Treatment of hyperbilirubinemia

Abstract

The present invention relates to a nucleic acid sequence suitable for use in the treatment of hyperbilirubinemia, in particular the treatment of Crigler-Najjar syndrome. More particularly, the nucleic acid sequence of the present invention is a codon-optimized UGT1A1 coding sequence.

Description

[0001] This application is a divisional application of the international application date of April 27, 2015, international application number PCT / EP2015 / 059099, which entered the Chinese national phase on October 20, 2016, application number 201580020784.7, and invention title "Treatment of Hyperbilirubinemia". Technical Field

[0002] This invention relates to a nucleic acid sequence suitable for treating hyperbilirubinemia, particularly Crigler-Najjar syndrome. More specifically, the nucleic acid sequence of this invention is a codon-optimized human UGT1A1 coding sequence. Background Technology

[0003] Krieger-Najjar syndrome (CN) is an autosomal recessive disorder (OMIM#218800) characterized by severe unconjugated hyperbilirubinemia due to a deficiency of the bilirubin UDP-glucuronyltransferase isoenzyme 1A1 (UGT1A1), encoded by the UGT1A1 gene. CN has a birth incidence of approximately 1 in 1,000,000 individuals, making it an extremely rare disease. Current treatment for CN relies on phototherapy to prevent elevated serum bilirubin levels. For the mild form of the disease (also known as type II CN), phenobarbital can be used to reduce hyperbilirubinemia. However, patients are at risk of potentially reaching life-threatening peaks of bilirubin in the blood, and liver transplantation remains the only curative treatment. In the most severe form of the disease, it is fatal due to bilirubin-induced neurological damage unless phototherapy is initiated from birth. Despite the availability of treatments, CN remains an unmet medical need for many reasons, including the loss of efficacy of light therapy during growth, poor compliance due to the inherent limitations of light therapy (which requires continuous administration for 10-12 hours daily), and pathological liver changes over time that may require liver transplantation.

[0004] Different animal models of the disease exist, including the naturally occurring Gunn rat and a more recent knock-in mouse model developed by Dr. Muro of ICGEB in Trieste, Italy, carrying the same mutation present in Gunn rats (Bortolussi et al., 2012). Gunn rats exhibit high serum bilirubin levels and have cerebellar hypoplasia; CN mice have a more severe phenotype and die shortly after birth if not treated rapidly with phototherapy or gene therapy (Bortolussi et al., 2012).

[0005] Previous studies aimed at developing gene-based therapies for CN have shown therapeutic efficacy using AAV vectors delivered to the liver (Bortolussi et al., 2012; Seppen et al., 2006). However, a need remains for more efficient treatment strategies.

[0006] Gilbert's syndrome (GS; OMIM#218800) is a hereditary liver disorder and the most common genetic cause of elevated bilirubin. It occurs in up to 3–12% of the population. GS is also caused by mutations in the UGT1A1 gene. Therefore, treatment strategies aimed at alleviating hyperbilirubinemia will also be advantageously implemented in the treatment of GS. Summary of the Invention

[0007] This invention relates to a codon-optimized UGT1A1 coding sequence derived from human UGT1A1 cDNA. More specifically, the codon-optimized UGT1A1 coding sequence has an increased GC content and / or a reduced number of variable open reading frames compared to the wild-type human coding sequence SEQ ID NO:1. For example, the nucleic acid sequence of this invention results in an increase of at least 2, 3, 4, 5, or 10% in the GC content of the UGT1A1 sequence compared to the wild-type human UGT1A1 sequence. In a particular embodiment, the nucleic acid sequence of this invention results in an increase of 2, 3, 4, or more preferably 5% or 10% (preferably 5%) in the GC content of the UGT1A1 sequence compared to the wild-type human UGT1A1 sequence. In one specific embodiment, the nucleic acid sequence encoding the codon-optimized human UGT1A1 protein of the present invention is "substantially identical" to sequence SEQ ID NO:2 or SEQ ID NO:3, i.e., having about 70% identity, more preferably about 80% identity, even more preferably about 90% identity, even more preferably about 95% identity, even more preferably about 97%, 98%, or even 99% identity. In one specific embodiment, the present invention relates to a nucleic acid sequence encoding the codon-optimized human UGT1A1 protein, wherein the nucleic acid sequence comprises the sequence shown in SEQ ID NO:2 or SEQ ID NO:3.

[0008] Advantageously, the codon-optimized nucleic acids of the present invention provide improved bilirubin level reduction and / or reduced immunogenicity.

[0009] This invention also relates to a nucleic acid construct comprising the nucleic acid sequence of this invention. The nucleic acid construct may correspond to an expression cassette comprising the nucleic acid sequence of this invention, said nucleic acid sequence being operatively linked to one or more expression control sequences or other sequences that improve the expression of transgenes. Such sequences are known in the art, such as promoters, enhancers, introns, polyadenylate signals, etc. Specifically, the expression cassette may include a promoter. The promoter may be a ubiquitous promoter or a tissue-specific promoter, particularly a liver-specific promoter. More specifically, the promoter is a liver-specific promoter, such as the α-1 antitrypsin promoter (hAAT) (SEQ ID NO:4), the transthyretin promoter, the albumin promoter, the thyroxine-binding globulin (TBG) promoter, etc. Other suitable liver-specific promoters are known in the art, such as those listed in the liver-specific gene promoter database compiled by Cold Spring Harbor Laboratory (http: / / rulai.cshl.edu / LSPD / ). Representative ubiquitous promoters include the cytomegalovirus enhancer / chicken β-actin (CAG) promoter, cytomegalovirus enhancer / promoter (CMV), PGK promoter, SV40 early promoter, etc. In one particular embodiment, the promoter is associated with enhancer sequences such as the ApoE control region, such as the human ApoE control region (or the human apolipoprotein E / CI locus, liver control region HCR-1–Genbank accession number U32510, shown as SEQ ID NO:11). In one particular embodiment, enhancer sequences such as ApoE sequences are associated with liver-specific promoters such as those listed above, and particularly such as the hAAT promoter.

[0010] In one specific embodiment, the nucleic acid construct includes introns, particularly introns positioned between the promoter and the coding sequence. Introns can be introduced to increase mRNA stability and protein production. In one specific embodiment, the nucleic acid construct includes human β-globulin b2 (or HBB2) introns, coagulation factor IX (FIX) introns, SV40 introns, or chicken β-globulin introns. In one specific embodiment, the nucleic acid construct of the present invention contains modified introns (particularly modified HBB2 or FIX introns) designed to reduce the number of variable open reading frames (ARFs) found in said introns or even completely remove ARFs. Preferably, ARFs spanning more than 50 bp and having a stop codon in the same frame as the start codon are removed. ARFs can be removed by modifying the sequence of the intron. For example, modification can be performed by nucleotide substitution, insertion, or deletion, preferably by nucleotide substitution. As an example, one or more nucleotides, particularly one nucleotide, in the ATG or GTG start codon present in the sequence of the target intron can be substituted to produce a non-start codon. For instance, within the sequence of the target intron, the ATG or GTG can be substituted by the CTG, which is not a start codon.

[0011] The classical HBB2 intron used in nucleic acid constructs is shown in SEQ ID NO:5. For example, this HBB2 intron can be modified by eliminating the start codons (ATG and GTG codons) within the intron. In a particular embodiment, the modified HBB2 intron included in the construct has the sequence shown in SEQ ID NO:6. The classical FIX intron used in nucleic acid constructs is derived from the first intron of human FIX and is shown in SEQ ID NO:7. The FIX intron can be modified by eliminating the start codons (ATG and GTG codons) within the intron. In a particular embodiment, the modified FIX intron included in the construct of the present invention has the sequence shown in SEQ ID NO:8. The classical chicken β-globulin intron used in nucleic acid constructs is shown in SEQ ID NO:9. The chicken β-globulin intron can be modified by eliminating the start codons (ATG and GTG codons) within the intron. In one particular embodiment, the modified chicken β-globulin intron contained in the construct of the present invention has the sequence shown in SEQ ID NO:10.

[0012] The inventors have demonstrated that the modified introns, particularly the modified HBB2 or FIX introns, possess advantageous properties and can significantly improve transgene expression. Furthermore, by reducing the number of ARFs within the introns included in the constructs of the present invention, the immunogenicity of the constructs is believed to be reduced as well.

