WO2019091457A1 - 肝脏特异性转录调控序列及其应用 - Google Patents

肝脏特异性转录调控序列及其应用 Download PDF

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WO2019091457A1
WO2019091457A1 PCT/CN2018/114844 CN2018114844W WO2019091457A1 WO 2019091457 A1 WO2019091457 A1 WO 2019091457A1 CN 2018114844 W CN2018114844 W CN 2018114844W WO 2019091457 A1 WO2019091457 A1 WO 2019091457A1
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gene
vector
liver
sequence
aav
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张琳
陈赛娟
王嫱
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上海交通大学医学院附属瑞金医院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Definitions

  • the present invention relates to the field of genetic engineering, and more particularly to a liver-specific transcriptional regulatory sequence and its use.
  • Hemophilia B belongs to a single-gene hereditary disease and is a sex-linked hereditary hemorrhagic disease caused by a defect in the human coagulation factor IX coding gene (hF9) on chromosome Xq27.1.
  • the incidence rate is about 1/25000.
  • the normal human F9 gene is about 34 kb in length, and the coding sequence cDNA is 1383 bp in length, located in the Xq27.1-q27.2 region of the long arm of the X chromosome, and contains 8 exons and 7 introns. Defects in the human F9 gene result in loss or dysfunction of FIX protein in plasma, which is the underlying cause of hemophilia B.
  • Gene therapy is currently one of the most promising strategies for curing hemophilia.
  • the therapeutic gene can be effectively transferred to gene-deficient cells to stably express normal-functioning blood coagulation factors for a long time, and the normal coagulation function of the patient can be restored, thereby achieving the purpose of lifelong healing.
  • the most mature and most curable technology is the in vivo gene therapy with adeno-associated virus (AAV). .
  • AAV adeno-associated virus
  • the patient's body produces an immune effect against the vector envelope protein. Although this immune effect can be controlled by immunosuppressive therapy, it causes the AAV vector to be cleared by the host body to some extent.
  • Another object of the present invention is to provide an adeno-associated virus vector comprising a liver-specific transcriptional regulatory sequence which can be used for gene therapy of hemophilia.
  • an isolated liver-specific transcriptional regulatory sequence is provided, characterized in that the regulatory sequence comprises an albumin regulatory sequence and a transthyretin regulatory sequence.
  • the albumin regulatory sequence comprises a binding site for the transcription factors HNF-1 and C/EBP.
  • the transthyretin regulatory sequence comprises a binding site for the transcription factors HNF-1, HNF-3, HNF-4, and C/EBP.
  • the transcription factor is a liver-specific transcription factor.
  • the liver-specific transcriptional regulatory sequence is a polynucleotide selected from the group consisting of:
  • the nucleotide sequence has a homology to the sequence of SEQ ID NO.: 1 ⁇ 90% (preferably ⁇ 95%, more preferably ⁇ 98%, optimally ⁇ 99%), and has a liver a polynucleotide that specifically transcribes activity;
  • the liver-specific transcriptional activity refers to an activity that drives specific transcription of downstream DNA fragments in liver cells.
  • the "specific transcription in liver cells” refers to transcriptional activity (or transcription amount) Z1 in the hepatocytes and transcriptional activity in non-liver cells (such as fibroblasts).
  • the polynucleotide selected from (b) or (c) retains at least ⁇ 50%, ⁇ 60% of the liver-specific transcriptional activity of the polynucleotide of SEQ ID NO.:1, ⁇ 70%, ⁇ 80%, ⁇ 90%, ⁇ 100%.
  • the regulatory sequence is an artificial recombinant regulatory sequence.
  • the regulatory sequence is derived from a human or non-human mammal.
  • nucleic acid construct comprising a foreign gene or an exogenous DNA fragment and operably linked to said foreign gene or DNA fragment is provided.
  • the regulatory sequence of the first aspect of the invention is provided.
  • the exogenous gene is selected from the group consisting of a therapeutic gene, a selection marker gene, a resistance gene, an antigenic protein gene, an RNAi gene, a microRNA gene, or a combination thereof.
