitamins.

[0107] Table 3 discloses non-limiting examples of nucleotide sequences that can be part of the polynucleotides of the present disclosure. [Table 3]

[0108] The polypeptide sequence may be the altered codon usage of SEQ ID NO: 15-16 or the altered codon usage of SEQ ID NO: 1 Engineered to have a desired altered codon usage, such as an altered codon usage of 7 to 18 For example, computer software can be used to generate codon usage sequences for SEQ ID NO:14. The polypeptide sequence may be the amino acid sequence of an endogenous polypeptide. A polypeptide sequence may share 100% homology with an amino acid sequence of an endogenous polypeptide. at most 10% homology to the nucleic acid sequence, at most 20% homology, at most 30% homology, at most 40% homology, at most 50% homology, at most 60% homology, at most 70% homology, at most 80% homology, at most 90% homology, or at most 99 % homology. NCBI BLAST, Clustal W, MAFFT, C lustal Omega, AlignMe, Praline or another suitable method or uses various methods and software programs, such as algorithms, to The homology between the peptides can be determined.

[0109] immunogenicity Contains compositions containing molecules of various sizes (polynucleotides, proteins or enzymes) Many agents that inhibit the immune system can provoke an immune response when administered to a subject. The immune system recognizes the composition as foreign and neutralizes its pharmacological effect. The leukocytes and compositions may have low immunogenicity or may be non-immunogenic, It elicits a smaller or no immune response by the immune system.

[0110] Immunogenicity can also be determined by measuring, for example, TNF-α and IL-8 levels and the binding ability to TLR-3, TLR-7 , TLR-8 and helicase RIG-1. To confirm whether the polynucleotide has the desired low immunogenicity, the amount of one or more factors can be measured after administration of the polynucleotide to a subject. The immunogenicity of a polypeptide can be determined in relation to an increase in the number of white blood cells upon administration of the polypeptide to a subject. In some cases, upon administration of the composition to a subject, the subject shows an increase in the number of white blood cells of less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30 %, less than 20%, or less than 10%. The polynucleotides of the present disclosure can induce a minimal or insignificant inflammatory or immunological response.

[0111] Various methods can be used to determine the immunogenicity of a polynucleotide. A very suitable method is the measurement of inflammatory markers in cells or simply the measurement of the number of white blood cells as a response to the administration of the polynucleotide. Such methods are described in the examples. For example, cytokines associated with inflammation such as TNF-α, IFN-α, IFN-β, IP-10, IL-8, IL-6 and / or IL-12 can be measured. The expression of dendritic cell activation markers can also be used for the evaluation of immunogenicity. Further indicators of an immunological response can be the detection of binding to Toll-like receptors TLR-3, TLR-7 and TLR-8 and helicase RIG-1.

[0112] The immunogenicity of polyribonucleotides was compared with pre-administration levels of polyribonucleotides. This can be measured as an overall increase in the levels of inflammatory markers or white blood cell count. For example, unmodified or modified engineered polyribonucleotides can be administered to cells. or to a subject, and a prescribed amount of polyribonucleotide is produced in response to administration of the polyribonucleotide. The secretion of inflammatory markers at defined time intervals can be measured.

[0113] composition In some cases, the disclosure provides a composition comprising a nucleic acid construct encoding axonemal dynein intermediate chain 1. The nucleic acid construct further comprises a complementary deoxyribonucleic acid sequence encoding axonemal dynein intermediate chain 1. The composition comprises a nucleic acid and is formulated for administration to a subject. provides a composition comprising a nucleic acid construct encoding axonemal dynein intermediate chain 1, The method further comprises the steps of: detecting intracellular cytoplasmic secretion in a subject having or at risk of having primary ciliary dyskinesia; The present invention provides a method for the production of axonemal dynein intermediate chain 1 protein or a mutant thereof, comprising: In some cases, the present disclosure provides a codon encoding a nuclear codon that encodes axonemal dynein intermediate chain 1. The present invention provides a composition comprising an acid construct, wherein the acid construct comprises 30 sequences of a nucleic acid encoding axonemal dynein intermediate chain 1. Less than % are nucleic acid analogs such as pseudouridine or 1-methylpseudouridine In some cases, the coding sequences of these constructs are protein-modified to enhance their stability. The mutant is engineered to have altered nucleotide usage in the protein coding region.

[0114] In some cases, the codons of the construct are selected from the group consisting of mammalian or human axonemal dynein intermediate chain 1 tandem codons. The construct also expresses axonemal dynein intermediate chain 1 (ADC1) and other proteins that are at least 70% homologous to the protein. The 3' or 5' non-coding region adjacent to the codon sequence encoding the protein of interest may be included, and this non-coding region enhances the expression of the protein in the cells of the subject. The 3' non-coding region adjacent to the codon may include a 3'-cap-independent translation enhancer (3'-CITE) or a 3'-stem-loop region derived from the nucleotide sequence of a histone protein or may include a 3'-triple helix structure derived from the nucleotide sequence of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). The 3' non-coding region adjacent to the codon can contain a polyadenosine tail, where the number of adenosines in the polyadenosine tail improves the translation efficiency or half-life of the protein of interest, such as the axonemal dynein intermediate chain 1 protein. In some cases, the length of the polyadenosine tail is at most 200 adenosines. In some cases, a proportion of the polyadenosine tails contain nucleic acid analogs. Less than 50%, 40%, 30%, 20%, 10% or 5% of the nucleic acids in the polyadenosine tail can be nucleic acid analogs. including, and this non-coding region enhances the expression of the protein in the cells of the subject. The codon adjacent 3' non-coding region may include a 3'-cap-independent translation enhancer (3'-CIT E) or a 3'-stem-loop region derived from the nucleotide sequence of a histone protein or also includes a 3'-triple helix structure derived from the nucleotide sequence of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). The 3' non-coding region adjacent to the codon can contain a polyadenosine tail, where the number of adenosines in the polyadenosine tail improves the translation efficiency or half-life of the protein of interest, such as the axonemal dynein intermediate chain 1 protein. In some cases the length of the polyadenosine tail is at most 200 adenosines. In some cases a proportion of the polyadenosine tails contain nucleic acid analogs. The nucleic acids in the polyadenosine tail less than 50%, 40%, 30%, 20%, 10% or 5% can be nucleic acid analogs .

[0115] If the composition contains a proportion of nucleotide analogs, the nucleotide analogs can be selected from the group consisting of pseudouridine, 1-methylpseudouridine, 2-thiouridine, 5-methyluridine, 5- methoxyuridine, 5-methylcytidine, 2'-amino-2'-deoxycytidine, 2' -fluoro-2'-deoxycytidine. In some cases, the nucleic acid analog is pseudouridine or 1-methylpseudouridine. In some cases the nucleic acid analog is 5-methoxyuridine. In some cases, the composition is a nucleic acid encoding axonemal dynein intermediate chain 1 and / or a nucleic acid

[0116] In some cases, the composition is a nucleic acid encoding axonemal dynein intermediate chain 1 and / or a nucleic acid It contains analogs. The composition may further contain at least one additional nucleic acid construct. This at least one additional nucleic acid construct may encode a protein selected from the group consisting of armadillo repeat-containing 4 (ARMC4), chromosome 21 open reading frame 59 (C21orf59), coiled-coil domain-containing 103 (CCDC103), coiled-coil domain-containing 114 (CCDC114), coiled-coil domain-containing 39 (CCDC39), coiled-coil domain-containing 40 (CCDC40), coiled-coil domain-containing 65 (CCDC65), dynein (axoneme) assembly factor 1 (DNAAF1), dynein (axoneme) assembly factor 2 (DNAAF2), dynein (axoneme) assembly factor 3 (DNAAF3), dynein (axoneme) assembly factor 5 (DNAAF5), axonemal dynein heavy chain 11 (DNAH11), axonemal dynein heavy chain 5 (DNAH5), axonemal dynein heavy chain 8 (DNAH8), axonemal dynein intermediate chain 2 (DNAI2), axonemal dynein light chain 1 (DNAL1), dynein regulatory complex subunit 1 (DRC1), dyslexia susceptibility 1 candidate 1 (DYX1C1), axoneme central pair apparatus protein (HYDIN), leucine-rich repeat-containing 6 (LRRC6), NME / NM23 family member 8 (NME8), oral-facial-digital syndrome 1 (OFD1), retinitis pigmentosa GTPase regulator (RPGR), radial spoke head 1 homolog (Chlamydomonas) (RSPH1), radial spoke head 4 homolog A (Chlamydomonas) (RSPH4A), radial spoke head 9 homolog (Chlamydomonas) (RSPH9), sperm-associated antigen 1 (SPAG1), and zinc finger MYND-type-containing 10 (ZMYND10).

[0117] The composition may comprise an engineered polynucleotide, vector or nucleic acid construct. A "naked" polynucleotide composition can be successfully administered to a subject without the aid of a carrier, stabilizer, diluent, dispersant, suspending agent, thickening agent and / or excipient, and can be taken up by the cells of the subject (Wolff et al. 1990, Science, 247, 14 65-1468). However, in many cases, encapsulation of the polynucleotide with a formulation that can increase endocytic uptake can enhance the effectiveness of the compositions of the present disclosure. To overcome this problem, in some cases, the composition comprises a nucleic acid construct, vector or isolated nucleic acid encoding axonemal dynein intermediate chain 1, the nucleic acid construct comprises a complementary deoxyribonucleic acid encoding axonemal dynein intermediate chain 1, and the composition is formulated for administration to a subject. Another technical problem underlying the delivery of polynucleotides to multicellular organisms is to identify a composition that provides efficient delivery of the polynucleotide to be translated within the cells or tissues of the subject. Administration of naked nucleic acids can be extremely inefficient and it has been recognized that it cannot provide an appropriate approach for the administration of polynucleotides to multicellular organisms. To solve this problem, a composition comprising an engineered polynucleotide can be encapsulated or formulated with a pharmaceutical carrier. The formulations can be nanoparticles, poly(lactic-co-glycolic acid) (PLGA) microspheres, lipidoids, lipoplexes, liposomes

[0118] Another technical problem underlying the delivery of polynucleotides to multicellular organisms is to identify a composition that provides efficient delivery of the polynucleotide to be translated within the cells or tissues of the subject. Administration of naked nucleic acids can be extremely inefficient and it has been recognized that it cannot provide an appropriate approach for the administration of polynucleotides to multicellular organisms. To solve this problem, a composition comprising an engineered polynucleotide can be encapsulated or formulated with a pharmaceutical carrier. The formulations can be nanoparticles, poly(lactic-co-glycolic acid) (PLGA) microspheres, lipidoids, lipoplexes, liposomes

[0119] To solve this problem, a composition comprising an engineered polynucleotide can be encapsulated or formulated with a pharmaceutical carrier. The formulations can be nanoparticles, poly(lactic-co-glycolic acid) (PLGA) microspheres, lipidoids, lipoplexes, liposomes , polymers, carbohydrates (including monosaccharides), cationic lipids, fibrin gels, fibrin hydrogels Drugs, fibrin glue, fibrin sealant, fibrinogen, thrombin, rapid Excluding lipid nanoparticles (reLNP) and combinations thereof, but not limited thereto. Compositions containing the engineered polynucleotides disclosed herein may contain from about 1 weight / volume % to about 99 weight / volume % of a carrier system. The amount of carrier present in the carrier system is based on several different factors or choices made by the formulator, such as the final concentration of the polynucleotide and the amount of solubilizing agent. A variety of carriers have been shown to be useful in the delivery of various classes of therapeutic agents in. Among these carriers, biodegradable nanoparticles formulated from the biocompatible polymers poly(D,L-lactide-co-glycolide) (PLGA) and polylactide (PLA) have shown potential for the sustained intracellular delivery of various therapeutic agents .

[0120] The loading weight percentage of the engineered polynucleotide in the composition is at least 0.05 %, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 4%, 5%, 6 %, 7%, 8%, 9% or 10%. The encapsulation efficiency of the modified m RNA in PLGA microspheres is at least 50%, at least 70%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%.

[0121] The present disclosure describes nanoparticles, oligomers, polymers or lipidoids containing alternating non-identical alkyleneamine units useful for delivering polynucleotides, in some cases engineered polynucleotides, to cells or tissues. (alkyleneamine). . The compositions disclosed herein can be stable for at least about 1 minute, 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days , 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 2 weeks, 4 weeks, 6 weeks , 8 weeks, 10 weeks, 12 weeks, 3 months, 4 months, 5 months, 6 months, 7 months , 8 months, 9 months, 10 months, 11 months or 1 year. The formulations disclosed herein can be stable, for example, at a temperature of at least about 0 °C, 5 °C, 10 °C, 15 °C, 20 °C, 25 °C , 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, 60 °C, 70 °C or 80 °C. The compositions of the present disclosure can have a desired density. The density of the composition can improve the properties of the composition, such as the rheology of the composition.