[0013] Therefore, the present invention also relates to an intron intended for use in an expression cassette and modified to increase the expression efficiency of a transgene placed in said cassette. Specifically, the present invention relates to a modified intron derived from a known intron, but wherein the number of ARFs has been reduced, or wherein the ARFs have been completely removed. In one particular embodiment, the present invention relates to a modified HBB2 intron having a reduced number of ARFs or no ARFs. In another particular embodiment, the modified HBB2 intron is the intron shown in SEQ ID NO:6. In another embodiment, the present invention relates to a modified FIX intron having a reduced number of ARFs or no ARFs. In another particular embodiment, the modified FIX intron is the intron shown in SEQ ID NO:8. In another embodiment, the present invention relates to a modified chicken β-globulin intron having a reduced number of ARFs or no ARFs. In another particular embodiment, the modified chicken β-globulin intron is the intron shown in SEQ ID NO:10. Another aspect of the invention relates to nucleic acid constructs, vectors such as viral vectors (particularly AAV vectors), and cells comprising introns modified according to the invention. The nucleic acid construct may include additional expression control sequences such as promoters and / or enhancers, as described herein and others. Modified introns, as disclosed herein, increase the expression efficiency of transgenes placed in the nucleic acid construct, such as target genes (e.g., therapeutic genes). In this aspect of the invention, a "therapeutic gene" generally refers to a gene encoding a therapeutic protein suitable for treating a pathological condition. A therapeutic gene, when expressed, confers a beneficial effect on the cells or tissues in which it is present or on the patient where the gene is expressed. Examples of beneficial effects include improving signs or symptoms of a condition or disease, preventing or suppressing a condition or disease, or providing a desired characteristic. Therapeutic genes include genes that partially or completely correct a patient's genetic defects. Specifically, a therapeutic gene may be (but is not limited to) a nucleic acid sequence encoding a protein suitable for alleviating a defect in gene therapy, the defect being caused by the absence, deficiency, or under-optimal level of said protein in the subject's cells or tissues. Therefore, this invention relates to introns containing the modifications of the present invention for use in gene therapy, as well as nucleic acid constructs, vectors such as viral vectors (especially AAV vectors), and cells that further contain a target therapeutic gene. The invention can generally be applied to therapies for any disease that can be treated by expressing a therapeutic gene in the cells or tissues of a subject.These diseases include, for example, proliferative disorders (cancer, tumors, dysplasia, etc.), infectious diseases; viral diseases (e.g., induced by hepatitis B or C virus, HIV, herpes, retroviruses, etc.), genetic diseases (cystic fibrosis, muscular dystrophy, proteoglycanopathy, myopathy such as Duchenne Muscular Myopathy; myotubular myopathy; hemophilia; sickle cell anemia, sickle cell disease, Fanconi's anemia; diabetes; amyotrophic lateral sclerosis, motor neuron diseases such as spinal muscular atrophy, spinobulbar muscular atrophy, or Charcot-Marie-Tooth disease; arthritis; severe combined immunodeficiency (such as RS-SCID, ADA-SCID, or X-SCID), Wiskott-Aldrich syndrome). Syndrome, X-linked thrombocytopenia, X-linked congenital neutropenia, chronic granulomatous disease, etc.; cardiovascular diseases (restenosis, ischemia, dyslipidemia, homozygous familial hypercholesterolemia, etc.); or neurological diseases (psychiatric disorders, neurodegenerative diseases such as Parkinson's disease or Alzheimer's disease, Huntington's disease, addiction (e.g., to tobacco, alcohol, or drugs), epilepsy, Canavan's disease, adrenoleukodystrophy, etc.); eye diseases such as retinitis pigmentosa, Leber congenital amaurosis, Leber hereditary optic neuropathy, Stargardt disease; lysosomal storage diseases such as San Filippo syndrome; hyperbilirubinemia such as type I or II CN or Gilbert's syndrome, Pompe disease. (disease), etc. As mentioned above, and further elaborated in the following disclosure, to achieve the expression of transgenic genes, such as therapeutic genes, in recipient host cells, it is preferably operatively linked to its own promoter or a heterologous promoter. Many suitable promoters are known in the art, and the selection of one depends on the desired expression level of the product encoded by the therapeutic gene; whether constitutive expression, cell-specific expression, or tissue-specific expression is required, etc. When the target gene is a therapeutic gene as defined above, a nucleic acid construct containing modified introns, a vector containing said nucleic acid construct, or a cell containing said construct or said vector can be further used in gene or cell therapy.

[0014] In one specific embodiment, the nucleic acid construct of the present invention is an expression cassette comprising, in the 5' to 3' direction, a promoter optionally pre-embedded with an enhancer, the codon-optimized UGT1A1 coding sequence of the present invention, and a polyadenylation signal. In another specific embodiment, the nucleic acid construct of the present invention is an expression cassette comprising, in the 5' to 3' direction, a promoter optionally pre-embedded with an enhancer (such as an ApoE control region), introns (particularly introns as defined above), the codon-optimized UGT1A1 coding sequence of the present invention, and a polyadenylation signal. In yet another specific embodiment, the nucleic acid construct of the present invention is an expression cassette comprising, in the 5' to 3' direction, an enhancer such as an ApoE control region, a promoter, introns (particularly introns as defined above), the codon-optimized UGT1A1 coding sequence of the present invention, and a polyadenylation signal.

[0015] This invention also relates to a vector comprising nucleic acid sequences as disclosed herein. Specifically, the vectors of this invention are vectors suitable for gene therapy. For example, the vector may be a plasmid vector. More specifically, the vector is a viral vector suitable for gene therapy that targets liver tissue or cells. In this case, the nucleic acid construct of this invention also contains sequences suitable for producing highly efficient viral vectors, as well as are well known in the art. In another specific embodiment, the viral vector is an AAV vector, such as AAV vectors suitable for transducing liver tissue or cells, more particularly AAV-1, AAV-2, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-rh10, AAV-rh74, AAV-dj, etc.; or a retroviral vector, such as a lentiviral vector. In another embodiment, the AAV vector comprises a genome that is single-stranded or self-complementary double-stranded. Preferably, for carrying out this invention, the AAV genome is single-stranded. As is known in the art, depending on the specific viral vector intended for use, suitable sequences will be introduced into the nucleic acid construct of this invention to obtain a functional viral vector. Suitable sequences include AAV ITRs for AAV vectors or LTRs for lentiviral vectors. Therefore, the present invention also relates to an expression cassette as described above, with ITRs or LTRs on each side.

[0016] In a particularly preferred embodiment, the present invention relates to an AAV vector comprising the nucleic acid construct of the invention in a single-stranded genome or a double-stranded self-complementary genome (e.g., a single-stranded genome). In a particular embodiment, the nucleic acid construct comprises the sequence shown in SEQ ID NO:2 or SEQ ID NO:3. In one embodiment, the AAV vector is an AAV8 vector. In another particular embodiment, the nucleic acid is operatively linked to a promoter, particularly a pervasive promoter or a liver-specific promoter. According to a particular variation, the promoter is a pervasive promoter, such as the cytomegalovirus enhancer / chicken β-actin (CAG) promoter, the cytomegalovirus enhancer / promoter (CMV), the PGK promoter, and the SV40 early promoter. In a particular variation, the pervasive promoter is the CAG promoter. According to another variation, the promoter is a liver-specific promoter, such as the α-1 antitrypsin promoter (hAAT), the transthyretin promoter, the albumin promoter, and the thyroxine-binding globulin (TBG) promoter. In one particular variation, the liver-specific promoter is the hAAT liver-specific promoter of SEQ ID NO:4. In another particular embodiment, the nucleic acid construct incorporated into the genome of the AAV vector of the present invention further comprises introns as described above, such as introns placed between the promoter and the nucleic acid sequence encoding the UGT1A1 protein. Representative introns that may be included in the nucleic acid construct introduced into the genome of the AAV vector include (but are not limited to) human β-globulin b2 (or HBB2) introns, FIX introns, and chicken β-globulin introns. The introns in the genome of the AAV vector may be classical (or unmodified) introns or modified introns designed to reduce the number of variable open reading frames (ARFs) within the introns or even completely remove variable open reading frames (ARFs). Modified and unmodified introns that can be used to implement this embodiment in which the nucleic acid of the present invention is introduced into the AAV vector have been fully described above. In one particular embodiment, the AAV vector (particularly the AAV8 vector) of the present invention includes modified (or optimized) introns within its genome, such as the modified HBB2 intron of SEQ ID NO:7, the modified FIX intron of SEQ ID NO:8, and the modified chicken β-globulin intron of SEQ ID NO:10.

[0017] This invention also relates to cells, such as hepatocytes, transformed with the nucleic acid sequences of this invention. The cells of this invention can be delivered to a subject by injection into the liver or bloodstream of the subject in need. In one particular embodiment, this invention relates to introducing the nucleic acid sequences of this invention into hepatocytes, particularly into the hepatocytes of a subject to be treated, and to administering said hepatocytes, in which the nucleic acid has been introduced, to the subject.