  • the therapeutic gene is selected from the group consisting of human coagulation factor IX gene, human coagulation factor VIII, human alpha 1-antitrypsin, human arylsulfatase B (ARSB) gene, human low density Lipoprotein receptor (LDLR) gene, or a combination thereof.
  • the coagulation factor IX gene comprises a wild-type coagulation factor IX gene and a mutant coagulation factor IX gene.
  • the mutant coagulation factor IX gene comprises (i) a codon-optimized coagulation factor IX gene, and (ii) a point mutation coagulation factor IX gene, wherein the point mutation coagulation factor IX gene
  • the encoded polypeptide is mutated to leucine at position 338 corresponding to wild-type factor IX.
  • sequence of the codon-optimized Factor IX gene is set forth in SEQ ID NO.: 2.
  • sequence of the point mutation coagulation factor IX gene is as shown in SEQ ID NO.: 3.
  • a vector is provided, characterized in that the carrier comprises the construct of the second aspect of the invention.
  • the vector is a plasmid or a viral vector.
  • the vector is a lentiviral vector, an adeno-associated viral vector.
  • the vector is an AAV vector, preferably an AAV8 vector.
  • the vector is an AAV8 vector, and (on one side) the vector has the mutated ITR sequence of the eighth aspect of the invention.
  • a host cell comprising the vector of the third aspect of the invention or the regulatory sequence of the first aspect of the invention integrated with exogenous or A nucleic acid construct according to the second aspect of the invention.
  • the cell is an isolated cell, and/or the cell is a genetically engineered cell.
  • the cell is a human or non-human mammalian cell.
  • the cell is a liver cell.
  • the use of the regulatory sequence of the first aspect of the invention, the nucleic acid construct of the second aspect of the invention or the vector of the third aspect of the invention is provided, characterized in that To prepare a preparation or composition for regulating the specific expression of a foreign gene or an exogenous DNA fragment in liver cells or liver tissues.
  • a sixth aspect of the invention there is provided use of the regulatory sequence of the first aspect of the invention, characterized in that it is used for the construction of an expression vector for specificity in liver cells or liver tissues Expression of a foreign gene.
  • the exogenous gene comprises a structural gene, a non-structural gene (such as a non-structural gene encoding an antisense RNA, miRNA or siRNA).
  • a pharmaceutical composition characterized in that the pharmaceutical composition comprises the carrier according to the third aspect of the invention and a pharmaceutically acceptable excipient.
  • the pharmaceutical composition is in the form of an oral dosage form, or an injection.
  • a mutated ITR sequence (terminal inverted repeat) is provided, the mutated ITR sequence comprising 106 bases, and a 3' wild type ITR sequence (SEQ ID NO.: The base sequence between positions 25 to 130 of 5) is the same or complementary.
  • the mutated ITR sequence deletes positions 1-24 (5' end) and 131-145 (3' end) of the wild type ITR sequence corresponding to SEQ ID NO.: 5. .
  • the mutated ITR sequence is set forth in SEQ ID NO.: 4.
  • the mutated ITR sequence is a base sequence complementary to SEQ ID NO.: 4.
  • a vector is provided, characterized in that the vector contains the ITR sequence of the eighth aspect of the invention.
  • the vector is a plasmid or a viral vector.
  • the vector is a lentiviral vector, an adeno-associated viral vector.
  • the vector is an AAV vector, preferably an AAV8 vector.
  • the 5' ITR sequence of the AAV vector is the mutated ITR sequence of the eighth aspect of the invention.
  • the 3' ITR sequence of the AAV vector is the mutated ITR sequence of the eighth aspect of the invention.
  • Figure 1 shows the 1113 gene mutation types differentially diagnosed in hemophilia B patients by population analysis, and the distribution of these mutations in the exon, intron and UTR regions of the human coagulation factor 9 (hF9) gene.
  • each code contains the following: E, exon; Int, intron; UTR, upstream regulatory region.
  • Figure 2 shows a schematic representation of wild-type AAV virus and AAV vectors.
  • Figure 2A shows that the wild-type AAV vector is a non-enveloped DNA virus of 20-25 nm in diameter containing a single-stranded DNA genome of about 4.7 kb in length.