[0122] Nanoparticles The present disclosure also provides nanoparticle-based formulations of nucleic acid constructs, engineered polynucleotides or vectors that can translocate after administration to a subject. In some cases, the administration is via the pulmonary route and the engineered polynucleotide moves intact, either actively or passively, from the site of administration to the systemic blood supply and then can deposit in various cells or tissues such as, for example, the breast. For example, axonemal dynein intermediate chain 1 (DNAI1), armadillo repeat-containing 4 (ARMC4), chromosome 21 open reading frame 59 (C 21orf59), coiled-coil domain-containing 103 (CCDC103), coiled-coil domain-containing 114 (CCDC114), coiled-coil domain-containing 39 (CCDC 39), coiled-coil domain-containing 40 (CCDC40), coiled-coil domain Containing 65 (CCDC65), cyclin O (CCNO), dynein (axoneme) assembly factor 1 (DNAAF1), dynein (axoneme) assembly factor 2 (DNAAF2), dynein (axoneme) assembly factor 3 (DNAAF3), dynein (axoneme) assembly factor 5 (DN AAF5), axonemal dynein heavy chain 11 (DNAH11), axonemal dynein heavy chain 5 (DNAH5 ), axonemal dynein heavy chain 6 (DNAH6), axonemal dynein heavy chain 8 (DNAH8), axonemal dy nein intermediate chain 2 (DNAI2), axonemal dynein light chain 1 (DNAL1), dynein regulatory complex subunit 1 (DRC1), dyslexia susceptibility 1 candidate 1 (DYX1C1), growth arrest specific 8 (GAS8), axoneme central pair apparatus protein (HYDIN), leucine rich repeat containing 6 (LRRC6), NME / NM23 family member 8 (NME8), oral - facial - digital syndrome 1 (OFD1), retinitis pigmentosa GTPase regulator (RPGR), radial spokehead 1 homolog (Chlamydomonas) (RSPH1), radial spokehead 4 homolog A (Chlamydomonas) (RSPH4A), radial spokehead 9 homolog (Chlamydomonas) (RSPH9), sperm associated antigen 1 (SPAG1) and zinc finger MYND - type containing 10 (ZMYND10) or a functional fragment thereof, etc., this transfer of nanoparticles containing an engineered polynucleotide encoding a therapeutic protein constitutes non - invasive systemic delivery of an active pharmaceutical ingredient across the lung and results in the production of a functional protein in non - lung cells or tissues that are systemically accessible. The nanoparticles are about 10 nanometers (nm) to 5000 nm, 10 nm to 1000 nm,

[0123] ​​Or it may be particles with a particle size of 60 nm to 500 nm, or 70 nm to 300 nm. In one example, the nanoparticles have a particle size of about 60 nm to 225 nm. The nanoparticles can encapsulate one or more polynucleotides, which can be engineered polynucleotides. The nanoparticles may include an encapsulating agent (such as a coating) that encapsulates the polynucleotides. The nanoparticles may include engineered and / or naturally occurring polynucleotides. The encapsulating agent can be a polymeric material such as PEI or PEG.

[0124] Lipidoids or lipid nanoparticles that can be used as delivery agents include lipids selected from the group consisting of C12-200, MD1, 98N12-5, DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, DLin-MC3-DMA, PLGA, PEG, PEG-DMG, PEGylated lipids and their analogs. Suitable nanoparticles can contain one or more lipids in various ratios. For example, the compositions of the present invention can have a ratio of C12-200:DOPE: cholesterol:DMG-PEG200 of 40:30:25:5 or a ratio of HGT5001:DOPE:cholesterol:DMG-PEG2000 of 40:20:35:5. The nanoparticles can contain at least 1, 2, 3, 4, 5, 6, 7 8, 9, 10 types of lipids or another suitable number of lipids. The nanoparticles can be formed with any suitable ratio of lipids selected from the group consisting of C12-200, MD1, 98N12-5, DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, DLin-MC3-DMA, PLGA, PEG, PEG-DMG. formed with any suitable ratio of lipids selected from the group consisting of C12-200,

[0125] The average size of the nanoparticle formulation can include modified mRNA from 60 nanometers (nm) to 225 nm The polydispersity index PDI of the nanoparticle formulation containing modified mRNA can be from 0.03 to 0.1 5. The ζ potential of the nanoparticle formulation can be from -10 to +10 at pH 7.4. The formulation of modified mRNA can include a fusion-inducing lipid, cholesterol, and a PEG lipid. The formulation can have a molar ratio of 50:10:38.5:1.5 - 3.0 (cationic lipid:fusion-inducing lipid: cholesterol: polyethylene glycol (PEG) lipid). The PEG lipid can be selected from, but not limited to, PEG-c-DOMG, PEG-DMG. The fusion -inducing lipid can be DSPC. The lipid nanoparticles of the present disclosure can be formulated into any fibrin sealant but not limited to such sealants.

[0126] Oligo(alkyleneamine group) The encapsulation of polynucleotides using some formulations can increase the endocytosis uptake of the composition, but it is also recognized that the polynucleotides taken up by cells may not be effectively translated intracellularly. Some formulations can be effectively used for plasmid DNA and / or siRNA delivery, but are not practical for use in the delivery of polynucleotides. The present disclosure provides a formulation that can be used for the effective delivery and translation of polynucleotide compositions to a subject. The present disclosure provides a formulation that can be used for the effective delivery and translation of polynucleotide compositions to a subject. It is also recognized that although some formulations can be effectively used for plasmid DNA and / or siRNA delivery, they are not practical for use in the delivery of polynucleotides. The present disclosure provides a formulation that can be used for the effective delivery and translation of polynucleotide compositions to a subject. Some formulations can be effectively used for plasmid DNA and / or siRNA delivery, but are not practical for use in the delivery of polynucleotides. The present disclosure provides a formulation that can be used for the effective delivery and translation of polynucleotide compositions to a subject. Some formulations can be effectively used for plasmid DNA and / or siRNA delivery, but are not practical for use in the delivery of polynucleotides. The present disclosure provides a formulation that can be used for the effective delivery and translation of polynucleotide compositions to a subject. The compositions of the present disclosure can be designed to provide polynucleotides that are effectively translated intracellularly. The compositions of the present disclosure can have alternating lengths of alkyl in 3 or more units of groups

[0127] The compositions of the present disclosure can be designed to provide polynucleotides that are effectively translated intracellularly. The compositions of the present disclosure can be designed to provide polynucleotides that are effectively translated intracellularly. The compositions of the present disclosure can have alternating lengths of alkyl in 3 or more units of groups comprising the arrangement of the renamine units and containing ethyleneamine units in the composition for transfecting cells with any polynucleotide, e.g., engineered poly bonucleotide. The compositions of the present disclosure can provide more effective delivery of poly bonucleotides to cells than similar arrangements of alkyleneamine units of non-alternating lengths.

[0128] Formula (I):

Chemical formula

[0129] Oligomers, polymers or lipidoids sharing a common structural entity shown in can be provided.

[0130] The compositions of the present disclosure may comprise an oligo(alkyleneamine) selected from the following: a) an oligomer or polymer comprising a plurality of groups of formula (II) as side chains and / or end groups:

Chemical formula

[0131] [wherein the variables a, b, p, m, n and R 2 ~R 6 are each independently defined as follows for each group of formula (II) in a plurality of such groups: a is 1 and b is an integer from 2 to 4, or a is an integer from 2 to 4 and b is 1 ; p is 1 or 2; m is 1 or 2; n is 0 or 1, and m + n is 2 or more; and R 2 ~R 5 are, independently of each other, hydrogen; -CH2-CH(OH)-R 7 ​, -CH(R 7 )-CH2-OH, -CH2-CH2-(C=O)-O-R 7 , -CH2-CH2-(C =O)-NH-R 7 or -CH2-R 7 group (wherein R 7 is selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond); a protecting group for an amino group ; and a poly(ethylene glycol) chain, and is selected from ; R is hydrogen; -CH2-CH(OH)-R 6 , -CH(R 7 )-CH2-OH, -C 7 )-CH2-OH, -C H2-CH2-(C=O)-O-R 7 , -CH2-CH2-(C=O)-NH-R 7 also is -CH2-R 7 group (wherein R 7 is selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond); a protecting group for an amino group; -C(NH) -NH; a poly(ethylene glycol) chain; and a receptor ligand, and is selected from ; and one or more of the nitrogen atoms shown in formula (II) may be protonated to provide a cationic group of formula (II) . .

[0132] b) An oligomer or polymer containing a plurality of groups of formula (III) as repeating units:

Chemical formula

[0133] [wherein the variables a, b, p, m, n and R 2 ~R 5 are each independently defined as follows for each group of formula (III) in a plurality of such groups: a is 1, b is an integer from 2 to 4, or a is an integer from 2 to 4 and b is 1 ; p is 1 or 2; m is 1 or 2; n is 0 or 1, and m + n is 2 or more; and R 2 ~R 5 are, independently of one another, hydrogen; -CH2-CH(OH)-R 7 , -CH(R 7 )-CH2-OH, -CH2-CH2-(C=O)-O-R 7 , -CH2-CH2-(C =O)-NH-R 7 , -CH2-R 7 or a -CH2- group, where R 7 is selected from C3-C1 8 alkyl or C3-C18 alkenyl having one C-C double bond ); a protecting group for an amino group; and a poly(ethylene glycol) chain; and one or more of the nitrogen atoms shown in formula (III) may be protonated to provide a cationic group of formula (III).

[0134] c) a lipidoid having the structure of formula (IV):

Chemical formula

[0135] [wherein the variables a, b, p, m, n and R 2 ~R 6 are defined as follows: a is 1, b is an integer from 2 to 4, or a is an integer from 2 to 4 and b is 1 ; p is 1 or 2; m is 1 or 2; n is 0 or 1, and m + n is 2 or more; and R 2 ~R 6 ​are, independently of one another, hydrogen; -CH2-CH(OH)-R 7 , -CH(R 7 )-CH2-OH, -CH2-CH2-(C=O)-O-R 7 , -CH2-CH2-(C =O)-NH-R 7 or -CH2-R 7 group (wherein R 7 is selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond); a protecting group for an amino group ; a poly(ethylene glycol) chain; and a receptor ligand, provided that , at least two of the residues of R ~R 1 ~R 6 are -CH2-CH(OH)-R 7 , -CH (R 7 )-CH2-OH, -CH2-CH2-(C=O)-O-R 7 , -CH2-CH2 -(C=O)-NH-R 7 or -CH2-R 7 group (wherein R 7 is selected from C3-C18 al kyl or C3-C18 alkenyl having one C-C double bond); and one or more of the nitrogen atoms shown in formula (IV) may be protonated to provide a cationic group of formula (IV). Non-limiting examples of alkenyl groups and alkenylene groups include linear, branched and cyclic al kenyl groups. One or more olefins of the alkenyl group can be, for example, E, Z, cis

[0136] , trans, terminal or exomethylene. The alkenylene group is, for example, substituted or unsubstituted C2, C3, C4, C5, C6, C7, C8, C9, C kenyl groups are included. One or more olefins of the alkenyl group can be, for example, E, Z, cis , trans, terminal or exomethylene. The alkenylene group is, for example, substituted or unsubstituted C2, C3, C4, C5, C6, C7, C8, C9, C or non-substituted C2, C3, C4, C5, C6, C7, C8, C9, C 10 , C 11 , C1 2, C13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , C 20 , C 21 , C2 2, C 23 , C 24 , C 25 , C 26 , C 27 , C 28 , C 29 , C 30 , C 31 , C3 2, C 33 , C 34 , C 35 , C 36 , C 37 , C 38 , C 39 , C 40 , C 41 , C4 2, C 43 , C 44 , C 45 , C 46 , C 47 , C 48 , C 49 or C 50 may be a group.

[0137] The oligo(alkyleneamine) structures of formulas (II), (III) and (IV) are characterized by the ability to alternately combine shorter (also referred to as "S" for purposes of illustration) ethyleneamine units (i.e., where a or b is 1) and longer (also referred to as "L" for purposes of illustration) alkyleneamine units (i.e., where the other of a or b is an integer from 2 to 4). Such an arrangement of protonable units provides an advantage with respect to the compatibility of the resulting groups for providing a vehicle for delivering polynucleotides into cells.

[0138] The compositions of the present disclosure may contain a plurality of oligo(alkyleneamine) groups of formula (II) as side chains or terminal groups: -NR 2 {CH2-(CH2) a -NR 3 -[CH2-(CH2) b -NR 4 p} m -[CH2-(CH2) a -NR 5 n -R 6 (II) [wherein the variables a, b, p, m, n and R 2 ~R 6 are each independently defined as follows for each group of formula (II): a is 1, b is an integer from 2 to 4, or a is an integer from 2 to 4 and b is 1 ; p is 1 or 2; m is 1 or 2; n is 0 or 1, and m + n is 2 or more; and R 2 ~R 5 are, independently of one another, hydrogen; -CH2-CH(OH)-R 7 , -CH(R 7 )-CH2-OH, -CH2-CH2-(C=O)-O-R 7 , -CH2-CH2-(C =O)-NH-R 7 or -CH2-R 7 group (wherein R 7 is selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond); a protecting group for an amino group ; -C(NH)-NH2-; and a poly(ethylene glycol) chain selected; R 6 is hydrogen; -CH2-CH(OH)-R 7 , -CH(R 7 ​​)-CH2-OH, -C H2-CH2-(C=O)-O-R 7 , -CH2-CH2-(C=O)-NH-R 7 Also is -CH2-R 7 group (where R 7 is selected from C3-C16 alkyl or C3-C16 alkenyl having one C-C double bond); a protecting group for an amino group; -C(NH) -NH; a poly(ethylene glycol) chain; and a receptor ligand].