[0018] The present invention also provides a pharmaceutical composition comprising a combination of a nucleic acid selected from the present invention, a carrier of the present invention, or a cell active agent of the present invention and a pharmaceutically acceptable carrier.

[0019] This invention also relates to a method for treating hyperbilirubinemia caused by mutations in the UGT1A1 gene, comprising the step of delivering the nucleic acid, vector, pharmaceutical composition, or cells of the invention to a subject in need. In a particular embodiment, the hyperbilirubinemia is type I or II CN syndrome or Gilbert's syndrome.

[0020] The present invention also relates to nucleic acids, carriers, pharmaceutical compositions or cells of the present invention used as pharmaceutical agents.

[0021] This invention also relates to nucleic acids, vectors, pharmaceutical compositions, or cells of the invention in methods for treating hyperbilirubinemia caused by mutations in the UGT1A1 gene, particularly in methods for treating type I or II CN syndrome or Gilbert syndrome.

[0022] The present invention further relates to the use of the nucleic acids, vectors, pharmaceutical compositions or cell manufacturing of the present invention in a medicament suitable for treating hyperbilirubinemia caused by mutations in the UGT1A1 gene, particularly for treating type I or II CN syndrome or Gilbert syndrome. Attached Figure Description

[0023] Figure 1 The graph includes a view showing messenger RNA levels observed in Huh-7 cells transfected with plasmids expressing wild-type UGT1A1 or a two-codon optimized UGT1A1 sequence (Figure A) and a graph showing the quantification of UGT1A1 protein in the same sample by Western blotting (Figure B).

[0024] Figure 2 Figures show the effects of different intron optimizations on luciferase expression (Figure A) and the effects of HBB2 optimization on UGT1A1 RNA and protein expression levels (Figure B).

[0025] Figure 3 These are images of Western blot gels showing the expression of UGT1A1 protein from two vectors: one containing a codon-optimized UGT1A1 coding sequence and the other containing either a wild-type (UGT1A1 2.0) or an optimized (UGT1A1 2.1) HBB2 intron.

[0026] Figure 4 This is a diagram illustrating the electronic analysis of the variable reading frame (ARF) within the vectors of wild-type UGT1A1(A) and codon-optimized UGT1A1 v2.1(B).

[0027] Figure 5 This is a graph showing the total bilirubin (TB) levels measured weekly after injection of the codon-optimized UGT1A1 vector or PBS in different rat strains.

[0028] Figure 6 This demonstrates the effectiveness of injecting lower doses of the codon-optimized UGT1A1 vector into different rat strains (compared to...). Figure 5 The graph shows the total bilirubin (TB) levels measured weekly after PBS (data reported in the journal) or PBS.

[0029] Figure 7 Includes (A) a graph showing total bilirubin (TB) levels measured weekly after injection of the three UGT1A1 vectors (compared to... Figure 8 (a) Data reported in the report; (b) Photographs of Western blots of liver extracts obtained from rats treated with the three vectors and their relative quantifications; and (c) A graph presenting a long-term assessment of the efficacy of AAV8-v2.1 UGT1A1 in both male and female animals 4 months after continuous injection.

[0030] Figure 8 This is a graph showing the ability of different constructs to correct severe hyperbilirubinemia (total bilirubin, expressed in mg / dl) in a mouse model of Krieger-Najjar syndrome. Untreated animals (UNTR) are also reported. Detailed Implementation

[0031] The term "UGT1A1" refers to the wild-type human UDP-glycosyltransferase 1 family 1 polypeptide A (UGT1A1) cDNA shown in SEQ ID NO:1 (accession number NM_000463.2, i.e., the reference sequence of the CDS of human UGT1A1 mRNA; OMIM reference number 191740).

[0032] The term "codon optimization" refers to changing a codon that exhibits human bias (i.e., is common in human genes but not in other mammalian or non-mammalian genes) into a synonymous codon that does not exhibit human bias (a codon encoding the same amino acid). Therefore, the codon change does not result in any change in the amino acid encoded in the protein.

[0033] The sequences shown in SEQ ID NO:2 or SEQ ID NO:3, especially the sequence shown in SEQ ID NO:2, are preferred embodiments of the codon-optimized nucleic acid sequences of the present invention.

[0034] The DNA sequence changes resulting from codon optimization in SEQ ID NO:2 and SEQ ID NO:3 led to an increase of approximately 5% and approximately 10% in the GC content of the UGT1A1 sequence, respectively.

[0035] The present invention also covers a nucleic acid sequence of the present invention encoding a codon-optimized human UGT1A1 protein, which is “substantially identical” to sequence SEQ ID NO:2 or SEQ ID NO:3, i.e., having about 70% identity, more preferably about 80% identity, even more preferably about 90% identity, even more preferably about 95% identity, even more preferably about 97%, 98% or even 99% identity.

[0036] "Identity" refers to the sequence identity between two nucleic acid molecules. When a position in two compared sequences is occupied by the same base, for example, if a position in each of two DNA molecules is occupied by adenine, then the molecules are identical at that position. The percentage of identity between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of comparison positions multiplied by 100. For example, if 6 / 10 of the positions in two sequences match, then the two sequences are 60% identical. Typically, comparisons are made when the two sequences are aligned to produce maximum identity. Various bioinformatics tools known to those skilled in the art can be used to align nucleic acid sequences, such as BLAST or FASTA.

[0037] The term "reduced immunogenicity," as applied to codon-optimized UGT1A1 coding sequences or modified introns of this invention, means that, compared to wild-type cDNA or other UGT1A1 cDNA variants, this codon-optimized gene or modified intron contains a reduced number of potential variable open reading frames (ARFs) within the intron or coding sequence, or both, thereby limiting the number of potential translational protein byproducts, particularly from the encoded mRNA. Specifically, the reduced ARFs are those that span more than 50 bp and have a stop codon in the same frame as the start codon.

[0038] In the context of this invention, the term "gene therapy" refers to treatment of a subject involving the delivery of genes / nucleic acids into the individual's cells for the purpose of treating a disease. Gene delivery is typically achieved using delivery vectors, also known as vectors. Viral and non-viral vectors can be used to deliver genes into the patient's cells. AAV vectors, particularly AAV8 vectors, are particularly preferred.

[0039] It should be understood that the nucleic acids of the present invention may include one or more polyadenylation signals that are typically located at the 3'-terminus of the molecule.

[0040] Preferred vectors for delivering the nucleic acids of the present invention are viral vectors, such as retroviral vectors, for example lentiviral vectors; or non-pathogenic parvoviruses, more preferably AAV vectors. Human parvovirus adeno-associated virus (AAV) is a naturally replication-defective virus that can integrate into the genome of infected cells to produce latent infection. This last property appears to be unique among mammalian viruses because the integration occurs in the human genome at a specific site called AAVS1, located on chromosome 19 (19q13.3-qter).

[0041] Therefore, AAV has attracted considerable attention as a potential vector for human gene therapy. Among the advantageous properties of this virus are its lack of association with any human disease, its ability to infect both dividing and non-dividing cells, and its wide range of infectable cell lines derived from different tissues.

[0042] Of the AAV serotypes isolated from and fully characterized from humans or non-human primates (NHPs), human serotype 2 was the first AAV to be developed as a gene transfer vector. Other currently used AAV serotypes include AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAVrh74, and AAVdj. Furthermore, non-naturally engineered variants and chimeric AAVs are also suitable.

[0043] AAV viruses can be engineered using conventional molecular biology techniques, making it possible to optimize these particles to achieve cell-specific delivery of nucleic acid sequences, minimizing immunogenicity, tuning stability and particle lifetime, achieving efficient degradation, and accurately delivering them to the nucleus.

[0044] Suitable AAV fragments for assembly into vectors include cap proteins (including vp1, vp2, vp3, and hypervariable regions), rep proteins (including rep 78, rep 68, rep 52, and rep 40), and sequences encoding these proteins. These fragments are readily available for use in a variety of vector systems and host cells.

[0045] AAV-based recombinant vectors lacking the Rep protein integrate into the host genome inefficiently and exist primarily as stable circular exosomes that can persist in target cells for many years.

[0046] As an alternative to using natural AAV serotypes, artificial AAV serotypes can be used in the context of this invention, including (but not limited to) AAVs having capsid proteins that are not naturally present. The artificial capsid can be generated by any suitable technique using a combination of a selected AAV sequence (e.g., a fragment of the vp1 capsid protein) and a heterologous sequence obtainable from different selected AAV serotypes, discontinuous portions of the same AAV serotype, non-AAV viral sources, or non-viral sources. The artificial AAV serotype can be (but is not limited to) a chimeric AAV capsid, a recombinant AAV capsid, or a “humanized” AAV capsid.