  • the AAV genome has two sets of coding genes, the rep gene encodes the protein required for viral replication and assembly, and the cap gene encodes three proteins to form a 60-mer viral envelope.
  • the coat protein of the AAV vector is identical or very similar to the wild-type AAV virus and contains a therapeutically relevant expression gene having a transcriptional drive element. 74% of the molecular weight of the AAV vector consists of protein and the maximum DNA carrying capacity is 5 kb.
  • Figure 2B shows that the wild-type AAV virus was engineered into a recombinant AAV vector in which the viral self-encoding gene was replaced by a therapeutic gene.
  • Figure 3 shows a schematic representation of AAV vector-mediated specific targeting of hepatocyte coagulation factor gene therapy for hemophilia B.
  • Figure 4 shows a comparison of the AAV vector structure map and the hFIX coding sequence before and after optimization.
  • Figure 4A shows a schematic view of the structure of an AAV vector.
  • Figure 4B shows a comparison before and after optimization of the human FIX coding sequence, including optimization parameters (left), GC content (middle), and cDNA secondary structure (right).
  • Figure 5 shows that sequence optimization of human FIX cDNA helps to increase the activity and protein levels of hFIX in HB mice.
  • Figure 5A shows the results of an ELISA method for determining hFIX antigen levels.
  • a DNA plasmid carrying wild-type hFIX, optimized hFIX (cohFIX), or an optimized hFIX positive control (+ve ctrl) expression frame was injected into hemophilia B mice (HB) by high pressure injection.
  • Mouse plasma was collected after 48 hours and hFIX antigen levels were determined by ELISA. Results are expressed as mean ⁇ standard deviation.
  • Figures 5B and 5C show the results of the ELISA method for determining hFIX activity and antigen levels, respectively.
  • the AAV8 vector carrying the GFP, optimized hFIX (cohFIX), or optimized hFIX positive control (+ve ctrl) expression frame was introduced into HB mice by tail vein injection. From the first week, mouse plasma was collected every four weeks, and hFIX activity and antigen levels were measured by chromogenic substrate method and ELISA method, respectively, and the results were expressed as mean ⁇ standard deviation.
  • Figure 6 shows that carrying a high-clotting activity mutant hFIX cDNA helps to effectively reduce the biological dose of the AAV vector.
  • the AAV8 vector expressing the hFIXR338L mutant was introduced into HB mice by tail vein injection. From the first week, mouse plasma was collected every four weeks, and hFIX activity and antigen levels were measured by chromogenic substrate method and ELISA method, respectively, and the results were expressed as mean ⁇ standard deviation.
  • FIG 7 shows the results of gel electrophoresis analysis of double-stranded AAV vectors and single-stranded AAV vectors.
  • the ITR m1 is an ITR structure contained in a vector published by Nathwani et al.
  • the ITR m2 contains an engineered ITR mutant structure in the present project, both of which mediate the formation of a vector containing a double-stranded DNA core (scAAV).
  • the ITR control contained a control ITR construct that was packaged to form a vector containing a single-stranded DNA core (ssAAV).
  • the AAV vector retains its single/double-stranded structure under alkaline gel electrophoresis and exhibits a single-stranded or double-stranded molecular weight. In the non-deformed gel, the double-stranded structure of scAAV is destroyed, thereby exhibiting the molecular weight of single-stranded DNA. .
  • Figure 8 shows the comparison of liver-specific regulatory element activity in HB mice.
  • the AAV vector carrying the cohFIX expression framework of different liver-specific regulatory sequences was injected into the hemophilia B mouse (HB) via the tail vein.
  • HB hemophilia B mouse
  • mouse plasma was collected every four weeks, and hFIX activity and antigen levels were measured by chromogenic substrate method and ELISA method, respectively, and the results were expressed as mean ⁇ standard deviation.
  • Figure 9 shows a dose escalation assay in vivo in HB mice carrying the AAV8 vector carrying the pSyn.cohFIX expression framework.
  • the AAV.GFP vector (2 x 10 12 /kg/head) was used as a negative control.