[0139] In some cases, R 2 ~R 5 is hydrogen, and R 6 is selected from hydrogen, a protecting group for an amino group, -C( NH)-NH2 and a poly(ethylene glycol) chain. In some cases, R 2 ~R 6 is hydrogen. In some cases, R 7 is C8-C18 alkyl or C8-C18 alkenyl having one C-C double bond, or C8-C12 alkyl or C8-C12 alkenyl having one C-C double bond, or C10-C12 alkyl or C10-C12 alkenyl having one C-C double bond. The present disclosed composition may contain one or more alkylene groups of formula (II)-(IV).

[0140] In some cases, the oligomers or polymers that can be used in the compositions according to the present disclosure contain a plurality of oligo(alkyleneamine) groups of formula (III) as repeating units: NR 2 {CH2-(CH2) a -NR 3 -[CH2-(CH2) b -NR 4 p} m -[[-END]]​ [CH2-(CH2) a -NR 5 n -(III) [wherein the variables a, b, p, m, n and R 2 ~R 5 are, for each group in the plurality of such groups, independently defined as follows according to the formula (III): a is 1, b is an integer from 2 to 4, or a is an integer from 2 to 4 and b is 1 ; p is 1 or 2; m is 1 or 2; n is 0 or 1, and m + n is 2 or more; and R 2 ~R 5 are, independently of one another, hydrogen; -CH2-CH(OH)-R 7 , -CH(R 7 )-CH2-OH, -CH2-CH2-(C=O)-O-R 7 , -CH2-CH2-(C =O)-NH-R 7 , -CH2-R 7 or a -CH2- group (wherein R 7 is selected from C3~C1 8 alkyl or C3~C18 alkenyl having one C-C double bond ); a protecting group for an amino group; -C(NH)-NH2; a poly(ethylene glycol) chain; and an endosome escape effector and a receptor ligand. In some cases , R 2 ~R 5 are hydrogen. In some cases, R 7 is selected from C8~C18 alkyl or C8~C18 alkenyl having one C-C. R 7 can be selected from C8~C12 alkyl or C8~C12 alkenyl having one C-C. Alternatively , R 7 ​may be selected from C10-C12 alkyl or C10-C12 alkenyl having one C-C bond.

[0141] One or more of the nitrogen atoms shown in formula (III) may be protonated to provide a cationic group of formula (III).

[0142] Oligomers or polymers containing a plurality of groups of formula (III) as repeating units may further contain one or more oligo(alkyleneamine) groups of formula (II) as side chains and / or end groups.

[0143] In a plurality of groups of formula (III) as repeating units, two, three or more of the groups of formula (III) can be included in the oligomer or polymer. Generally, substances containing 2 to 9 repeating units are referred to herein as oligomers, and substances containing 10 or more repeating units are referred to as polymers. Thus, in polymers containing a plurality of groups of formula (III) as repeating units, 10 or more groups of formula (III) may be present. The groups of formula (III) may have the same structure within the polymer or oligomer, or may have two or more different structures within the scope of formula (III). In some cases, oligomers or polymers containing a plurality of groups of formula (III) as repeating units may be provided in the form of a library of sequence-defined polymers prepared in a controlled stepwise polymerization from different groups of formula (III).

[0144] According to the above formulas (II) and (III), the alkyleneamine unit is such that an oligo(alkyleneamine) moiety of the -S-L-L-S- or -L-S-S-L- type is obtained. It may be repeated once in the chain of interactions, where S represents a shorter ethyleneamine unit and L represents a longer alkyleneamine unit. In some cases, the groups of formula (II) and ( III) do not undergo repetition so that shorter or longer units do not appear in pairs, i.e., p is 1. The group of formula (II) can be an oligo (alkyleneamine) group of formula (IIa), and the group of formula (III) can be an oligo(alkylene amine) group of (IIIa): -NR {CH2-(CH2) 2 -NR a -CH2-(CH2) 3 -NR b} 4 -[C m H2-(CH2) -NR a 5 -R n (IIa) [wherein a, b, m, n and R ~R 2 ~R 6 are defined as in formula (II), and one or more of the nitrogen atoms shown in formula (IIa) can be protonated to provide a cationic oligomer or polymer structure]; -NR 2 {CH2-(CH2) a -NR 3 -CH2-(CH2) b -NR 4} m -[C H2-(CH2) a -NR 5 n -(IIIa) [wherein a, b, m, n and R 2 ~R 5 are defined as in formula (III), and one or more of the nitrogen atoms shown in formula (IIia) can be protonated to provide a cationic oligomer or polymer structure].​​

[0145] Furthermore, in some cases, the oligo(alkyleneamine) of formulas (II) and (III) groups may have an n of 1. In some cases, m is 1 and n is 1. In some cases the group of formula (II) is an oligo(alkyleneamine) group of formula (IIb), and the group of formula (III ) is an oligo(alkyleneamine) group of formula (IIIb): -NR 2 -CH2-(CH2) a -NR 3 -CH2-(CH2) b -NR 4 -CH2- (CH2) a -(NR 5 )-R 6 (IIb) [wherein a, b and R 2 ~R 6 are defined as in formula (II), and one or more of the nitrogen atoms shown in formula (IIb ) may be protonated to provide a cationic oligomer or polymer structure]; -NR 2 -CH2-(CH2) a -NR 3 -CH2-(CH2) b -NR 4 -CH2- (CH2) a -NR 5 -(IIIb) [wherein a, b and R 2 ~R 5 are defined as in formula (III), and one or more of the nitrogen atoms shown in formula (II Ib) may be protonated to provide a cationic oligomer or poly mer structure].

[0146] Formulas (II), (IIa), (IIb) and (III), (IIIa), (IIIb) Regarding the length of the alkyleneamine units in the oligo(alkyleneamine) group, the alternating one of the units can be an ethyleneamine unit (i.e., either a or b is 1 ). The other alternating unit can be a propyleneamine unit, a butyleneamine unit, or a pentyleneamine unit (i.e., the other of a or b can be an integer from 2 to 4). In some cases, the other of a or b can be 2 or 3, and in some cases, a is 1 and b is 2, or a is 2 and b is 1. In some cases, the oligo(alkyleneamine) group of formula (IIc) is used instead of or in addition to group (II), and and / or the oligo(alkyleneamine) group of formula (IIIc) is used instead of or in addition to group (III). The formulas for group (IIc) and group (IIIc) are as follows: : -NR -CH2-CH2-NR 2 -CH2-CH2-CH2-NR 3 -CH2-CH 4 2-NR -R 5 (IIc) 6 wherein R ~R 2 are as defined in formula (II), R 6 ~R 2 ~R 6 are hydrogen, and one or more of the nitrogen atoms shown in formula (IIc) can be protonated to provide a cationic oligomer or polymer structure]; -NR 2 -CH2-CH2-NR 3 -CH2-CH2-CH2-NR 4 -CH2-CH 2-NR 5 -(IIIc) wherein R 2 ~R 5is as defined in formula (III) and one or more of the nitrogen atoms shown in formula (IIIc) may be protonated to provide a cationic oligomer or polymer structure One or more of the nitrogen atoms shown may be protonated to provide a cationic oligomer or polymer structure

[0147] In some cases, R in formulas (II), (IIa), (IIb) and (IIc) 2 ~R 6 groups or R in formulas (III), (IIIa), (IIIb) and (IIIc) R 2 ~R 5 groups may be protecting groups for amino groups. Non-limiting examples of protecting groups include t-b butoxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc) or carbobenzyloxy (Cbz).

[0148] In some cases, R in formulas (II), (IIa), (IIb) and (IIIc) 1 ~R 6 groups or R in formulas (III), (IIIa), (IIIb) and (IIIc) R 2 ~R 5 groups are receptor ligands such as those described by Philipp and Wagner in 「Gene an d Cell Therapy-Therapeutic Mechanisms an d Strategy」, 3rd Edition, Chapter 15, CRC P ress, Taylor & Francis Group LLC, Boca Rat on 2009. Examples of receptor ligands that target lung tissue are described by Pfeifer et al. 2010, Ther. Deliv. 1(1):133-48. Receptor ligands include specific cell types Screening of a peptide library for cell surface structures or binding to specific cell types Synthetic cyclic or linear peptides, cyclic or linear RGD peptides, such as those induced to be synthesized or natural carbohydrates such as sialic acid, galactose or mannose, or synthetic ligands derived, for example, from reacting a peptide with a carbohydrate, antibodies that specifically recognize cell surface structures, folic acid, epidermal growth factor and peptides derived therefrom, transferrin, anti-transferrin receptor antibodies, nanobodies and antibody fragments, approved drugs and the like that can bind to cell surface molecules (e.g., cell surface receptors) may be included.

[0149] R in formulas (II), (IIa), (IIb) and (IIc) 1 ~R 6 groups or R in formulas (III), (IIIa), (IIIb) and (IIIc) 2 ~R 5 groups As long as any of them is a poly(ethylene glycol) chain, the molecular weight of the poly(ethylene glycol) chain is about 100 g / mol to 20,000 g / mol, about 1,000 g / mol to 10,000 g / mol or about 1,000 g / mol to 5,000 g / mol and can be.

[0150] In some cases, group (II) is an oligo(alkyleneamine) group of formula (IId) and can be obtained: -NH-CH2-CH2-NH-CH2-CH2-CH2-NH-CH2-CH2-N H-H (IId) [wherein one or more of the nitrogen atoms shown in formula (IId) may be protonated to provide a cationic polymer or dendrimer structure]. In some cases, group (III) is of formula ( ​The oligo(alkyleneamine) group of (IIId): -NH-CH2-CH2-NH-CH2-CH2-CH2-NH-CH2-CH2-N H-(IIId) [where one or more of the nitrogen atoms shown in formula (IIId) may be protonated to provide a cationic polymer or dendrimer structure].

[0151] Lipidoid The engineered polynucleotide can be encapsulated in a lipidoid formulation . The lipidoid formulation can be any substance having the properties of lipids such as fats, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E and K, etc.), monoglycerides, diglycerides, triglycerides, phospholipids, etc. For example, the lipid or lipidoid formulation can be cholesterol, DOPE, DOPC or DS PC and other lipids such as those referred to as helper lipids in the scientific literature and / or any other lipids useful for preparing PEGylated lipids or lipoplexes. The formulation containing the engineered polynucleotide can be nanoparticles that can contain at least one lipid. The lipidoid formulation can be lipid nanoparticles . The lipids can be selected from, but are not limited to, DOPE, DOPC, DSPC, cholesterol, DLin-DMA, DLi n-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLi n-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG and PEGylated lipids . In another aspect, the lipid can be a cationic lipid such as DLin-DM A, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA and

[0152] The composition containing lipidoid may be about 40 - 60% lipidoid, about 40 - 60% cholesterol, and about 5 - 20% PEG-lipid (weight percentage based on the total weight of the composition). The composition containing lipidoid may be about 50 - 60% lipidoid, about 40 - 50% cholesterol, and about 5 - 10% PEG-lipid. The composition containing lipidoid may be about 50 - 75% lipidoid, about 20 - 40% cholesterol, and about 1 - 10% PEG-lipid. The composition containing lipidoid may be about 60 - 70% lipid oid, about 25 - 35% cholesterol, and about 5 - 10% PEG-lipid. The composition may be provided using techniques described, for example, in Akinc et al, 2007, Nat Biotech, 2 6, 561 - 569; Akinc et al, 2009, Mol Ther, 17, 8 72 - 9; Love et al, 2010, PNAS, 107, 1864 - 9; US Patent No. 8,450,298, International Publication No. WO2006 / 138380. The RNA / lipidoid complex can form particles useful for delivering any RNA, such as single-stranded RNA or mRNA, into cells. The composition of the present disclosure may be engineered polynucleotides encapsulated by a lipidoid of formula (IV):

[0153] R -NR R 1 -NR 2 {CH2-(CH2) a -NR 3 -[CH2-(CH2) b -NR 4 p } m -[CH2-(CH2) a -NR 5 n -R 6 ​​(IV) [In the formula, variables a, b, p, m, n, and R1 to R6 are defined as follows: a is 1, and b is an integer from 2 to 4, or a is an integer from 2 to 4 and b is 1; ; p is 1 or 2; m is 1 or 2; n is 0 or 1, and m + n is 2 or more; and R 1 ~R 6 are, independently of one another, hydrogen; -CH2-CH(OH)-R 7 , -CH(R 7 7 =O)-NH-R 7 or -CH2-R 7 group (where R 7 is selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond); a protecting group for an amino group; -C(NH)-NH2; a poly(ethylene glycol) chain; and a receptor ligand 1 selected from; provided that at least two of the residues of R 6 7 7 7 7 (OH)-R 7 7 7 R 7 7 7 or -CH2-R 7 group (where R 7 is selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond)].