[0047] Therefore, this invention relates to an AAV vector comprising the nucleic acid of the invention, which is a codon-optimized UGT1A1 coding sequence. In the case of this invention, the AAV vector comprises an AAV capsid capable of transducing target cells, particularly hepatocytes. According to one specific embodiment, the AAV vector has serotypes such as AAV-1, AAV-2, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-rh10, AAV-rh74, and AAV-dj. In another specific embodiment, the AAV vector is a pseudotype vector, i.e., its genome and capsid are derived from different serotypes of AAV. For example, a pseudotype AAV vector may be a vector whose genome is derived from serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAVrh74, or AAVdj, and whose capsid is derived from another serotype. For example, the genome of a pseudotype vector may be derived from serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV10, AAVrh10, AAVrh74, or AAVdj, and its capsid may be derived from serotypes AAV8 or AAV9, particularly AAV8.

[0048] In another embodiment, the capsid is a modified capsid. In the context of this invention, a "modified capsid" may be a chimeric capsid or a capsid comprising one or more variant VP capsid proteins derived from one or more wild-type AAV VP capsid proteins.

[0049] In one particular embodiment, the AAV vector is a chimeric vector, i.e., its capsid contains VP capsid proteins derived from at least two different AAV serotypes, or contains at least one chimeric VP protein that combines VP protein regions or domains derived from at least two AAV serotypes. Examples of chimeric AAV vectors suitable for transducing hepatocytes are described in Shen et al., Molecular Therapy, 2007 and Tenney et al., Virology, 2014. For example, a chimeric AAV vector may be obtained by combining an AAV8 capsid sequence with sequences of serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV9, AAV10, AAVrh10, AAVrh74, or AAVdj. In another embodiment, the capsid of the AAV vector contains one or more variant VP capsid proteins, such as those described in WO2015013313, particularly the RHM4-1, RHM15-1, RHM15-2, RHM15-3 / RHM15-5, RHM15-4 and RHM15-6 capsid variants, which exhibit high hepaticity.

[0050] In another embodiment, the modified capsid may also be obtained by capsid modification via error-prone PCR and / or peptide insertion (e.g., as described in Bartel et al., 2011). Furthermore, capsid variants may include single-amino acid changes, such as tyrosine mutants (e.g., as described in Zhong et al., 2008).

[0051] Furthermore, the genome of the AAV vector can be a single-stranded genome or a self-complementary double-stranded genome (McCarty et al., Gene Therapy, 2003). Self-complementary double-stranded AAV vectors are generated by deleting the terminal dissociation site (trs) from an AAV terminal repeat sequence. These modified vectors, whose replicative genome is half the length of the wild-type AAV genome, have a tendency to package DNA dimers. In a preferred embodiment, the AAV vector used in carrying out the invention has a single-stranded genome and further preferably contains an AAV8, AAV2, or AAV5 capsid, more preferably containing an AAV8 capsid.

[0052] In addition to the specific delivery systems illustrated in the examples below, various delivery systems are known and can be used to administer the nucleic acids of the present invention, such as those encapsulated in liposomes, microparticles, microcapsules, recombinant cells capable of expressing codon-optimized UGT1A1 coding sequences, receptor-mediated endocytosis, and constructing therapeutic nucleic acids as part of retroviral vectors or other vectors. Methods of administering the nucleic acids include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The nucleic acid sequences of the present invention, whether or not vectorized, can be administered via any suitable route, such as by infusion or bolus injection, by absorption through epithelial or mucosal skin linings (e.g., oral mucosa, rectal mucosa, and intestinal mucosa), and can be administered together with other bioactive agents. Administration can be systemic or local. Furthermore, it is desirable to introduce the pharmaceutical compositions of the present invention into the liver of a subject via any suitable route. Additionally, naked DNA such as small loops and transposons can be used to deliver lentiviral vectors. In addition, gene editing technologies such as zinc finger nucleases, megabase-wide nucleases, TALEN, and CRISPR can also be used to deliver the coding sequences of this invention.

[0053] In one particular embodiment, it may be desirable to apply the pharmaceutical composition of the present invention topically to the area requiring treatment, namely the liver. This can be achieved, for example, by means of an implant, which is a porous, non-porous, or gel-like material, including membranes (such as silicone rubber membranes) or fibers.

[0054] In another embodiment, the nucleic acid of the present invention can be delivered in vesicles, particularly liposomes.

[0055] In another embodiment, the nucleic acid of the present invention can be delivered in a controlled release system.

[0056] This invention also provides pharmaceutical compositions comprising the nucleic acids of this invention, the carriers of this invention, or the cells of this invention. The compositions comprise a therapeutically effective amount of a therapeutic agent (the nucleic acid, carrier, or cell of this invention) and a pharmaceutically acceptable carrier. In a particular embodiment, the term "pharmaceutical acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia or European Pharmacopoeia or other generally recognized pharmacopoeia for use in animals and humans. The term "carrier" refers to a diluent, adjuvant, excipient, or medium with which the therapeutic agent is administered. The pharmaceutical carrier may be a sterile liquid, such as water and oil, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextran and glycerol solutions may also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, etc.

[0057] If necessary, the composition may also contain small amounts of wetting agents, emulsifiers, or pH buffers. These compositions may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. Oral formulations may include standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable drug carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. The composition will contain a therapeutically effective amount of a preferably purified therapeutic agent and a suitable carrier to provide a form suitable for appropriate administration to a subject. In one particular embodiment, the nucleic acid, carrier, or cell of the invention is formulated into a composition comprising phosphate-buffered saline supplemented with 0.25% human serum albumin. In another specific embodiment, the nucleic acids, vectors, or cells of the present invention are formulated into a composition comprising a ringer lactate solution and a nonionic surfactant such as pluronic F68, wherein the nonionic surfactant is at a final concentration of 0.01-0.0001% by weight of the total composition, such as at a concentration of 0.001%. The formulation may further comprise serum albumin, particularly human serum albumin, such as 0.25% human serum albumin. Other suitable formulations for storage or administration are known in the art, particularly from WO 2005 / 118792 or Ally et al., 2011.

[0058] In a preferred embodiment, the composition is formulated according to conventional procedures to be a pharmaceutical composition suitable for intravenous administration to humans. Typically, the composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizer and a local anesthetic such as lidocaine to reduce pain at the injection site.

[0059] The amount of the therapeutic agent (i.e., nucleic acid, vector, or cell) of the present invention that will effectively treat CN syndrome can be determined using standard clinical techniques. In addition, in vivo and / or in vitro assays may optionally be used to help predict the optimal dose range. The precise dose to be used in the formulation will also depend on the route of administration and the severity of the disease, and should be determined based on the practitioner's judgment and the individual patient's condition. The dose of nucleic acid, vector, or cell administered to the subject in need will vary based on several factors, including (but not limited to) the route of administration, the specific disease being treated, the subject's age, or the expression level necessary to achieve the desired therapeutic effect. Those skilled in the art can readily determine the required dose range based on these and other factors based on their knowledge in the art. In cases involving the administration of a viral vector, such as an AAV vector, to the subject, a typical dose of the vector is at least 1 x 10⁻⁶ per kilogram of body weight. 8 One vector genome (vg / kg), such as at least 1x10 9 vg / kg, at least 1x10 10 vg / kg, at least 1x10 11 vg / kg, at least 1x10 12 At least 1 x 10 vg / kg 13 vg / kg or at least 1x10 14 vg / kg.

[0060] Scope: Throughout this disclosure, various aspects of the invention may be presented in the form of scope. It should be understood that the description in the form of scope is for convenience and brevity only and should not be construed as rigidly limiting the scope of the invention. Therefore, a description of scope should be regarded as explicitly disclosing all possible sub-scopes and individual values ​​within that scope. For example, a description of a scope such as 1 to 6 should be regarded as explicitly disclosing sub-scopes such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and individual values ​​within that scope such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the scope.

[0061] All patents, patent applications and publications cited in this article are incorporated herein in their entirety by reference.

[0062] Without further description, it is believed that those skilled in the art can prepare and utilize the compounds of the present invention and implement the claimed methods using the previously described and the following illustrative examples.

[0063] Example

[0064] The invention will be further described in detail with reference to the following experimental examples and accompanying drawings. These examples are provided for illustrative purposes only and are not intended to be limiting.