  • the plasma was collected once in the first week and every four weeks after the injection, and the plasma activity and antigen level of hFIX were measured by the chromogenic substrate method and the ELISA method, respectively, and the results were expressed as mean ⁇ standard deviation.
  • Figure 10 shows a comparison of RNA expression levels in liver, spleen and thymus tissues of each group of HB mice in the AAV vector dose increase test.
  • 2-3 of the mice in each group were collected for liver, spleen, and thymus tissue to extract RNA and reverse transcribed to generate cDNA.
  • the cDNA levels of hFIX and GAPDH were semi-quantitatively determined by real-time PCR, and the difference in RNA levels in each tissue was compared.
  • the RNA level of human coagulation factor IX is expressed as the relative RNA copy number (2- ⁇ (hFIX-GAPDH) ). Results are shown as individual mouse data and mean ⁇ standard deviation.
  • Figure 11 shows the biological distribution of AAV vectors in the tissues of mice in each group in a dose escalation test.
  • 2-3 mice in each group were collected from the liver, spleen, and thymus tissues at 12 weeks after injection of the AAV vector to extract DNA from the liver, spleen, and thymus tissues.
  • the semi-quantitative AAV vector was determined by real-time PCR. The concentration of DNA in each tissue cell. Results are shown as mean ⁇ standard deviation.
  • Figure 12 shows the measurement of liver function indexes of mice in each group in the dose increase test. Plasma concentrations of AST and ALT were measured by chromogenic substrate method in 2-3 mice of each group before injection of AAV vector and at 4 and 12 weeks after injection. The results showed mean ⁇ standard deviation.
  • Figure 13 shows the measurement of the high coagulation activity index of each group of mice in the dose increase test.
  • the plasma concentrations of D-dimer were detected by ELISA in 2-3 mice before injection of AAV vector and at 4 and 12 weeks after injection. The results showed mean ⁇ standard deviation.
  • the inventors have extensively and intensively studied for the first time to unexpectedly find a liver-specific transcriptional regulatory sequence that can drive enhanced specific expression of downstream genes in liver cells.
  • the regulatory sequences of the present invention can enhance the specific expression of foreign genes (such as human coagulation factor IX) in liver cells, and their transcriptional regulation efficiency is high (it has been verified in the hemophilia B mouse model).
  • the regulatory sequences of the present invention are also useful for specifically expressing other therapeutic genes in liver cells, and are of great importance for gene therapy strategies expressed by hepatocytes.
  • regulatory sequence As used herein, the terms “regulatory sequence”, “transcriptional regulatory sequence”, “pSyn regulatory sequence” are used interchangeably and refer to a nucleic acid sequence that accurately and efficiently initiates the transcriptional function of a gene.
  • the regulatory sequences of the invention include transcriptional regulatory sequences derived from albumin and transthyretin.
  • the regulatory sequence is a polynucleotide selected from the group consisting of:
  • the nucleotide sequence has a homology to the sequence of SEQ ID NO.: 1 ⁇ 90% (preferably ⁇ 95%, more preferably ⁇ 98%, optimally ⁇ 99%), and has a liver a polynucleotide that specifically transcribes activity;
  • the pSyn-regulated nucleotide sequence can drive enhanced specific expression of downstream genes in liver cells.
  • it is mainly used to enhance the specific expression of human coagulation factor IX in liver cells, and its transcriptional regulation efficiency and other liver transcriptional regulatory sequences have been compared in the hemophilia B mouse model.
  • pSyn regulatory sequences can also be used to specifically express other therapeutic genes in liver cells. Therefore, this sequence is very important for gene therapy strategies expressed by hepatocytes.
  • the regulatory sequence includes a variant of the regulatory sequence shown in SEQ ID NO.: 1, which can be obtained by inserting or deleting a regulatory region, performing random or site-directed mutagenesis or the like.
  • the present invention also includes 50% or more (preferably 60% or more, 70% or more, 80% or more, more preferably 90% or more, more preferably 95% or more, and most preferably 98% or more, such as 99%) of the regulatory sequence of the present invention.
  • a nucleic acid of homology that also has a function of specifically regulating the expression of a tissue on the plant that initiates the plant.