[0154] In some cases, R 1 ~R 6 are, independently, hydrogen; -CH2-C(OH)H-R 7Also is -CH(R 7 )-CH2-OH (where R 7 is selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond); a protecting group for an amino group; and is selected from a poly(ethylene glycol) chain; provided that R 1 ~R 6 at least two of the residues are -CH2-C(OH)H-R 7 or -CH(R 7 )-CH2-OH group (where R 7 is selected from C3-C18 alkyl or C3- C18 alkenyl having one C-C double bond). In some cases, R 1 ~R 6 are independently hydrogen; and -CH2-CH(OH)-R 7 or -CH(R 7 )-CH2-OH group (where R 7 is selected from C3-C16 alkyl or C3-C16 alkenyl having one C-C double bond); provided that R at least two of the residues of R 1 ~R 6 are -CH2-CH(OH)-R or -CH(R 7 )-CH2-OH group (where 7 R is selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond). In some cases, R 7 and R are independently hydrogen 1 ; and -CH2-CH(OH)-R 6 or -CH(R )-CH2-OH group (where 7 R 7 )-CH2-OH group (where R 7is a C3-C18 alkyl or a C3-C18 alkoxy group having one C-C double bond. and R 2 ~R 5 are all -CH2-CH(O H)-R 7 or -CH(R 7 )-CH2-OH group (where R 7 C3 to C18 Al alkenyl or C3-C18 alkenyl having one C-C double bond). In some cases, R 7 has a C8-C16 alkyl or one C-C double bond C8-C18 alkenyl, C8-C12 alkyl or one CC double bond C8-C12 alkenyl having one bond, or C10-C12 alkyl or one C- Selected from C10 to C12 alkenyl having a C double bond.

[0155] One or more of the nitrogen atoms shown in formula (IV) may be protonated to form a cationic ligand of formula (IV). A peptide may be provided.

[0156] According to formula (IV) above, the alkyleneamine unit is -SLLS- or -L In alternating chains, so as to obtain -SSL-type oligo(alkyleneamine) moieties may be repeated once, where S represents a shorter ethyleneamine unit and L represents a longer In some cases, the lipidoid of formula (IV) may have a shorter alkyleneamine unit. or one that does not repeat so that longer units do not appear in pairs, i.e. p 1. The lipidoid of formula (IV) may be a lipidoid of formula (IVa): R 1 -NR 2 {CH2-(CH2) a -NR 3-CH2-(CH2) b -NR} m - CH2-(CH2) a -NR 5 n -R 6 (IVa) [wherein, a, b, m, n and R 1 ~R 6 are defined as in formula (IV), and one or more of the nitrogen atoms shown in formula (IVa) may be protonated to provide a cationic lipidoid ; In some cases, the lipidoid is a lipidoid of formula (IV). In some cases, "n" is 1 in the lipidoid of formula (IV). In some cases, "m" is 1 and n is 1 in the lipid oid of formula (IV). In some cases, the lipidoid of formula (IV) is a lipidoid of formula (IVb): R-NR 2 -CH2-(CH2) a -NR 3 -CH2-(CH2) b -NR 4 -CH2 -(CH2) a -NR 5 -R 6 (IVb) [wherein, a, b and R 1 ~R 6 are defined as in formula (IV), and one or more of the nitrogen atoms shown in formula (IVb) may be protonated to provide a cationic lipidoid].

[0157] Regarding the length of the alkyleneamine units in the lipidoids of formulas (IV), (IVa) and (IVb) It is understood that one of the alternating units needs to be an ethyleneamine unit (i.e., either a or b is 1). The other alternating unit is a propyleneamine unit ​units, butyleneamine units, pentyleneamine units, or other suitable units (i.e., either a or b is an integer from 2 to 4). In some cases, the lipid ide of formula (IV) is the lipidide of formula (IVc): R 1 -NR 2 -CH2-CH2-NR 3 -CH2-CH2-CH2-NR 4 -CH2- CH2-NR 5 -R S (IVc) [wherein R 1 ~R 6 is as defined in formula (IV), and one or more of the nitrogen atoms shown in formula (IVc) may be protonated to provide a cationic lipidide].

[0158] In some cases, the groups R 1 ~R 6 in formulas (IV), (IVa), (IVb) and (IVc) are protecting groups for amino groups. Non-limiting examples of protecting groups include t-butoxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc) or carbobenzyloxy (Cbz).

[0159] The groups R 1 ~R 6 in formulas (IV), (IVa), (IVb) and (IVc) are as described by Philipp and Wagner in "Gene and Cell Therapy - Therapeutic Mechanisms and Strategy", 3rd Edition, Chapter 15, CRC Press, Taylor & Francis Group LLC, Boca Raton 2009 and are incorporated herein by reference. ​​As long as it is a receptor ligand such as the receptor ligand being received. Examples of receptor ligands targeting lung tissue are described in Pfeifer et al. 2010, Ther. Deliv. 1(1) :133-48. Receptor ligands include synthetic cyclic or linear peptides, cyclic or linear RGD peptides, sialic acid, synthetic or natural carbohydrates such as galactose, lactose or mannose, or synthetic ligands derived from reacting, for example, peptides and carbohydrates, antibodies that specifically recognize cell surface structures, folic acid, epidermal growth factor and peptides derived therefrom, transferrin, anti-transferrin receptor antibodies, nanobodies and antibody fragments, approved drugs that can bind to cell surface molecules (such as cell surface receptors), etc. that are induced by screening peptide libraries for binding to specific cell surface structures or specific cell types :133-48. Receptor ligands include synthetic cyclic or linear peptides, cyclic or linear RGD peptides, sialic acid, synthetic or natural carbohydrates such as galactose, lactose or mannose, or synthetic ligands derived from reacting, for example, peptides and carbohydrates, antibodies that specifically recognize cell surface structures, folic acid, epidermal growth factor and peptides derived therefrom, transferrin, anti-transferrin receptor antibodies, nanobodies and antibody fragments, approved drugs that can bind to cell surface molecules (such as cell surface receptors), etc. that are induced by screening peptide libraries for binding to specific cell surface structures or specific cell types :133-48. Receptor ligands include synthetic cyclic or linear peptides, cyclic or linear RGD peptides, sialic acid, synthetic or natural carbohydrates such as galactose, lactose or mannose, or synthetic ligands derived from reacting, for example, peptides and carbohydrates, antibodies that specifically recognize cell surface structures, folic acid, epidermal growth factor and peptides derived therefrom, transferrin, anti-transferrin receptor antibodies, nanobodies and antibody fragments, approved drugs that can bind to cell surface molecules (such as cell surface receptors), etc. that are induced by screening peptide libraries for binding to specific cell surface structures or specific cell types :133-48. Receptor ligands include synthetic cyclic or linear peptides, cyclic or linear RGD peptides, sialic acid, synthetic or natural carbohydrates such as galactose, lactose or mannose, or synthetic ligands derived from reacting, for example, peptides and carbohydrates, antibodies that specifically recognize cell surface structures, folic acid, epidermal growth factor and peptides derived therefrom, transferrin, anti-transferrin receptor antibodies, nanobodies and antibody fragments, approved drugs that can bind to cell surface molecules (such as cell surface receptors), etc. that are induced by screening peptide libraries for binding to specific cell surface structures or specific cell types :133-48. Receptor ligands include synthetic cyclic or linear peptides, cyclic or linear RGD peptides, sialic acid, synthetic or natural carbohydrates such as galactose, lactose or mannose, or synthetic ligands derived from reacting, for example, peptides and carbohydrates, antibodies that specifically recognize cell surface structures, folic acid, epidermal growth factor and peptides derived therefrom, transferrin, anti-transferrin receptor antibodies, nanobodies and antibody fragments, approved drugs that can bind to cell surface molecules (such as cell surface receptors), etc. that are induced by screening peptide libraries for binding to specific cell surface structures or specific cell types :133-48. Receptor ligands include synthetic cyclic or linear peptides, cyclic or linear RGD peptides, sialic acid, synthetic or natural carbohydrates such as galactose, lactose or mannose, or synthetic ligands derived from reacting, for example, peptides and carbohydrates, antibodies that specifically recognize cell surface structures, folic acid, epidermal growth factor and peptides derived therefrom, transferrin, anti-transferrin receptor antibodies, nanobodies and antibody fragments, approved drugs that can bind to cell surface molecules (such as cell surface receptors), etc. that are induced by screening peptide libraries for binding to specific cell surface structures or specific cell types :133-48. Receptor ligands include synthetic cyclic or linear peptides, cyclic or linear RGD peptides, sialic acid, synthetic or natural carbohydrates such as galactose, lactose or mannose, or synthetic ligands derived from reacting, for example, peptides and carbohydrates, antibodies that specifically recognize cell surface structures, folic acid, epidermal growth factor and peptides derived therefrom, transferrin, anti-transferrin receptor antibodies, nanobodies and antibody fragments, approved drugs that can bind to cell surface molecules (such as cell surface receptors), etc. that are induced by screening peptide libraries for binding to specific cell surface structures or specific cell types :133-48. Receptor ligands include synthetic cyclic or linear peptides, cyclic or linear RGD peptides, sialic acid, synthetic or natural carbohydrates such as galactose, lactose or mannose, or synthetic ligands derived from reacting, for example, peptides and carbohydrates, antibodies that specifically recognize cell surface structures, folic acid, epidermal growth factor and peptides derived therefrom, transferrin, anti-transferrin receptor antibodies, nanobodies and antibody fragments, approved drugs that can bind to cell surface molecules (such as cell surface receptors), etc. that are induced by screening peptide libraries for binding to specific cell surface structures or specific cell types :133-48. Receptor ligands include synthetic cyclic or linear peptides, cyclic or linear RGD peptides, sialic acid, synthetic or natural carbohydrates such as galactose, lactose or mannose, or synthetic ligands derived from reacting, for example, peptides and carbohydrates, antibodies that specifically recognize cell surface structures, folic acid, epidermal growth factor and peptides derived therefrom, transferrin, anti-transferrin receptor antibodies, nanobodies and antibody fragments, approved drugs that can bind to cell surface molecules (such as cell surface receptors), etc. that are induced by screening peptide libraries for binding to specific cell surface structures or specific cell types

[0160] For R in formulas (IV), (IVa), (IVb) and (IVc) 1 ~R 6 As long as the groups are poly (ethylene glycol) chains, the molecular weight of the poly(ethylene glycol) chain can be about 1 00 g / mol to 20,000 g / mol, about 1,000 g / mol to 10,000 g / mol or about 1,000 g / mol to 5,000 g / mol. In some cases the molecular weight of the PEG chain can provide a composition with the desired density

[0161] Multiple lipidoid molecules can be bound to the engineered polynucleotide For example, the composition can be 1 engineered polynucleotide to 100 lipidoid molecules, 1 engineered polynucleotide to 1,000 lipidoid molecules, 10 engineered Polyribonucleotides per 1,000 lipidoid molecules or 100 engineered polynucleotides per 1,000 lipidoid molecules. The engineered polynucleotide can contain up to 10,000 lipidoid molecules. The complex of ribonucleotides and lipidoids can form particles. Particle diameter can range, for example, from 10 nanometers to 1200 nanometers. In some cases, the particle diameters range from 10 nanometers to 500 nanometers. The particles have diameters between 20 nanometers and 150 nanometers.

[0162] Administration to subjects A polynucleotide (e.g., a polyribonucleotide, a nucleic acid construct, or a vector) is subjected to Methods for administering to the body are further described herein. Via a delivery agent such as a particle or capsule having an encapsulating agent that encapsulates the nucleotide. The delivery agent can be a therapeutic agent. The subject is suffer from a condition (e.g. primary ciliary dyskinesia (PCD), Kartagener syndrome or cancer) The delivery agent can be administered to the subject at a predetermined dosage (e.g., (e.g., self-administered or administered by a third party such as a healthcare provider), and the dose increases over time The dosage can be increased, decreased over time, or kept constant. The time course of the disease can be altered based on the progression or regression of a disease in a subject, such as a cancer or tumor. do.

[0163] The polyribonucleotides of the present disclosure may be administered to a subject using one or more pharma- ceutically acceptable In some cases, the polyribonucleotide may be formulated with a carrier that is It can be formulated for targeted delivery to a target cell or cell population. In some cases the polynucleotide can be formulated for non-targeted delivery to a cell or cell population and then the encoded polypeptide product of the polynucleotide is transcribed and accumulates within the recipient cell.

[0164] The composition can be a combination of any engineered polynucleotide described herein and other chemical components such as carriers, stabilizers , diluents, dispersants, suspending agents, thickening agents and / or excipients. The composition facilitates the administration of the compound to an organism. The pharmaceutical composition can be administered in a therapeutically effective amount as a pharmaceutical composition by various forms and routes including, for example, intravenous, subcutaneous, intramuscular, oral, rectal, aerosol, parenteral, ocular, pulmonary, transdermal, vaginal, otic, nasal and topical administration. For example, intravenous, subcutaneous, intramuscular, oral, rectal, aerosol, parenteral, ocular, pulmonary, transdermal, vaginal, otic, nasal and topical administration. It can be administered in a therapeutically effective amount as a pharmaceutical composition by various forms and routes including, for example, intravenous, subcutaneous, intramuscular, oral, rectal, aerosol, parenteral, ocular, pulmonary, transdermal, vaginal, otic, nasal

[0165] The composition can be administered locally or systemically, for example, by direct injection of the compound into an organ, and may be administered as a depot formulation or a sustained release formulation. The pharmaceutical composition can be provided in the form of an immediate release formulation, a sustained release formulation, or an intermediate release formulation. The composition can be administered locally or systemically, for example, by direct injection of the compound into an organ, and may be administered as a depot formulation or a sustained release formulation. The pharmaceutical composition can be provided in the form of an immediate release formulation, a sustained release formulation, or an intermediate release formulation. The immediate release formulation can provide immediate release. The sustained release formulation can provide controlled release or delayed sustained release. The immediate release formulation can provide immediate release. The sustained release formulation can provide controlled release or delayed sustained release. The immediate release formulation can provide immediate release. The sustained release formulation can provide controlled release or delayed sustained release.