[0065] Materials and methods

[0066] Codon optimization and AAV vector construction:

[0067] UGT1A1 underwent codon optimization according to several different algorithms. Additionally, the removal of cryptic transcription start sites was implemented throughout the construct. The resulting construct was introduced into expression plasmids or packaged into AAV serotype 8 vectors, and its potency was tested in vitro and in vivo (rat and mouse).

[0068] The following abbreviations will be used for these constructs throughout this experimental section:

[0069] -WT.0: Wild-type UGT1A1 transgene and wild-type HBB2 intron (SEQ ID NO:5);

[0070] -WT: Wild-type UGT1A1 transgenic and optimized form of HBB2 intron with some ARF removed (SEQ ID NO:6);

[0071] -v2 (or v2.0): Contains the codon-optimized UGT1A1 transgenic form 2.0 (SEQ ID NO:2) and the wild-type HBB2 intron (SEQ ID NO:5);

[0072] -v2.1: Contains codon-optimized UGT1A1 transgenic form 2.0 (SEQ ID NO:2) and HBB2 introns of optimized form with some ARFs removed (SEQ ID NO:6);

[0073] -v3: Contains codon-optimized UGT1A1 transgenic form 3 (SEQ ID NO:3) and HBB2 introns of an optimized form with some ARFs removed (SEQ ID NO:6).

[0074] -AAV8-hAAT-wtUGT1A1: An AAV8 vector containing the WT construct, which, under the control of the hAAT promoter, transgenerates wild-type UGT1A1;

[0075] -AAV8-hAAT-coUGT1A1v2: An AAV8 vector containing the v2 construct, under the control of the hAAT promoter;

[0076] -AAV8-hAAT-coUGT1A1v2.1: An AAV8 vector containing the v2.1 construct, under the control of the hAAT promoter;

[0077] -AAV8-hAAT-coUGT1A1v3: An AAV8 vector containing the v3 construct, wild-type under the control of the hAAT promoter.

[0078] In vitro assay:

[0079] Human hepatocyte cell line Huh7 was transduced at increasing multiples of infection (MOI) of 0, 5000 (5), 10000 (10), or 25000 (25), or transfected with indicated plasmid vectors and cationic lipofectamine. Forty-eight hours after transduction, cells were collected, lysed, and microsomal extracts were prepared and loaded onto Western blots, with a polyclonal antibody against human UGT1 used for protein detection. Constitutively expressed protein calnexin was used as a loading control.

[0080] A portion of the cells used for microsome preparation was used to extract mRNA with triazole. The extracted mRNA was treated with DNase, reverse transcribed, and analyzed by RT-PCR using oligonucleotide primers specific to the UGT1A1 sequence. Oligonucleotide primers specific to human serum alkaline phosphatase were used for normalization.

[0081] Electronic Analysis:

[0082] Variable reading frame (ARF) analysis was performed on the coding strands of the two UGT1A1 sequences using the ORF analysis tool available in VectorNTI software (Life Technologies). Typical start and stop sites for eukaryotic cells were used (ATG as the start site and TAA, TGA, and TAG as the stop sites, respectively). ARFs were considered when their length exceeded 50 bp and they had a stop codon in the same frame as the start codon.

[0083] animal:

[0084] Gunn rats exhibiting a UGT1A1 gene defect were injected with the vector at 6–8 weeks of age. The vector was delivered via the tail vein in a 0.5 ml volume. Serum samples were collected weekly to monitor total bilirubin (TB) levels. Untreated affected animals and wild-type or healthy littermates served as controls.

[0085] Ugt1 mutant mice with a C57Bl / 6 background have been previously generated (Bortolussi et al., 2012). Wild-type littermates were used as controls. Mice were housed and treated in accordance with institutional guidelines and experimental procedures approved by the local ethics committee and relevant regulatory authorities, taking into full consideration EU Directive 2010 / 63 / EU on animal experiments. The genetic mutation in the Ugt1a gene was transferred into the FVB / NJ mouse strain. Animals used in this study were obtained after more than nine backcrosses with C57Bl / 6 mice and FVB / NJ mice, respectively, and had at least 99.8% C57Bl / 6 or FVB / NJ genetic background. Mice were kept in a temperature-controlled environment with a 12 / 12-hour light-dark cycle. They were given a standard diet and water with free access. The vector was injected intraperitoneally on day 2 after birth (P2), and bilirubin levels were measured 4 weeks after the injection.

[0086] AAV dosage:

[0087] The dosage of the applied carrier is indicated in the legend.

[0088] Rat serum preparation:

[0089] Blood samples were collected weekly via puncture of the posterior orbital sinus into a dry syringe. The blood was centrifuged at 8000 rpm at 4°C, aliquoted, and frozen at -20°C.

[0090] Preparation of mouse plasma:

[0091] Four weeks after injection into both mutant and wild-type littermates, blood samples were collected via cardiac puncture into an EDTA collection syringe. Blood was centrifuged at 2500 rpm, plasma was collected, aliquoted, and frozen at -80°C. All procedures were performed in the dark to prevent bilirubin degradation.

[0092] Bilirubin determination in rats:

[0093] Serum total bilirubin was determined using the bilirubin assay kit (Abnova, reference number KA1614) as described by the manufacturer. We used a 50 μL volume of serum for analysis. Absorbance values ​​were obtained at 530 nm using a multi-plate reader (PerkinElmerEnSpire).

[0094] Bilirubin determination in mice:

[0095] As described by the manufacturer, plasma total bilirubin was determined using the Direct and Total Bilirubin Kit (BQ Kits, San Diego, CA) with the following minor modifications: the reaction was scaled down and the reaction was performed with only 10 μl of plasma at a final volume of 300 μl (instead of 6000 μl). Three commercial bilirubin reference standards (control serum I, control serum II, and bilirubin calibrator, Diazyme Laboratories, Poway, CA, USA) were included as quality controls in each assay. Absorbance values ​​at 560 nm were obtained using a multi-plate reader (Perkin Elmer Envision plate reader, Walthman, MA, USA).

[0096] Western blot of liver extract:

[0097] Rapidly homogenized frozen livers obtained from animals injected with one of three vectors were homogenized. The homogenate was used for microsome preparation. The microsomal extract was then loaded onto a Western blot, with a polyclonal antibody against human UGT1 used for protein detection. Protein bands were quantified.

[0098] result

[0099] The codon-optimized human UGT1A1 coding sequence was generated and introduced into an expression plasmid. Two optimized UGT1A1 coding sequences (v2 and v3 sequences) and the wild-type sequence were transfected into Huh-7 cells. The results are reported in [Journal Name]. Figure 1 This experiment shows that the two-codon optimized sequence is translated more efficiently in vitro in human cells than the wild-type sequence.

[0100] exist Figure 2 Figure A shows the luciferase levels produced in Huh-7 cells by transfection with a plasmid expressing luciferase under transcriptional control of the hAAT promoter. Different intron sequences were cloned at the 5' of the luciferase coding sequence. Two of them, the HBB2 and FIX introns, were optimized by removing an ARF from the sequence, which was performed by replacing a nucleotide in the ATG codon identified in the wild-type sequence of the intron. Expression of the optimized construct in hepatocyte lines showed that removal of the ARF from the intron sequence increased luciferase expression in vitro in both cases, with the optimized HBB2 intron being particularly effective. In Figure B, two plasmids expressing UGT1A1 under transcriptional control of the hAAT promoter are compared. V2.0 contains the wild-type HBB2 intron, while v2.1 contains the optimized form. The data shown indicate that plasmid v2.1 expresses 50% more UGT1A1 than v2.0, without any increase in mRNA levels.

[0101] In vitro testing was conducted on codon-optimized UGT1A1 forms 2.0 and 2.1AAV8 vectors (UGT1A1 2.0 and UGT1A1 2.1, respectively). The UGT1A1 2.0 and UGT1A1 2.1 vectors differ only in that they contain either a wild-type HBB2 intron (SEQ ID NO:5) or a modified HBB2 intron with ARF removed (SEQ ID NO:6), respectively. The results are reported in [Journal Name]. Figure 3 This experiment shows that the codon-optimized UGT1A1 vector form 2.1 is more effective in human cells in vitro than form 2.0.

[0102] Figure 4 The results of electronic analysis of variable reading frames (ARFs) within the wild-type UGT1A1(A) and the codon-optimized UGT1A1v2.1(B) vectors are shown. Compared to the wild-type sequence, the v2.1 vector has only a limited number of ARFs, and they are mainly in the opposite direction relative to the promoter. Furthermore, in Figure 4 In the diagram, we can see that ARF9 and ARF10, which are normally present in the HBB2 intron (used in the wild-type UGT1A1 construct represented in A), have been removed from the modified HBB2 intron of SEQ ID NO: 6 introduced into the UGT1A1v2.1 optimized vector.