[0166] In the case of administration by inhalation, the active compound can be in the form of an aerosol, mist, vapor, spray or powder . The pharmaceutical composition can be conveniently in the form of an aerosol spray from a pressurized pack or a nebulizer using a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. It is delivered. In the case of pressurized aerosols, the dosage unit can be determined by providing a valve to deliver a metered amount. For example, gelatin capsules and cartridges for use in inhalers or injectors can be formulated to contain a powder mixture of the compound and a suitable powder base such as lactose or starch.

[0167] The eye contains several structurally and functionally distinct vascular beds that supply important eye components for maintaining vision. These beds include the retinal and choroidal vascular systems that supply the inner and outer parts of the retina, respectively, and the limbal vascular system located around the cornea.

[0168] A pharmaceutical composition containing an engineered polynucleotide can be administered to the eye by any suitable form or route including, for example, topical, oral, systemic, intravitreal, intracameral, subconjunctival, sub-Tenon's, retrobulbar, intraocular, juxtascleral, peribulbar, subretinal and suprachoroidal administration. The composition can be administered by injecting the formulation into any part of the eye including the anterior chamber, posterior chamber, vitreous cavity (intravitreal), subretinal space and / or subretinal subspace. The composition can also be delivered by non-invasive methods. Non-invasive methods of delivering the formulation can include using a needle-free injector device. Multiple administration routes can be used for efficient delivery of the pharmaceutical composition. The engineered polynucleotides of the present disclosure can be, for example, endothelial cells such as vascular endothelial cells, cells of the retina such as retinal pigment epithelium (RPE), corneal cells, fibroblasts, astrocytes, glial cells, pericytes, iris epithelial cells, cells of neural origin, ciliary epithelial cells, Müller cells, cells of the lateral rectus muscle

[0169] Cells surrounding the eye, such as muscle cells, orbital fat cells, sclera, and episclera cells that are attached to the eye and can be delivered to any suitable eye cells, including trabecular meshwork cells and connective tissue cells .

[0170] When the compositions disclosed herein are administered to a subject, they can have a transfection efficiency of at least about 80%, 90%, or 95% by the cells of the subject. In some cases the transfection efficiency of the encapsulated composition, when administered to a subject, is at least about 50%, 60%, 70%, 80%, 9 0%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160 %, 170%, 180%, 190%, 200%, 225%, 250%, 275%, 300 %, 325%, 350%, 375%, 400%, 450%, or 500% compared to non-encapsulated polynucleotides. In some situations, the transfection efficiency of a composition comprising a modified polynucleotide (and in some cases also a non-modified polynucleotide ) is at least about 50%, 60%, 70%, 80 %, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 450%, or 500% compared to a composition comprising only non-modified polyribonucleotides. 160%, 170%, 180%, 190%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 450%, or 500%. The transfection efficiency of the composition can be enhanced by adding a carrier such as a cell-penetrating peptide or a cationic coating to the outer layer of the composition. The transfection efficiency of the composition can be adjusted by the density of the composition.

[0171] ​​ Method for preparing a composition comprising an engineered polynucleotide described herein comprises formulating the compound with one or more inert pharmaceutically acceptable excipients or carriers to form a solid, semi-solid or liquid composition. Solid compositions include, for example, powders, tablets, dispersible granules, capsules, cachets and suppositories. Liquid compositions include, for example, solutions in which the compound is dissolved, emulsions containing the compound, or solutions containing liposomes, micelles or nanoparticles containing the compounds disclosed herein . Semi-solid compositions include, for example, gels, suspensions and creams. The composition can be a liquid solution or suspension, a solid form suitable for a solution or suspension in a liquid prior to use, or an emulsion . These compositions can also include small amounts of non-toxic auxiliary substances such as wetting agents or emulsifying agents, pH buffering agents, and other pharmaceutically acceptable additives . Non-limiting examples of pharmaceutically acceptable excipients are, for example, Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company

[0172] , 1995); Hoover, John E., Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and ​​​​​​and Pharmaceutical Dosage Forms and Drug De livery Systems, Seventh Ed. (Lippincott Wi lliams & Wilkins 1999), each of which is incor porated herein by reference in its entirety.

[0173] Compositions containing polynucleotides (e.g., polyr ibonucleotides) can be provided in various dosages. The dosage of the polynucleotide or po lyribonucleotide can be from about 1 μg to about 1000 μg, from about 1 μg to about 500 μg, from about 1 μg to about 1000 μg, from about 10 μg to about 500 μg, from about 20 μg to about 500 μg, from about 25 μg to about 500 μg, from about 30 μg to about 500 μg, from about 40 μg to about 500 μg, from about 50 μg to about 500 μg, from about 10 μg to about 250 μg, from ab out 20 μg to about 250 μg, from about 30 μg to about 250 μg, from about 40 μg to about 250 μg, from about 50 μg to about 250 μg, from about 1 μg to about 200 μg, from about 10 μg to about 50 mg, about 40 mg to about 50 mg, about 50 mg to about 100 mg, about 1 mg to about 25 mg , about 2 mg to about 25 mg, about 3 mg to about 25 mg, about 4 mg to about 25 mg, about 5 mg to about 25 mg, about 1 mg to about 20 mg, about 1 mg to about 20 mg, about 2 mg to about 20 mg, about 3 mg to about 20 mg, about 4 mg to about 20 mg, or about 5 mg to about 20 mg of the manipulative polyribo nucleotide.

[0174] The proportion of polyribonucleotide in the composition (for example, in the encapsulating agent) is 0.25 wt % polyribonucleotide, 0.5 wt% polyribonucleotide, 0.75 wt% polyribo nucleotide, 1 wt% polyribonucleotide, 1.25 wt% polyribonucleotide, 1.5 wt% polyribonucleotide, 1.75 wt% polyribonucleotide, 2 wt% p olyribonucleotide, 2.25 wt% polyribonucleotide, 2.5 wt% polyribonucleo tide, 2.75 wt% polyribonucleotide, 3 wt% polyribonucleotide, 3. 25 wt% polyribonucleotide, 3.5 wt% polyribonucleotide, 3.75 wt% polyribonucleotide, 4 wt% polyribonucleotide, 4.25 wt% polyribonucle otide, 4.5 wt% polyribonucleotide, 4.75 wt% polyribonucleotide, 5 wt% polyribonucleotide, 5.25 wt% polyribonucleotide, 5.5 wt% poly ribonucleotide, 5.75 wt% polyribonucleotide, 6 wt% polyribonucleoti de, 6.25 wt% polyribonucleotide, 6.5 wt% polyribonucleotide, 6.7 5 wt% polyribonucleotide, 7 wt% polyribonucleotide, 7.25 wt% polyri bonucleotide, 7.5 wt% polyribonucleotide, 7.75 wt% polyribonucleo Thide, 8 wt% polynucleotide, 8.25 wt% polynucleotide, 8.5 wt% polynucleotide, 8.75 wt% polynucleotide, 9 wt% polynucleotide, 9.25 wt% polynucleotide, 9.5 wt% polynucleotide, 9.75 wt% polynucleotide, 10 wt% polynucleotide, 10.25 wt% polynucleotide, 10.5 wt% polynucleotide, 10.75 wt% polynucleotide, 11 wt% polynucleotide, 11.25 wt% polynucleotide, 11.5 wt% polynucleotide, 11.75 wt% polynucleotide, 12 wt% polynucleotide, 12.25 wt% polynucleotide, 12.5 wt% polynucleotide, 12.75 wt% polynucleotide, 13 wt% polynucleotide, 13.25 wt% polynucleotide, 13.5 wt% polyribo nucleotide, 13.75 wt% polynucleotide, 14 wt% polynucleotide, 14.25 wt% polynucleotide, 14.5 wt% polynucleotide, 14.75 wt% polynucleotide, 15 wt% polynucleotide, 15.25 wt% polynucleotide, 15.5 wt% polynucleotide, 15.75 wt% polynucleotide, 16 wt% polynucleotide, 16.25 wt% polynucleotide, 16.5 wt% polynucleotide, 16.75 wt% polynucleotide, 17 wt% polynucleotide, 17.25 wt% polynucleotide, 17.5 wt% polynucleotide, 17.75 wt% polynucleotide, 18 wt% polynucleotide, 18.25 wt% polynucleotide, 18.5 wt% polyribo nucleotide, 18.75 wt% polynucleotide, 19 wt% polynucleotide, 19.25 wt% polynucleotide, 19.5 wt% polynucleotide, 19.75 wt% polynucleotide, 20 wt% polynucleotide, 20.25 wt% polynucleotide, 20.5 wt% polynucleotide, Polynucleotide, 18.25% by weight of polynucleotide, 18.5% by weight of poly nucleotide, 18.75% by weight of polynucleotide, 19% by weight of polynucleo tide, 19.25% by weight of polynucleotide, 19.5% by weight of polynucleotide, 19.75% by weight of polynucleotide, 20% by weight of polynucleotide, 20.5% by weight of polynucleotide, 21% by weight of polynucleotide, 21.5% by weight of poly nucleotide, 22% by weight of polynucleotide, 22.5% by weight of polynucleotide , 23% by weight of polynucleotide, 23.5% by weight of polynucleotide, 24% by weight of polynucleotide, 24.5% by weight of polynucleotide, or 25% by weight of poly nucleotide or more. Alternatively, the proportion of polynucleotide in the formulation (e.g., in the encapsulating agent) is less than about 25% of polynucleotide, 24.5% of polynucleotide, 24% of polynucleotide, 23.5% of polynucleotide, 23% of poly ribonucleotide, 22.5% of polynucleotide, 22% of polynucleotide, 21.5% of polynucleotide, 21% of polynucleotide, 20.5% of poly ribonucleotide, 20% of polynucleotide, 19.5% of polynucleotide, 19% of poly ribonucleotide, 18.5% of polynucleotide, 18% of polynucleotide, 17.5% of polynucleotide, 17% of polynucleotide, 16.5% of poly ribonucleotide, 16% of polynucleotide, 15.5% of polynucleotide, 15% of poly ribonucleotide, 14.5% of polynucleotide, 14% of polynucleotide, 13.5% of polynucleotide, 13% of polynucleotide, 12.5% of poly ribonucleotide, 13% of polynucleotide, 12.5% of polynucleotide Nucleotide, 12% polyribonucleotide, 11.5% polyribonucleotide, 11% poly ribonucleotide, 10.5% polyribonucleotide, 10% polyribonucleotide, 9.5% polyribonucleotide, 9% polyribonucleotide, 8.5% polyribonucleo tide, 8% polyribonucleotide, 7.5% polyribonucleotide, 7% polyribonucle otide, 6.5% polyribonucleotide, 6% polyribonucleotide, 5.5% polyribo nucleotide, 5% polyribonucleotide, 4.5% polyribonucleotide, 4% polyri bonucleotide, 3.5% polyribonucleotide, 3% polyribonucleotide, 2.5% polyribonucleotide, 2% polyribonucleotide, 1.5% polyribonucleotide, 1 % polyribonucleotide, 0.5% polyribonucleotide, or 0.1% polyribonucle otide may be.

[0175] In some cases, the encapsulated composition of the present disclosure is to increase the plasma, serum or blood concentration of the polyribonucleotide, pharmaceutical carrier, encapsulating agent or polymer material (such as polyethylene glycol or polyethyleneimine) in a subject within about 1 second to about 30 minutes, about 1 second to 20 minutes, about 1 second to 10 minutes, about 1 second to 5 minutes, about 1 second to 2 minutes, about 1 second to 1 minute, about 1 second to about 30 seconds, about 30 seconds to 30 minutes, about 30 seconds to 20 minutes, about 30 seconds to 10 minutes, about 30 seconds to 5 minutes about 30 seconds to 2 minutes, about 30 seconds to about 1 minute, about 1 minute to about 30 minutes, is achievable. The plasma, serum or blood concentration of a polynucleotide, pharmaceutical carrier, encapsulating agent or polymer material (e.g., polyethylene glycol or polyethyleneimine) can be the peak concentration or the average concentration.

[0176] [Examples] [Example 1] Preparation of DNAI1 RNA-containing This experiment demonstrates the preparation of a DNAI1 complementary deoxyribonucleic acid construct.

[0177] Method: DNAI1 was synthesized by GenScript. pUC57 / DNAI1 was digested with Hin dIII and EcoRI HF restriction enzymes. Furthermore, the digested pVAX120 vector and DNAI1 cDNA were gel-purified and ligated (the ORF of DNAI1 is codon-optimized). For RNA generation using unmodified nucleotides, standard in vitro translation procedures were used. The capping reaction was carried out using the vaccinia virus capping system and cap 2'-O-methyltransferase. Figure 1 is an agarose gel showing the generation of capped and uncapped DNAI1 RNA. Note that in this experiment, DNAI1 cDNA was ligated into pVAX120 to provide a construct containing a poly(A) tail.

[0178] [Example 2] Expression of DNAI ribonucleic acid in mammalian cells This experiment demonstrates the expression (translation) of DNAI1 in HEK-293 cells. Figure 2 shows 293 cells at 6 hours, 24 hours and 48 hours after transfection. It is a Western blot showing the translation of DNAI1 mRNA. For this experiment, 5×10 293 cells / well in a well plate were transfected with 2.5 μg of DNAI1 RNA using 3.75 μl of messenger max transfection reagent. 6, 24, and 48 hours after transfection, the cells were scraped from the wells, pelleted, and the pellet was lysed in RIPA buffer. This blot was probed with anti-DNAI1 ab166912 from Abcam. C-terminal FLAG-tagged DNAI1 5 plasmid DNA was transfected as a control, and the difference in MW between the plasmid and the mRNA is probably due to the FLAG tag in the pENTRY vector.

[0179] [Example 3] Formulation of a composition containing engineered polynucleotides for the treatment of human subjects suffering from primary ciliary dyskinesia.

[0180] The composition is formulated as follows: A nucleic acid construct encoding the NCBI reference sequence: NM_012144 which is the DNAI1 gene sequence is prepared as described in Example 1. Branched polyethyleneimine is purchased from Sigma Aldrich (trademark). Linear in vivo jetPEI (registered trademark) (poly ethyleneimine) is purchased from Polyplus transfection (registered trademark) (Ill kirch, France) and used without further purification. According to the manufacturer's protocol, jetPEI is combined with the sterile 10% glucose solution provided by the manufacturer and HP purchased from Sigma-Aldrich (St. Louis, MO) ​​​​​​​​​Dilute with LC-grade water to 5% glucose (final concentration). After diluting the nucleic acid construct in 5% glucose (final concentration), combine / mix the RNA and jetPEI solutions in a 1:1 ratio to a final N / P ratio of 8. Then administer the mRNA by intranasal instillation. Alternatively, the nucleic acid construct can also be formulated for administration by spraying or sniffing the lipoplex formulation.

[0181] [Example 4] Effect of post-transcriptional polyadenylation reaction time on RNA quality The effect of post-transcriptional polyadenylation reaction time on RNA quality was tested. The polyadenylation reaction time after in vitro transcription (IVT) is typically 60 - 90 minutes in length and usually provides a polyA length of at most about ~200 As. Since mRNA is prone to hydrolysis, it is often degraded over time during the post-transcriptional polyadenylation reaction. To maintain the optimal length of the DNAI1 polyA tail and maximize RNA quality, a nucleic acid construct encoding axonemal dynein intermediate chain 1 protein or a variant thereof having a polyA tail already included in the template was

[0182] constructed. The outlines of the nucleic acid constructs encoding the DNAI1 gene sequences with and without

Table 4

[0183] Figure 3 shows the DNAI1-pCMV 6Entry plasmid polyadenylated post-transcriptionally at reaction times of 0 - 60 minutes to Shows the fragment analyzer data. This demonstrates that the length of the transcript increases with a longer polyadenylation reaction time. Figure 4 shows the fragment analyzer data for examining the quality of these DNAI1 mRNAs polyadenylated after transcription with a reaction time of 0 - 60 minutes. These results indicate that as the polyadenylation reaction progresses, the RNA undergoes degradation, as shown by the decrease in peak % and the increase in pre-peak smear % with longer reaction times. Figure 5 shows the length of the polyA sequence in the DNAI1-pVAX plasmid template determined by 8% PAGE. Example 5 RNA Production and Quality Control in Vitro

[0184] To compare the effects of incorporating specific chemically modified nucleotides at various ratios on translation efficiency in different cell types and immunogenicity, the following experiments were conducted. The experiments included: 1) in vitro transcription of nucleic acid constructs; 2) in vitro capping of nucleic acid constructs; 3) analysis of the integrity of the transcribed RNA; 4) immunodot blot assay of dsRNA; and 5) analysis of the nucleotide composition of the transcribed RNA. With some modifications, the general protocols for in vitro transcription (IVT) and capping of RNA were followed. The IVT reaction of the nucleic acid construct encoding the DNAI1 gene was carried out at 37°C for 6 hours in the presence of 20 mM MgCl2 and 7.5 mM of each ribonucleotide. Table 5 shows various specific chemically modified nucleotides transcribed in vitro from the nucleic acid construct encoding the axonemal dynein intermediate chain 1.

[0185]

[0186] [Table 5]​​​​​​​​​​

[0187] Result: UV measurement [Table 6]

[0188] Template poly(A) length - Fragment Analyzer Poly(A) tail length analysis of the DNAI1 nucleic acid construct (SEQ ID NO: 5) used as a template for in vitro transcription showed that the number of A residues was maintained compared to the original cloning vector (pVAX - A120). The first vector contained 120 adenosine nucleotides, and a band of 100 - 150 bp was detected in this nucleic acid construct (Figure 5). The template was digested with EcoRI and Not I to remove the poly(A) fragment: 1 2 non - poly(A) nucleotides are expected to be part of the fragment. G*AAT TCtgcag - poly(A) - GC*GGCCGC = 12 nucleotides + poly(A) in the EcoRI / NotI generated fragment.

[0189] RNA smear analysis - Fragment Analyzer For all transcripts generated for DNAI1, a peak of approximately 2,000 nucleotides was detected. Evaluation of in vitro generated transcripts on the Fragment Analyzer showed that capped transcripts maintained good integrity: limited detection of smear content (an indicator of RNA degradation and / or hydrolysis) was observed in repeated experiments. Briefly, 2 μL of a 200 ng / μL sample was loaded onto the Fragment Analyzer (DNF - 471 standard sensitivity RN ​​​​​Analyzed using an A analysis kit (lower marker of 15 nucleotides). Data analysis was performed using PROS ize 2.0 software. The sizing accuracy is within approximately ±5%, the sizing reproducibility is within approximately 5% CV, the quantification accuracy is within approximately ±20%, and the quantification reproducibility is approximately 10% CV. Figure 6 shows the fragment analyzer data for in vitro reactions containing only standard nucleotides, namely adenosine 5'-triphosphate, guanosine 5'-triphosphate, cytidine 5'-triphosphate, and uridine 5'-triphosphate only. Figure 7 shows the fragment analyzer data for in vitro reactions containing a 50% / 50% mixture of pseudouridine and uridine 5'-triphosphate. Figure 8 shows the fragment analyzer data for in vitro reactions containing 1 00% pseudouridine 5'-triphosphate. Figure 9 shows the fragment analyzer data for in vitro reactions containing 100% 1-methyl-pseudouridine 5'-triphosphate . Table 7 summarizes the results of RNA smear analysis:

Table 7

[0190] The double-stranded RNA content detected by dot blot showed reactivity with the J2 antibody . Figure 10 shows the double-stranded RNA content detected by dot blot analysis of in vitro transcribed RNA from a nucleic acid construct encoding axonemal dynein intermediate chain 1, as well as the double-stranded RNA content of the transcribed DNAI1 constructs with various specific modifications shown in Table 5.

[0191] Nucleoside composition analysis Tables 8 to 10 show the nucleotide composition analysis of in vitro transcribed RNA from nucleic acid constructs encoding axonemal dynein intermediate chain 1. Various specific nucleotide modifications are shown in Table 5. Figures 1 1 to 13 show the corresponding HPLC chromatograms of the individual ribonucleotides obtained after nuclease digestion of the transcript and subsequent dephosphorylation.

[0192] Table 8 shows the nucleotide composition analysis of in vitro transcribed RNA from a nucleic acid construct encoding axonemal dynein intermediate chain 1 with unmodified nucleotides. Figure 11 shows the corresponding HPLC chromatogram.

Table 8

[0193] Table 9 shows the nucleotide composition analysis of in vitro transcribed RNA from a nucleic acid construct encoding axonemal dynein intermediate chain 1 with 50% pseudouridine. Figure 12 shows the corresponding HPLC chromatogram. The retention time of hydrophobic 1-methyl-pseudouridine under the same reverse-phase HPLC conditions averages about 9.5 minutes and is well separated from all other ribonucleotides examined (data not shown).

Table 9

[0194] Table 10 shows the nucleotide composition analysis of in vitro transcribed RNA from a nucleic acid construct encoding axonemal dynein intermediate chain 1 with 100% pseudouridine. Figure 13 shows the corresponding HPLC chromatogram.

Table 10

[0195] [Example 6] Translation efficiency The translation efficiency of the above DNAI1 transcript was evaluated in three cell lines: 1) HEK-293 human embryonic kidney cells ; 2) A549 adenocarcinoma human alveolar basal epithelial cells; and 3) MLE-15 mouse lung epithelial cells Each cell line was transfected in triplicate with each DNAI1 transcript, and the resulting cell extracts were analyzed for DNAI1 protein expression by Western blotting . Briefly, 1×10 (HEK-293, MLE-15) or 2 6 (A549) cells were plated per well and transfected in 6-well plates 18 hours later ×10 6 . Cells were transfected with approximately 100 ng of each RNA using MessengerMax transfection reagent at an RNA:MessengerMax ratio of 1:37.5 . Cells were harvested 6 hours after transfection and whole cell extracts were prepared in RIPA buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1% Triton X-1 00, 0.1% SDS, 0.5% sodium taurocholate) . 3.5 μg of total protein from each extract was prepared in 1×LDS sample buffer containing 2.5% β-mercaptoethanol and loaded onto a 4-12% Bis-Tris SDS-PAG E gel . The gel was then run at a constant voltage of 30 V for 30 minutes, followed by 150 V for 1 hour . Proteins were transferred to a PVDF membrane at 25 V for 1 hour in 1×NuPAGE transfer buffer containing 10% methanol . After transfer, Western blotting was performed using an anti-DNAI1 antibody . ​DNAI1 protein was detected by lot and developed using an alkaline phosphatase chemiluminescent substrate. Western blot values were normalized using Sypro Ruby total protein staining as a loading control and represented as relative expression to unmodified RNA. Each data point is the mean ± standard deviation of three biological (transfection) replicates. Figures 14, 15, and 16 show the expression of DNAI1 protein in HEK-293, A549, and MLE-15 cells, respectively. DNAI1 was expressed as a 699 amino acid, 79

[0196] .3 kDa protein. Pseudouridine (Ψ)-containing transcripts were well-expressed in all three cell types at unmodified RNA or greater expression levels. Similarly, 1-methylpseudouridine (m Ψ)-containing transcripts resulted in unmodified RNA or greater expression levels in A549 and MLE15 cells (expression in HEK-293 cells was not tested for this transcript). Importantly, the expression levels of each transcript and their relative ranking were similar in each cell line, indicating no cell-type specific effect on DNAI1 translation. Figure 17 is a graph showing the relative expression of DNAI1 protein in HEK-293, A549, and ML E-15 cells. Western blot signal values were normalized using total protein staining and plotted as mean expression ± standard deviation compared to unmodified DNAI1 m RNA. 1 Table 11 is a summary of the relative expression of DNAI1 protein in each of the above cell lines.

[0197]

Table 11

[0198] [Example 7] Immunogenicity of a nucleic acid construct encoding human DNAI1 in vitro The immunogenicity of the above transcript was tested by measuring cytokine production in two cell lines, namely A549 adenocarcinoma human alveolar basal epithelial cells and HepG2 human hepatocarcinoma cells IL-6 production in response to the transcript was measured in A549 cells, and IP-10 production was measured in H epG2 cells. Each cell line was transfected in triplicate with each RNA at a certain titer Briefly, either 20,000 (A549) or 40,00 0 (HepG2) cells per well were plated and transfected in 96-well plates after 24 hours The cells were then transfected with each transcript at a certain titer: RNA:MessengerMax ratio 1:1.5 using MessengerMax reagent For unmodified, 50% Ψ and 100% Ψ transcripts, 250 ng - 7 n g per well and for 100% m1Ψ mRNA, 1000 ng - 32 ng per well

[0199] Culture supernatants were harvested 18 hours after transfection. The CellTiter-Glo assay kit (Prome ga), which measures ATP levels as an indicator of metabolically active cells, was used to measure cell viability immediately after supernatant collection. For IL-6 detection, A54 9 cell culture supernatants were diluted 1:20 with assay buffer and IL-6 levels were measured using an IL-6 high-sensitivity human ELISA kit (Abcam ab46042). Human IP-1 0 ELISA kit SimpleStep (Abcam ab173194) was used to , IP-10 was detected in undiluted HepG2 cell culture supernatants.

[0200] Figures 18 and 19 show the induction of IL-6 in A549 cells treated with DNAI1 transcripts. For the assay shown in FIG. 18, cells were incubated with RNA-MessengerMax After 18 hours of exposure to the complex, the assay shown in Figure 19 showed that RNA-MessengerMax The complexes were removed 2 hours after transfection. In both cases, the Cell culture supernatants were collected at 18 hours for detection of IL-6. 18 and 19, as measured using the Titer-Glo assay. Figure 22 shows the cell viability. For this assay, cells were incubated with RNA-MessengerMax complex. The mice were exposed to 18 h of genomic DNA for 18 h. Then, the mice were induced with various amounts of each DNAI1 mRNA. IP-10 expression was measured by ELISA. 23 shows cell viability measured using the assay shown in FIG. 22.