[0103] Next, in 5x10 12 The codon-optimized AAV8-hAAT-coUGT1A1v2.1 vector was administered at a dose of vg / kg. The vector was administered via tail vein injection in 6-week-old homozygous Gunn rats (UGT1A1- / -). Figure 5 The figure shows total bilirubin (TB) levels measured weekly in wild-type (WT, gray line), heterozygous (UGT1A1+ / -, dotted line), and homozygous (black line) Gunn rats after injection and after PBS injection. All data are expressed as mean ± SE. Injection of codon-optimized vectors resulted in complete correction of the disease phenotype.

[0104] Also in 5x10 11 The AAV8-hAAT-UGT1A1v2.1 vector was administered at a dose of vg / kg. Administration was carried out via tail vein injection in 6-week-old homozygous Gunn rats (UGT1A1- / -). Figure 6 The figure shows total bilirubin (TB) levels measured weekly in wild-type (WT, gray line), heterozygous (UGT1A1+ / -, dotted line), and homozygous (black line) Gunn rats after injection and after PBS injection. All data are expressed as mean ± SE. Injection of codon-optimized vectors resulted in complete correction of the disease phenotype.

[0105] In 5x10 11 Two codon-optimized vectors (v2.1 and v3) and the wild-type AAV8-hAAT-UGT1A1 vector were further administered at a dose of vg / kg. The vectors were administered via tail vein injection in 6-week-old homozygous Gunn rats (UGT1A1- / -). Figure 7 Graph A shows the total bilirubin (TB) levels measured weekly after injection. All data are expressed as mean ± SE. Figure 7 As shown in Figure A, injection of the three vectors resulted in complete correction of the disease phenotype. Two months after injection, the animals were sacrificed, and the UGT1A1 protein level in the liver homogenate was quantified by Western blotting. Figure B shows a photograph of the Western blot obtained using an antibody specific for UGT1A1 protein. Quantification of the bands showed an increase in the amount of UGT1A1 protein in rats treated with AAV8-hAAT-coUGT1A1v2.1, even though the difference was not significant due to the high variability in expression levels observed in different animals.

[0106] Already using 5x10 12 Long-term efficacy was evaluated in 2-month-old Gunn rats injected with the AAV8-v2.1 UGT1A1 vector at a dose of vg / kg. Four months post-injection, the mean serum bilirubin level was 1.75 mg / dL in male rats (initial level at D0: 7.49, a 77% reduction) and 0.85 mg / dL in female rats (initial level at D0: 6.15 mg / dL, an 86% reduction). This result, indicating a long-term correction to the phenotype, is particularly striking compared to previous studies by Pastore et al., who reported only a 50% reduction in baseline bilirubin levels in female rats using different constructs. In summary, the data presented indicate that the vector produced by the method of this invention applied to AAV8-hAAT-coUGT1A1v2.1 has better in vivo efficacy than other vectors developed to cure CN.

[0107] We also tested the efficacy of total bilirubin correction in a mouse model of Krieger-Najjar syndrome. Figure 8 This is a graph showing the total bilirubin (TB) level one month after injection. Animals were injected with a dose of 3E10 vg / mouse on day 2 after birth (P2).

[0108] Untreated affected animals that survived 15 days of light therapy were used as controls (UNTR(PT)).

[0109] This experiment showed that, among all vectors, the 2.1 form produced the highest TB correction level. All data are expressed as mean ± SD. Each point represents a single animal.

[0110] References

[0111] Allay et al. Hum Gene Ther. May 2011; 22(5):595-604

[0112] Bartel et al., Front Microbiol. October 4, 2011; 2:204

[0113] Bortolussi et al., FASEB J. March 2012; 26(3):1052-63

[0114] McCarty et al., Gene Ther. December 2003; 10(26):2112-8

[0115] Pastore et al., Mol Ther., May 2013; Vol. 21; Supplement 1; S192-3 (Abstract No. 499)