[0201] [Example 8] Translation of DNAI1 mRNA in HEK293 cells FIG. 24 shows the expression of the axonemal dynein intermediate chain 1 (DNAI1) in HEK293 cells. The peak expression of the nucleic acid or nucleic acid control is shown in FIG. Although translation of DNAI1 nucleic acid constructs in HEK293 cells peaks at 6 hours, , and still exists after 48 hours.

[0202] [Example 9] Unfractionated IgG1 mRNA after administration of 100% m1Ψ-containing DNAI1 mRNA in a lipoplex Expression of DNAI1 protein in undifferentiated and fully differentiated human airway epithelial cells (HAEC) and mouse tracheal epithelial cells (MTEC).

[0203] Expression of DNAI1 protein in primary human airway epithelial cells and mouse tracheal epithelial cells after treatment with lipoplex-formulated DNAI1-HA mRNA was evaluated by Western blot. The 100% m1Ψ-containing transcript used in this experiment was generated from an alternative codon usage template of DNAI1 (SEQ ID NO: 15) containing the HA epitope tag. Primary human epithelial cells were maintained in liquid culture for undifferentiated cultures or at the air-liquid interface and differentiated into fully differentiated ciliated epithelium over approximately 3 weeks. Next, 12 or 24 μg of lipoplex-formulated DNAI1-HA mRNA was applied to the apical side of fully differentiated cultures or directly to undifferentiated liquid cultures. Cells were treated once or twice daily for 1 day. Cells were then harvested 24 or 48 hours after the final treatment and whole cell extracts were prepared in RIPA buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% sodium taurocholate). Total protein from each extract was separated on a 4-12% Bis-Tris SDS-PAGE gel and transferred to a PVDF membrane. The DNAI1-HA protein was detected by Western blot using an anti-HA antibody and developed using an enhanced chemiluminescence substrate. As shown in FIGS. 2 and 5, the DNAI1-HA protein was expressed at high levels in both undifferentiated and fully differentiated ciliated human airway epithelial cells and mouse tracheal epithelial cells.

[0204] [Example 10]​​​​​​​​​​​​​​​​ The altered nucleotide usage frequency in the coding region enhances the stability of the mRNA for transcript therapy. Enhance stability.

[0205] A scheme of altered nucleotide usage frequency aimed at reducing the number of more reactive dinucleotides within and across codons of the modified mRNA partially alleviates the limitations imposed by the inherent chemical instability of RNA. At the same time, reducing the U content in the RNA transcript decreases its immunogenicity. Traditional codon optimization (CO) can be carried out before the removal of (+) reactive dinucleotides and the reduction of (+)U, resulting in an open reading frame (ORF) generally referred to as CO++. A scheme of altered nucleotide usage frequency aimed at reducing the number of more reactive dinucleotides within and across codons of the modified mRNA partially alleviates the limitations imposed by the inherent chemical instability of RNA. At the same time, reducing the U content in the RNA transcript decreases its immunogenicity. Traditional codon optimization (CO) can be carried out before the removal of (+) reactive dinucleotides and the reduction of (+)U, resulting in an open reading frame (ORF) generally referred to as CO++. A scheme of altered nucleotide usage frequency aimed at reducing the number of more reactive dinucleotides within and across codons of the modified mRNA partially alleviates the limitations imposed by the inherent chemical instability of RNA. At the same time, reducing the U content in the RNA transcript decreases its immunogenicity. Traditional codon optimization (CO) can be carried out before the removal of (+) reactive dinucleotides and the reduction of (+)U, resulting in an open reading frame (ORF) generally referred to as CO++. A scheme of altered nucleotide usage frequency aimed at reducing the number of more reactive dinucleotides within and across codons of the modified mRNA partially alleviates the limitations imposed by the inherent chemical instability of RNA. At the same time, reducing the U content in the RNA transcript decreases its immunogenicity. Traditional codon optimization (CO) can be carried out before the removal of (+) reactive dinucleotides and the reduction of (+)U, resulting in an open reading frame (ORF) generally referred to as CO++. A scheme of altered nucleotide usage frequency aimed at reducing the number of more reactive dinucleotides within and across codons of the modified mRNA partially alleviates the limitations imposed by the inherent chemical instability of RNA. At the same time, reducing the U content in the RNA transcript decreases its immunogenicity. Traditional codon optimization (CO) can be carried out before the removal of (+) reactive dinucleotides and the reduction of (+)U, resulting in an open reading frame (ORF) generally referred to as CO++. A scheme of altered nucleotide usage frequency aimed at reducing the number of more reactive dinucleotides within and across codons of the modified mRNA partially alleviates the limitations imposed by the inherent chemical instability of RNA. At the same time, reducing the U content in the RNA transcript decreases its immunogenicity. Traditional codon optimization (CO) can be carried out before the removal of (+) reactive dinucleotides and the reduction of (+)U, resulting in an open reading frame (ORF) generally referred to as CO++.

[0206] Figure 26 shows the overall quality improvement in DNAI1 expressing the polynucleotide of SEQ ID NO: 15 (B) compared to the polynucleotide of SEQ ID NO: 14 (A). The overall quality improvement is determined by the increase in the percentage of the major RNA peak in the fragment analyzer trace for the polynucleotide engineered with the altered codon usage frequency strategy. Figure 26 shows the overall quality improvement in DNAI1 expressing the polynucleotide of SEQ ID NO: 15 (B) compared to the polynucleotide of SEQ ID NO: 14 (A). The overall quality improvement is determined by the increase in the percentage of the major RNA peak in the fragment analyzer trace for the polynucleotide engineered with the altered codon usage frequency strategy. Figure 26 shows the overall quality improvement in DNAI1 expressing the polynucleotide of SEQ ID NO: 15 (B) compared to the polynucleotide of SEQ ID NO: 14 (A). The overall quality improvement is determined by the increase in the percentage of the major RNA peak in the fragment analyzer trace for the polynucleotide engineered with the altered codon usage frequency strategy. Figure 26 shows the overall quality improvement in DNAI1 expressing the polynucleotide of SEQ ID NO: 15 (B) compared to the polynucleotide of SEQ ID NO: 14 (A). The overall quality improvement is determined by the increase in the percentage of the major RNA peak in the fragment analyzer trace for the polynucleotide engineered with the altered codon usage frequency strategy. Furthermore, the DNAI1 mRNA characterized by the CO++ optimized open reading frame, i.e., the altered codon usage frequency, shows an improvement in translation efficiency in transfected A549 cells compared to the traditionally optimized transcript (CO) (see Figure 27). Here, 1.25x10 Furthermore, the DNAI1 mRNA characterized by the CO++ optimized open reading frame, i.e., the altered codon usage frequency, shows an improvement in translation efficiency in transfected A549 cells compared to the traditionally optimized transcript (CO) (see Figure 27). Here, 1.25x10 Furthermore, the DNAI1 mRNA characterized by the CO++ optimized open reading frame, i.e., the altered codon usage frequency, shows an improvement in translation efficiency in transfected A549 cells compared to the traditionally optimized transcript (CO) (see Figure 27). Here, 1.25x10 Furthermore, the DNAI1 mRNA characterized by the CO++ optimized open reading frame, i.e., the altered codon usage frequency, shows an improvement in translation efficiency in transfected A549 cells compared to the traditionally optimized transcript (CO) (see Figure 27). Here, 1.25x10 6 cells were plated per well and transfected in a 6-well plate after 18 hours. Cells were transfected with MessengerMax transfection reagent at an RNA:MessengerMax ratio of 1:12 at approximately 100n cells were plated per well and transfected in a 6-well plate after 18 hours. Cells were transfected with MessengerMax transfection reagent at an RNA:MessengerMax ratio of 1:12 at approximately 100n cells were plated per well and transfected in a 6-well plate after 18 hours. Cells were transfected with MessengerMax transfection reagent at an RNA:MessengerMax ratio of 1:12 at approximately 100n Transfected with each RNA of g and harvested 6 hours after transfection. Anti-D By Western blotting using the NAI1 antibody, the expression of the DNAI1 protein as a 699-amino acid, 79.3 kDa protein was revealed. The relative translation efficiency is shown as the average of three biological (transfection) replicates.

[0207] As shown in Figure 28, changes in reactivity with the J2 antibody-sensing double-stranded RNA content were observed Based on comparison with known concentrations of poly-IC, the dsRNA content of the mRNA encoding DNA I1, characterized by the CO++ ORF, is on average 39 ng, while the RNA having the CO ORF When 200 ng of in vitro transcribed mRNA was dotted, it was estimated to contain 68 ng of d sRNA contaminants.

[0208] [Example 11] Purification of unmodified and 100% m 1 Ψ-containing DNAI1 mRNA by HPLC.

[0209] Using reverse-phase high-performance liquid chromatography (HPLC) of DNAI1 mRNA, full-length RNA was purified to remove contaminants such as long dsRNA generated during in vitro transcription using T7 RNA polymerase. Triethylammonium acetate (TEAA) as an ion-pairing agent and a mobile phase containing a gradually increasing acetonitrile content, non-porous RNAS ep C18 semi-prep (100 mm × 21.2 mm, column volume (CV) approximately 2.4 m L) column, the fractions obtained and the purification results are shown in Figure 29. As judged by fragment analyzer evaluation of the purified fractions, overall using the semi-prep RNASep column Using the semi-prep RNASep column, overall Quality improvement and full-length RNA enrichment were observed. This quality improvement was achieved with both unmodified (A, B) and and 100% m 1 Ψ-containing DNAI1 mRNA species (C, D).

[0210] As shown in Figure 30, a moderate improvement in translation activity was observed in the full-length unmodified mRNA transcript enrichment fractions in A549 cells (A and B). Here, 1 × 10 6 A549 cells per well were plated and transfected in 6-well plates 18 hours later. Transfection was performed using the MessengerMax transfection reagent at an RNA:MessengerMax ratio of 1:12, and the cells were transfected with approximately 100 ng of each RNA and harvested 6 hours after transfection. The expression of the DNAI1 protein as a 699 amino acid, 79 .3 kDa protein was revealed by Western blotting using a 1:2000 rabbit anti-DNAI1 (A bCam ab166912, rabbit monoclonal anti-DNAI1 against the recombinant DNAI1 fragment) antibody (C). The relative translation efficiency is shown as the mean ± standard deviation of three biological (transfection) replicates.

[0211] Importantly, HPLC readily removes the dot blot reaction species that elute slowly on a semi-prep scale. The detectable double-stranded RNA content reacting with the J2 antibody was observed only in the slowly eluting fractions F7 and the unpurified control transcript, but not in any of the other HPLC-purified DNAI1 mRNA fractions F1 - F6 (D). The immunogenicity of the unmodified HPLC-purified transcripts was assessed by the production of IL-6 in response to the transfected mRNA. ​​By observation, further tests were conducted on A549 cells. Each cell line was replicated in triplicate and transfected with each RNA at a certain titer. Briefly, 20, 000 cells per well were plated and transfected in a 96-well plate 18 hours later . Then, using the MessengerMax reagent at an RNA:MessengerMax ratio of 1:1.5, cells were transfected with each transcript at a certain titer at 250 ng to 7 ng per well . For the HPLC-purified fraction F3 that produced the highest relative DNAI1 protein level, a decrease in the IL-6 response was observed (for cells transfected with 125 ng of RNA, unpurified reference DNAI1 mRNA: (727 ± 109 pg / m L) > F3 (73 ± 30 pg / mL)) (E).

[0212] Preferred embodiments of the present invention have been shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided merely as examples. The present invention is not limited by the specific examples provided herein. Although the present invention has been described with reference to the above specification, the description and examples of the embodiments herein are not intended to be construed in a limiting sense . Without departing from the present invention, numerous variations, modifications, and substitutions will occur to those skilled in the art. Furthermore, it should be understood that all aspects of the present invention are not limited to the specific expressions, shapes, or relative ratios described herein, which depend on various conditions and variables . It should be understood that various alternatives to the embodiments of the present invention described herein can be used in the practice of the present invention . Therefore, the present invention is intended to include such alternatives, modifications, variations, or equivalents. The following claims are the ​ Define the scope of the invention, and it is intended that the methods and structures within these claims and their equivalents be encompassed thereby.

Claims

1. Methods for treating a subject having or at risk of having primary ciliary dyskinesia The present invention relates to a method for the preparation of axonemal dynein intermediate chain 1 proteins, comprising the steps of: and administering to the subject a composition comprising the nucleic acid construct, wherein the nucleic acid construct is expressed within a cell of the subject. Heterologous expression or enhanced expression of said axonemal dynein intermediate chain 1 protein or a mutant thereof and thereby have or have primary ciliary dyskinesia. The method comprises treating said subject at risk of having a pulmonary embolism.

2. The method of claim 1 , wherein the nucleic acid construct is a complementary deoxyribonucleic acid construct.

3. The nucleic acid construct encodes an axonemal dynein intermediate chain 1 protein or a mutant thereof. compared to the levels in cells exposed to a composition comprising a nucleic acid construct that does not contain the codon. At least about 1.5-fold increased levels of silk dynein intermediate chain 1 protein or a mutant thereof The method of claim 1 , wherein the nucleic acid sequence is encoded by

4. The method of claim 3, wherein the multiple is at least about 5.

5. The codons of the construct are at least The method of claim 1, which is 70% homologous.

6. The codons of the construct have at least 70 identical sequences to human axonemal dynein intermediate chain 1 mRNA. % homology.