[0116] Seppen et al., Mol Ther. June 2006; 13(6):1085-92

[0117] Shen et al., Mol Ther. November 2007; 15(11):1955-62

[0118] Tenney et al., Virology. April 2014; 454-455:227-36

[0119] Zhong et al., Proc Natl Acad Sci US A. June 3, 2008; 105(22):7827-32. sequence list <110> Genethanon International Center for Genetic Engineering and Biotechnology <120> Treatment of hyperbilirubinemia <130> SPI216269-91 <160> 11 <170> PatentIn version 3.5 <210> 1 <211> 1602 <212> DNA <213> Homo sapiens <400> 1 atggctgtgg agtcccaggg cggacgccca cttgtcctgg gcctgctgct gtgtgtgctg 60 ggcccagtgg tgtcccatgc tgggaagata ctgttgatcc cagtggatgg cagccactgg 120 ctgagcatgc ttggggccat ccagcagctg cagcagaggg gacatgaaat agttgtccta 180 gcacctgacg cctcgttgta catcagagac ggagcatttt acaccttgaa gacgtaccct 240 gtgccattcc aaagggagga tgtgaaagag tcttttgtta gtctcgggca taatgttttt 300 gagaatgatt ctttcctgca gcgtgtgatc aaaacataca agaaaataaa aaaggactct 360 gctatgcttt tgtctggctg ttcccactta ctgcacaaca aggagctcat ggcctccctg 420 gcagaaagca gctttgatgt catgctgacg gaccctttcc ttccttgcag ccccatcgtg 480 gcccagtacc tgtctctgcc cactgtattc ttcttgcatg cactgccatg cagcctggaa 540 tttgaggcta cccagtgccc caacccattc tcctacgtgc ccaggcctct ctcctctcat 600 tcagatcaca tgaccttcct gcagcgggtg aagaacatgc tcattgcctt ttcacagaac 660 tttctgtgcg acgtggttta ttccccgtat gcaacccttg cctcagaatt ccttcagaga 720 gaggtgactg tccaggacct attgagctct gcatctgtct ggctgtttag aagtgacttt 780 gtgaaggatt accctaggcc catcatgccc aatatggttt ttgttggtgg aatcaactgc 840 cttcaccaaa atccactatc ccaggaattt gaagcctaca ttaatgcttc tggagaacat 900 ggaattgtgg ttttctcttt gggatcaatg gtctcagaaa ttccagagaa gaaagctatg 960 gcaattgctg atgctttggg caaaatccct cagacagtcc tgtggcggta cactggaacc 1020 cgaccatcga atcttgcgaa caacacgata cttgttaagt ggctacccca aaacgatctg 1080 cttggtcacc cgatgacccg tgcctttatc acccatgctg gttcccatgg tgtttatgaa 1140 agcatatgca atggcgttcc catggtgatg atgcccttgt ttggtgatca gatggacaat 1200 gcaaagcgca tggagactaa gggagctgga gtgaccctga atgttctgga aatgacttct 1260 gaagatttag aaaatgctct aaaagcagtc atcaatgaca aaagttacaa ggagaacatc 1320 atgcgcctct ccagccttca caaggaccgc ccggtggagc cgctggacct ggccgtgttc 1380 tgggtggagt ttgtgatgag gcacaagggc gcgccacacc tgcgccccgc agcccacgac 1440 ctcacctggt accagtacca ttccttggac gtgattggtt tcctcttggc cgtcgtgctg 1500 acagtggcct tcatcacctt taaatgttgt gcttatggct accggaaatg cttggggaaa 1560 aaagggcgag ttaagaaagc ccacaaatcc aagacccatt ga 1602 <210> 2 <211> 1602 <212> DNA <213> Artificial <220> <223> Optimized UGT1A1 v2.1 <400> 2 atggctgtgg aatcacaagg aggtagacca ctggttctcg gacttttgct ttgcgtgctg 60 gggcccgtgg tgtcgcatgc cggaaagatc ctgctgatcc cggtggatgg atcacactgg 120 ctgtccatgc tgggtgccat ccaacagctc cagcagcggg gccacgaaat tgtggtcctg 180 gccccggacg cttccctgta tattcgggac ggagcgttct acactctcaa gacctaccct 240 gtccccttcc aaagggagga cgtgaaggaa agctttgtgt cgctggggca taatgtgttc 300 gagaacgaca gcttcctcca aagggttatt aaaacctaca agaagatcaa aaaggattcg 360 gccatgctcc tttccggatg ttcacacctg ttgcataaca aggaattgat ggccagcctg 420 gcagaatcca gctttgacgt catgcttact gacccgttct tgccttgctc cccgattgtg 480 gcccaatacc tgtcgctccc aaccgtgttc ttcctgcacg ccttgccttg ttcgctggaa 540 ttcgaagcga ctcagtgtcc caatccgttc tcctacgtcc cgcgcccgct ttcaagccat 600 tcggatcaca tgactttcct ccagcgcgtc aagaacatgc tcattgcgtt cagccagaac 660 tttctgtgcg acgtggttta ctcaccttac gctaccttgg cttctgagtt cctgcagaga 720 gaagtgactg tgcaagatct gctgtcctca gcgtccgttt ggttgttccg gtctgacttc 780 gtcaaggact acccgcgccc gatcatgccg aatatggtct ttgtgggcgg tatcaactgc 840 ctgcatcaaa acccactgag ccaggagttt gaggcgtaca tcaacgcctc gggagagcat 900 ggaatcgtgg tgttctccct cggttccatg gtgtccgaga tcccggaaaa gaaggcaatg 960 gccatcgcag atgccctggg caaaatcccg cagaccgtgc tctggcgcta cacgggtact 1020 cggcctagca atttggcaaa caacaccatc ctggtgaaat ggctgccgca gaacgacctc 1080 ctgggccacc caatgactcg cgctttcatt acccatgcgg gctcgcacgg agtctacgaa 1140 tccatctgca atggagtccc gatggtgatg atgccacttt tcggagatca gatggataat 1200 gcaaaaagaa tggaaaccaa gggggccgga gtgacgctga acgtgcttga aatgacctcg 1260 gaagatctgg agaacgctct caaagcggtg atcaacgaca agtcctacaa ggaaaacatc 1320 atgcgcctga gctccctcca caaggaccga ccagtggaac cgctggacct cgcggtcttt 1380 tgggtggagt tcgtgatgag gcacaagggc gccccccacc tcagacccgc agctcatgac 1440 ctcacttggt accagtacca ttcgctggat gtcatcggct ttctcctggc ggtcgtgctc 1500 accgtggcgt tcatcacctt caagtgctgc gcctacggat atcgcaaatg cttggggaag 1560 aaaggacggg tgaagaaggc acacaagtca aagacgcact ga 1602 <210> 3 <211> 1602 <212> DNA <213> Artificial <220> <223> Optimized UGT1A1 v3 <400> 3 atggccgtgg aatctcaggg cggcagacct ctggtgctgg gcctgctgct gtgtgtgctg 60 ggacctgtgg tgtctcacgc cggcaagatc ctgctgatcc ccgtggatgg cagccactgg 120 ctgtctatgc tgggcgccat tcagcagctg cagcagaggg gccacgagat cgtggtgctg 180 gcccctgatg ccagcctgta catcagagat ggcgccttct acaccctgaa aacctacccc 240 gtgcccttcc agcgcgagga cgtgaaagaa agcttcgtgt ccctgggcca caacgtgttc 300 gagaacgaca gcttcctgca gagagtgatc aagacctaca agaagatcaa gaaagacagc 360 gccatgctgc tgagcggctg ctcccatctg ctgcacaaca aagaactgat ggcctccctg 420 gccgagagca gcttcgacgt gatgctgacc gacccattcc tgccctgcag ccctatcgtg 480 gcccagtacc tgagcctgcc taccgtgttc ttcctgcacg ccctgccttg ctccctggaa 540 ttcgaggcca cccagtgccc caaccccttc agctacgtgc ccagaccact gagcagccac 600 agcgaccaca tgacctttct gcagcgcgtg aagaacatgc tgatcgcctt cagccagaac 660 ttcctgtgcg acgtggtgta cagcccctac gctaccctgg ccagcgaatt cctgcagcgg 720 gaagtgaccg tgcaggacct gctgtctagc gccagcgtgt ggctgttccg cagcgacttc 780 gtgaaggact accccagacc catcatgccc aacatggtgt tcgtgggcgg catcaactgc 840 ctgcaccaga accccctgag ccaggaattt gaggcctaca tcaacgccag cggcgagcac 900 ggcatcgtgg tgtttagcct gggcagcatg gtgtccgaga tccccgagaa aaaggccatg 960 gctatcgccg acgccctggg aaagatcccc cagacagtgc tgtggcggta caccggcacc 1020 agacccagca acctggccaa caacaccatc ctcgtgaaat ggctgcccca gaacgacctg 1080 ctgggccacc ctatgacccg ggcctttatc acacacgccg gctcccatgg cgtgtacgag 1140 agcatctgca acggcgtgcc catggtcatg atgcccctgt tcggcgacca gatggacaac 1200 gccaaggga tggaaaaa gggcgctggc gtgaccctga acgtgctgga aatgaccagc 1260 gaggcctgg aaaacgccct gaaggccgtg atcaacgaca agagctacaa agaaaacatc 1320 atgcggctgt ccagcctgca caaggacaga cccgtggaac ccctggacct ggccgtgttc 1380 tgggtggaat tcgtgatgcg gcacaagggc gctccccatc tgaggcctgc agctcacgac 1440 ctgacctggt atcagtacca cagcctggac gtgatcggct tcctgctggc agtggtgctg 1500 accgtggcct tcatcacctt caagtgctgc gcctacggct accggaagtg cctgggcaag 1560 aaaggcagag tgaagaaggc ccacaagagc aagacccact ga 1602 <210> 4 <211> 397 <212> DNA <213> Homo sapiens <400> 4 gatcttgcta ccagtggaac agccactaag gattctgcag tgagagcaga gggccagcta 60 agtggtactc tcccagagac tgtctgactc acgccacccc ctccaccttg gacacaggac 120 gctgtggttt ctgagccagg tacaatgact cctttcggta agtgcagtgg aagctgtaca 180 ctgcccaggc aaagcgtccg ggcagcgtag gcgggcgact cagatcccag ccagtggact 240 tagcccctgt ttgctcctcc gataactggg gtgaccttgg ttaatattca ccagcagcct 300 cccccgttgc ccctctggat ccactgctta aatacggacg aggacagggc cctgtctcct 360 cagcttcagg caccaccact gacctgggac agtgaat 397 <210> 5 <211> 441 <212> DNA <213> Artificial <220> <223> HBB2 intron <400> 5 gtacacatat tgaccaaatc agggtaattt tgcatttgta attttaaaaa atgctttctt 60 cttttaatat acttttttgt ttatcttatt tctaatactt tccctaatct ctttctttca 120 gggcaataat gatacaatgt atcatgcctc tttgcaccat tctaaagaat aacagtgata 180 atttctgggt taaggcaata gcaatatttc tgcatataaa tatttctgca tataaattgt 240 aactgatgta agaggtttca tattgctaat agcagctaca atccagctac cattctgctt 300 ttattttatg gttgggataa ggctggatta ttctgagtcc aagctaggcc cttttgctaa 360 tcatgttcat acctcttatc ttcctcccac agctcctggg caacgtgctg gtctgtgtgc 420 tggcccatca ctttggcaaa g 441 <210> 6 <211> 441 <212> DNA <213> Artificial <220> <223> Modified HBB2 intron <400> 6 gtacacatat tgaccaaatc agggtaattt tgcatttgta attttaaaaa atgctttctt 60 cttttaatat acttttttgt ttatcttatt tctaatactt tccctaatct ctttctttca 120 gggcaataat gatacaatgt atcatgcctc tttgcaccat tctaaagaat aacagtgata 180 atttctgggt taaggcaata gcaatatttc tgcatataaa tatttctgca tataaattgt 240 aactgatgta agaggtttca tattgctaat agcagctaca atccagctac cattctgctt 300 ttattttctg gttgggataa ggctggatta ttctgagtcc aagctaggcc cttttgctaa 360 tcttgttcat acctcttatc ttcctcccac agctcctggg caacctgctg gtctctctgc 420 tggcccatca ctttggcaaa g 441 <210> 7 <211> 1438 <212> DNA <213> Artificial <220> <223> FIX intron <400> 7 ggtttgtttc cttttttaaa atacattgag tatgcttgcc ttttagatat agaaatatct 60 gatgctgtct tcttcactaa attttgatta catgatttga cagcaatatt gaagagtcta 120 acagccagca cgcaggttgg taagtactgg ttctttgtta gctaggtttt cttcttcttc 180 atttttaaaa ctaaatagat cgacaatgct tatgatgcat ttatgtttaa taaacactgt 240 tcagttcatg atttggtcat gtaattcctg ttagaaaaca ttcatctcct tggtttaaaa 300 aaattaaaag tgggaaaaca aagaaatagc agaatatagt gaaaaaaaat aaccacatta 360 tttttgtttg gacttaccac tttgaaatca aaatgggaaa caaaagcaca aacaatggcc 420 ttatttacac aaaaagtctg attttaagat atatgacatt tcaaggtttc agaagtatgt 480 aatgaggtgt gtctctaatt ttttaaatta tatatcttca atttaaagtt ttagttaaaa 540 cataaagatt aacctttcat tagcaagctg ttagttatca ccaacgcttt tcatggatta 600 ggaaaaaatc attttgtctc tatgtcaaac atcttggagt tgatatttgg ggaaacacaa 660 tactcagttg agttccctag gggagaaaag cacgcttaag aattgacata aagagtagga 720 agttagctaa tgcaacatat atcactttgt tttttcacaa ctacagtgac tttatgtatt 780 tcccagagga aggcatacag ggaagaaatt atcccatttg gacaaacagc atgttctcac 840 aggaagcatt tatcacactt acttgtcaac tttctagaat caaatctagt agctgacagt 900 accaggatca ggggtgccaa ccctaagcac ccccagaaag ctgactggcc ctgtggttcc 960 cactccagac atgatgtcag ctgtgaaatc gacgtcgctg gaccataatt aggcttctgt 1020 tcttcaggag acatttgttc aaagtcattt gggcaaccat attctgaaaa cagcccagcc 1080 agggtgatgg atcactttgc aaagatcctc aatgagctat tttcaagtga tgacaaagtg 1140 tgaagttaac cgctcatttg agaactttct ttttcatcca aagtaaattc aaatatgatt 1200 agaaatctga ccttttatta ctggaattct cttgactaaa agtaaaattg aattttaatt 1260 cctaaatctc catgtgtata cagtactgtg ggaacatcac agattttggc tccatgccct 1320 aaagagaaat tggctttcag attatttgga ttaaaaacaa agactttctt aagagatgta 1380 aaattttcat gatgttttct tttttgctaa aactaaagaa ttattctttt acatttca 1438 <210> 8 <211> 1438 <212> DNA <213> Artificial <220> <223> Modified FIX intron <400> 8 ggtttgtttc cttttttaaa atacattgag tatgcttgcc ttttagatat agaaatatct 60 gatgctgtct tcttcactaa attttgatta catgatttga cagcaatatt gaagagtcta 120 acagccagca cgcaggttgg taagtactgg ttctttgtta gctaggtttt cttcttcttc 180 atttttaaaa ctaaatagat cgacattgct tttgttgcat ttatgtttaa taaacactgt 240 tcagttcatg atttggtcat gtaattcctg ttagaaaaca ttcatctcct tggtttaaaa 300 aaattaaaag tgggaaaaca aagaaatagc agaatatagt gaaaaaaaat aaccacatta 360 tttttgtttg gacttaccac tttgaaatca aattgggaaa caaaagcaca aacaatggcc 420 ttatttacac aaaaagtctg attttaagat atatgacatt tcaaggtttc agaagtatgt 480 aatgaggtgt gtctctaatt ttttaaatta tatatcttca atttaaagtt ttagttaaaa 540 cataaagatt aacctttcat tagcaagctg ttagttatca ccaacgcttt tcatggatta 600 ggaaaaaatc attttgtctc tttgtcaaac atcttggagt tgatatttgg ggaaacacaa 660 tactcagttg agttccctag gggagaaaag cacgcttaag aattgacata aagagtagga 720 agttagctat tgcaacatat atcactttgt tttttcacaa ctacagtgac tttttgtatt 780 tcccagagga aggcatacag ggaagaaatt atcccatttg gacaaacagc ttgttctcac 840 aggaagcatt tatcacactt acttgtcaac tttctagaat caaatctagt agctgacagt 900 accaggatca ggggtgccaa ccctaagcac ccccagaaag ctgactggcc ctgtggttcc 960 cactccagac atgatgtcag ctgtgaaatc gacgtcgctg gaccataatt aggcttctgt 1020 tcttcaggag acatttgttc aaagtcattt gggcaaccat attctgaaaa cagcccagcc 1080 agggtgttgg atcactttgc aaagatcctc attgagctat tttcaagtgt tgacaaagtg 1140 tgaagttaac cgctcatttg agaactttct ttttcatcca aagtaaattc aaatatgatt 1200 agaaatctga ccttttatta ctggaattct cttgactaaa agtaaaattg aattttaatt 1260 cctaaatctc catgtgtata cagtactgtg ggaacatcac agattttggc tccatgccct 1320 aaagagaaat tggctttcag attatttgga ttaaaaacaa agactttctt aagagatgta 1380 aaattttctt gttgttttct tttttgctaa aactaaagaa ttattctttt acatttca 1438 <210> 9 <211> 881 <212> DNA <213> Artificial <220> <223> Chicken beta-globin intron <400> 9 gcgggagtcg ctgcgttgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc 60 gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc 120 tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga 180 aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg 240 cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg 300 ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg 360 gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt 420 gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca 480 cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg 540 tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc 600 ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg 660 cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc 720 gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac 780 cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga 840 gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct c 881 <210> 10 <211> 881 <212> DNA <213> Artificial <220> <223> Modified chicken β-globin intron <400> 10 gcgggagtcg ctgcgttgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc 60 gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc 120 tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga 180 aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg 240 cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg 300 ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg 360 gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt 420 gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca 480 cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg 540 tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc 600 ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg 660 cggctgtcga ggcgcggcga gccgcagcca ttgccttttt tggtaatcgt gcgagagggc 720 gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac 780 cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaat tgggcgggga 840 gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct c 881 <210> 11 <211> 322 <212> DNA <213> Artificial <220> <223> ApoE control region <400> 11 aaggctcaga ggcacacagg agtttctggg ctcaccctgc ccccttccaa cccctcagtt 60 cccatcctcc agcagctgtt tgtgtgctgc ctctgaagtc cacactgaac aaacttcagc 120 ctactcatgt ccctaaaatg ggcaaacatt gcaagcagca aacagcaaac acacagccct 180 ccctgcctgc tgaccttgga gctggggcag aggtcagaga cctctctggg cccatgccac 240 ctccaacatc cactcgaccc cttggaattt cggtggagag gagcagaggt tgtcctggcg 300 tggtttaggt agtgtgagag gg 322

Claims

1. A modified HBB2 intron, the nucleic acid sequence of which is shown in SEQ ID NO:

6.

2. A nucleic acid construct comprising the introns according to claim 1.

3. The nucleic acid construct according to claim 2, further comprising a target gene and one or more additional expression control sequences.

4. The nucleic acid construct according to claim 2, further comprising a promoter and / or an enhancer.

5. The nucleic acid construct according to claim 4, wherein the promoter is a ubiquitous promoter or a tissue-specific promoter.

6. The nucleic acid construct according to claim 4, wherein the promoter is a liver-specific promoter.

7. A vector comprising an intron according to claim 1 or a nucleic acid construct according to any one of claims 2 to 6.

8. The vector according to claim 7 is a viral vector.

9. The vector according to claim 8 is a retroviral vector.

10. The vector according to claim 9, wherein it is a lentiviral vector.

11. The carrier according to claim 8, wherein it is an AAV carrier.

12. The vector according to claim 11 is a single-stranded or double-stranded self-complementary AAV vector.

13. The carrier according to claim 11 or 12, wherein the AAV carrier has a capsid derived from AAV, or has a mortise capsid.

14. The carrier according to claim 11 or 12, wherein the AAV carrier has an AAV-1, AAV-2, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-rh10, AAV-rh74, or AAV-dj capsid.

15. The carrier according to claim 11, wherein the AAV carrier has an AAV8 capsid.

16. The carrier according to claim 11 is a pseudo-AAV carrier.

17. The carrier according to claim 12, wherein it is a pseudo-AAV carrier.

18. The carrier according to claim 13 is a pseudo-AAV carrier.

19. The carrier according to claim 14 is a pseudo-AAV carrier.

20. The carrier according to claim 15 is a pseudo-AAV carrier.

21. An isolated cell, transformed with a nucleic acid construct according to any one of claims 2 to 6, or with a vector according to any one of claims 7 to 20.

22. The cell according to claim 21, wherein it is a liver cell or a muscle cell.

23. Use of the nucleic acid construct according to any one of claims 2 to 6, or the vector according to any one of claims 7 to 20, in the preparation of a medicament for gene or cell therapy, wherein the target gene is a therapeutic gene.

24. Use of the cell according to claim 21 or 22 in the preparation of a medicament for cell therapy, wherein the target gene is a therapeutic gene.

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