7. The construct further comprises a 3' codon sequence encoding the axonemal dynein intermediate chain 1. or a 5' non-coding region, wherein said non-coding region is a 5' non-coding region of said protein in a cell of said subject. The method of claim 1, which enhances expression of a protein.

8. The construct further comprises a 3' codon sequence encoding the axonemal dynein intermediate chain 1. a 3′ cap-independent translation enhancer ( 8. The method of claim 7, comprising the step of:

9. At least one of the codon sequence and the 3' non-coding region or the 5' non-coding region The method of claim 7, further comprising one intermediate sequence region.

10. The codon sequence according to claim 7, wherein the codon sequence comprises an open reading frame (ORF) sequence. Method of posting.

11. The construct further comprises a 3' codon sequence encoding the axonemal dynein intermediate chain 1. a 3' non-coding region, the 3' non-coding region comprising the nucleotide sequence of a histone protein; The method of claim 7, comprising a 3' stem-loop region derived from the sequence.

12. The 3' non-coding region adjacent to the codon sequence includes a polyadenosine tail, The number of adenosines in the adenosine tail is determined by the number of adenosines in the axonemal dynein intermediate chain 1 protein. The method of claim 7, which improves translation efficiency.

13. The 3' non-coding region adjacent to the codon sequence includes a polyadenosine tail, The number of adenosines in the adenosine tail determines the number of adenosines in the axonemal dynein intermediate chain 1 mRNA. The method of claim 7, which extends the half-life.

14. The codon sequence comprises an open reading frame (ORF) sequence.

14. The method according to claim 13.

15. 12, 13. The polyadenosine tail has a length of at most 200 adenosines. Or the method according to claim 14.

16. A proportion of the polyadenosine tail comprises nucleotide analogues.

15. The method according to claim 14.

17. wherein less than 20% of the nucleotides in the polyadenosine tail are nucleotide analogs. The method according to claim 12, 13 or 14.

18. The method of claim 1 , wherein the construct comprises a non-canonical nucleotide analogue.

19. Fewer than 30% of the nucleotides encoding axonemal dynein intermediate chain 1 are nucleotide analogs. The method of claim 18 .

20. The nucleotide analogues are pseudouridine, 1-methylpseudouridine and 5 -methoxyuridine.

21. 20. The method of claim 18, wherein the nucleotide analog is pseudouridine.

22. The method of claim 18, wherein the nucleotide analog is 1-methylpseudouridine. Law.

23. 19. The method of claim 18, wherein the nucleotide analog is 5-methoxyuridine.

24. The nucleotide analogs are pseudouridine and 1-methylpseudouridine. The method according to claim 18.

25. The composition comprises an armadillo repeat containing 4 (ARMC4), an open chromosome 21 (OPL21), Coiled-coil domain-containing frame 59 (C21orf59), coiled-coil domain-containing 103 (CC DC103), coiled-coil domain-containing 114 (CCDC114), coiled-coil Coiled-coil domain containing 39 (CCDC39), coiled-coil domain containing 40 (CCDC40) , coiled-coil domain containing 65 (CCDC65), cyclin O (CCNO), dye Dynein (axoneme) assembly factor 1 (DNAAF1), dynein (axoneme) assembly factor 2 ( DNAAF2), dynein (axoneme) assembly factor 3 (DNAAF3), dynein (axoneme) ) DNA assembly factor 5 (DNAAF5), axonemal dynein heavy chain 11 (DNAH11), axonemal dynein Dynein heavy chain 5 (DNAH5), axonemal dynein heavy chain 6 (DNAH6), axonemal dynein heavy chain 8 (DNAH8), axonemal dynein intermediate chain 2 (DNAI2), axonemal dynein light chain 1 (DNAL 1), dynein regulatory complex subunit 1 (DRC1), dyslexia susceptibility 1 candidate 1 (DY X1C1), growth arrest specific 8 (GAS8), axonemal central pair apparatus protein (HYDIN) , leucine-rich repeat containing 6 (LRRC6), NME / NM23 family member 8 (NME8), Orofacial-Digital Syndrome 1 (OFD1), Retinitis Pigmentosa GTPase Regulator child (RPGR), radial spoke head 1 homolog (Chlamydomonas) (RSPH1) , radial spoke head 4 homolog A (Chlamydomonas) (RSPH4A), radial Spokehead 9 homolog (Chlamydomonas) (RSPH9), sperm associated antigen 1 (SPA G1) and zinc finger MYND type-containing 10 (ZMYND10). and at least one additional nucleic acid construct encoding a protein of interest. Item 1. The method according to item 1.

26. The composition has at least 4 moles of phosphate groups in the modified polyribonucleotide. The method of claim 1 , comprising the ratio of moles of amine groups of the cationic polymer to the moles of amine groups of the cationic polymer.

27. The method of claim 1 , wherein the composition is formulated into nanoparticles or nanocapsules.

28. The composition is formulated into a cationic lipid, a cationic polymer, or a nanoemulsion. The method of claim 1 .

29. A composition comprising a nucleic acid construct encoding axonemal dynein intermediate chain 1, said nucleic acid construct wherein the composition comprises a complementary deoxyribonucleic acid encoding axonemal dynein intermediate chain 1, A composition formulated for administration to the body.

30. A composition comprising a nucleic acid construct encoding axonemal dynein intermediate chain 1, said nucleic acid construct in cells of a subject having or at risk of having primary ciliary dyskinesia. Heterologous or enhanced expression of axonemal dynein intermediate chain 1 protein or a mutant thereof A composition comprising the codons provided.

31. A composition comprising a nucleic acid construct encoding an axonemal dynein intermediate chain 1, A composition in which less than 30% of the nucleotides encoding interstrand 1 are nucleotide analogs. 。

32. The codons of the construct are identical to those of the mammalian human axonemal dynein intermediate chain 1 protein.

32. The composition of claim 29, 30 or 31, wherein said sequence is 70% homologous to said sequence.

33. The codons of the construct have at least 70% identical codon identity to human axonemal dynein intermediate chain 1 protein. The composition of claim 32 which is homologous.

34. The construct further comprises a 3' codon sequence encoding the axonemal dynein intermediate chain 1. or a 5' non-coding region, wherein said non-coding region is a 5' non-coding region of said protein in a cell of said subject.

32. The composition of claim 29, 30 or 31, which enhances expression of a protein.

35. The construct further comprises a 3' non-codon sequence adjacent to the codon sequence encoding the axonemal dynein intermediate chain 1. the 3' non-coding region comprises a 3' cap-independent translation enhancer (3' 32. The composition of claim 29, 30 or 31, comprising:

36. The construct further comprises a 3' codon sequence encoding the axonemal dynein intermediate chain 1. a 3' non-coding region, the 3' non-coding region comprising the nucleotide sequence of a histone protein; 32. The composition of claim 29, 30 or 31, comprising a 3' stem-loop region derived from the sequence. 。

37. The 3' non-coding region adjacent to the codon sequence includes a polyadenosine tail, The number of adenosines in the adenosine tail is determined by the number of adenosines in the axonemal dynein intermediate chain 1 protein.

32. The composition of claim 29, 30 or 31, which improves translation efficiency.

38. The 3' non-coding region adjacent to the codon sequence includes a polyadenosine tail, The number of adenosines in the adenosine tail determines the number of adenosines in the axonemal dynein intermediate chain 1 mRNA.

32. The composition of claim 29, 30 or 31, which has improved half-life.

39. 30. The polyadenosine tail has a length of at most 200 adenosines. Or the composition described in 31.

40. 29, 30 or 31, wherein a proportion of the polyadenosine tail comprises nucleotide analogs.

32. The composition according to claim 31.

41. 29. The method of claim 29, wherein less than 20% of the nucleic acid in the polyadenosine tail is a nucleotide analog.

32. The composition according to claim 30, 31 or 32.

42. 31. The construct of claim 29 or 30, wherein the construct comprises a proportion of nucleotide analogues. Composition.

43. Fewer than 30% of the nucleotides encoding axonemal dynein intermediate chain 1 are nucleotide analogs. The composition of claim 42.

44. Fewer than 20% of the nucleotides encoding axonemal dynein intermediate chain 1 are nucleotide analogs. The composition of claim 42.

45. 43. The method of claim 31 or 42, wherein the nucleotide analog is pseudouridine. Composition.

46. 31 or 42, wherein the nucleotide analog is 1-methylpseudouridine. The composition described in

47. 43. The method of claim 31 or 42, wherein the nucleotide analogue is 5-methoxyuridine. Composition of.

48. The nucleotide analogs are pseudouridine and 1-methylpseudouridine.

43. The composition of claim 31 or 42.

49. 29, 30 or 31, wherein the composition further comprises at least one additional nucleic acid construct.

32. The composition according to claim 31.

50. At least one additional nucleic acid construct is selected from the group consisting of armadillo repeat-containing 4 (ARMC4), 2 Chromosome 1 open reading frame 59 (C21orf59), coiled Main-containing 103 (CCDC103), coiled-coil domain-containing 114 (CCDC1 14), coiled-coil domain-containing 39 (CCDC39), coiled-coil domain-containing Coiled-coil domain-containing 40 (CCDC40), coiled-coil domain-containing 65 (CCDC65), CCNO, dynein (axoneme) assembly factor 1 (DNAAF1), dynein (axoneme) assembly factor 2 (DNAAF2), dynein (axoneme) assembly factor 2 (DNAAF2), dynein (axoneme) assembly factor 3 (DNAA F3), dynein (axonemal) assembly factor 5 (DNAAF5), axonemal dynein heavy chain 11 ( DNAH11), axonemal dynein heavy chain 5 (DNAH5), axonemal dynein heavy chain 6 (DNAH6 ), axonemal dynein heavy chain 8 (DNAH8), axonemal dynein intermediate chain 2 (DNAI2), axonemal dynein Dynein light chain 1 (DNAL1), dynein regulatory complex subunit 1 (DRC1), dyslexia sensitive 1 candidate 1 (DYX1C1), growth arrest specific 8 (GAS8), axonemal center pair apparatus Protein (HYDIN), leucine-rich repeat containing 6 (LRRC6), NME / NM 23 family member 8 (NME8), oral-facial-digital syndrome 1 (OFD1), retinal pigmentary dystrophy Role of GTPase Regulator (RPGR), Radial Spokehead 1 Homolog (Chlamydophila monas) (RSPH1), radial spoke head 4 homolog A (Chlamydomonas) (R SPH4A), radial spoke head 9 homolog (Chlamydomonas) (RSPH9), Sperm-associated antigen 1 (SPAG1) and zinc finger MYND type-containing 10 (ZMYND 50. The method of claim 49, wherein the gene encodes a protein selected from the group consisting of: 10).

51. 32. The method of claim 30 or 31, wherein the nucleic acid construct is further formulated for administration to a subject. The composition described.

52. The formulation comprises a therapeutically effective amount of the nucleic acid construct encoding axonemal dynein intermediate chain 1.

52. The composition of claim 51.

53. The nucleic acid construct encodes an axonemal dynein intermediate chain 1 protein or a mutant thereof.

32. The composition of claim 29, 30 or 31, comprising DNA.

54. 32. A vector comprising the nucleic acid construct of claim 29, 30 or 31.

55. 32. An isolated nucleic acid comprising the nucleic acid construct of claim 29, 30 or 31.

56. 1. A method for producing an exogenously encoded protein in a subject, comprising: Armadillo repeat containing 4 (ARMC4), an open region of chromosome 21, in the subject's cells Coiled-coil domain-containing 103 (C CDC103), coiled-coil domain-containing 114 (CCDC114), coiled coil domain coiled-coil domain-containing 39 (CCDC39), coiled-coil domain-containing 40 (CCDC40 ), coiled-coil domain-containing 65 (CCDC65), dynein (axoneme) assembly factor Dynein assembly factor 1 (DNAAF1), dynein (axoneme) assembly factor 2 (DNAAF2), dynein (axoneme) assembly factor 3 (DNAAF3), dynein (axoneme) assembly factor 5 (DNAF4), AAF5), axonemal dynein heavy chain 11 (DNAH11), axonemal dynein heavy chain 5 (DNAH5 ), axonemal dynein heavy chain 8 (DNAH8), axonemal dynein intermediate chain 2 (DNAI2), axonemal dynein Dynein light chain 1 (DNAL1), dynein regulatory complex subunit 1 (DRC1), dyslexia Sensitivity 1 candidate 1 (DYX1C1), axonemal central pair apparatus protein (HYDIN), leucine LRRC6, NME / NM23 family member 8 (NM E8), Orofacial-Digital Syndrome 1 (OFD1), Retinitis Pigmentosa GTPase Regulator (RP GR), radial spoke head 1 homolog (Chlamydomonas) (RSPH1), radial radial spoke head 4 homolog A (Chlamydomonas) (RSPH4A), radial spoke head 9 homolog (Chlamydomonas) (RSPH9), sperm associated antigen 1 (SPAG1) and and zinc finger MYND type-containing 10 (ZMYND10). administering to said subject a composition comprising a nucleic acid construct encoding a protein, The codons of the nucleic acid construct are optimized for expression of the protein in a cell of the subject. wherein, upon translation, said construct produces a polypeptide that treats said subject.

57. A nucleic acid construct encoding axonemal dynein intermediate chain 1 and comprising any one of SEQ ID NOs: 14 to 16. A composition comprising:

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