  • "Homology” refers to a similar level (ie, sequence similarity or identity) between two or more nucleic acids, as a percentage of the same position.
  • telomere length refers to the expression of a gene of interest at a particular time and/or in a particular tissue.
  • exogenous or “heterologous” refers to the relationship between two or more nucleic acid or protein sequences from different sources. For example, if the combination of a promoter and a gene sequence of interest is generally not naturally occurring, the promoter is foreign to the gene of interest. A particular sequence is “exogenous” to the cell or organism into which it is inserted.
  • a promoter of the invention can be operably linked to a foreign gene which can be foreign (heterologous) relative to the promoter.
  • the foreign gene also referred to as a gene of interest
  • a preferred exogenous gene is a foreign gene for gene therapy expressed by a liver cell, such as a human coagulation factor IX gene.
  • the invention also provides nucleic acid constructs and vectors comprising the regulatory sequences. Methods for preparing recombinant vectors are well known to those of ordinary skill in the art.
  • the expression vector can be a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus or other vector.
  • a particularly preferred vector is the AAV8 virus.
  • One of ordinary skill in the art can construct expression vectors containing the promoters and/or gene sequences of interest described herein using well known methods. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like.
  • transcriptional regulatory elements were screened and optimized.
  • the viral vector such as AAV8 has affinity for liver tissue and can successfully infect tissue cells, it may also infect other non-liver cells, and the structure will produce off-target expression and corresponding adverse reactions. Therefore, the primary requirement for the design of regulatory elements is to have hepatocyte-specific regulation.
  • the packaging capacity based on the double-stranded complementary AAV vector is only half of that of the wild type. In order to successfully assemble into a viral solid particle, the regulatory element also needs to contain as few bases as possible, otherwise it will exceed the packaging capacity of the viral particle. Invalid product.
  • the transcriptional regulatory sequences mainly comprise a promoter and an enhancer.
  • the promoter is responsible for the recognition and binding of RNA polymerase
  • the enhancer comprises a binding site that promotes regulation of transcription factors and can increase the transcriptional effects of downstream genes.
  • the regulatory element of the hFIX sequence is an enhancer of the human apolipoprotein E-Hepatocyte Control Region (ApoE-HCR) group, and human alpha antitrypsin.
  • the promoters combine to form a composite structure sequence and are sized to accommodate the packaging capacity of the AAV.
  • the vector carrying this regulatory element belongs to one of the most efficient expression vectors in liver tissue and has successfully demonstrated a certain therapeutic effect in clinical trials. However, this level of expression only relieves the patient's phenotype to a certain extent and does not fully meet the criteria for healing.
  • albumin is one of the most important members.
  • albumin enhancers and promoters have more liver-specific regulatory elements, which are closely related to transcriptional regulation systems in liver cells.
  • the promoter of albumin includes a region between the transcription initiation site and the upstream 217 bp, and its sequence contains binding sites for different transcription factors (Table 5), such as the liver specific transcription factor HNF-1 (Hepatocyte nuclear factor 1).
  • LF-B1, PAF, AFP1 At least three enhancers are present in the upstream region of the 5' end of the human albumin gene, including the -265 bp, -1.7 kb, and -6 kb regions upstream of the promoter.
  • These AT- and GC-rich regions are transcription factors, such as the major binding sites for HNF-1, which have a markedly promoting effect on the transcription of downstream genes.
  • Transthyretin also known as prealbumin, is one of the proteins produced mainly in hepatocytes.
  • the promoter length of the mouse TTR gene comprises a region from the transcription start site to the upstream of 5' of the base of 202, wherein the region from -108 to -202 belongs to the region necessary for TTR to specifically transcribe and express in liver cells.
  • a DNA fragment of about 100 bp between -1.86 kb and -1.96 kb upstream of the 5' has a strong enhancer effect, and promotes the expression efficiency of a proximal promoter driven in a liver cancer cell line. 10 times.
  • the promoter and enhancer regions of TTR have been identified to contain binding sites for at least four different transcription factors, such as HNF-1, HNF-3, HNF-4, and C/EBP. Most of these factors belong to liver-specific transcription factors and have a strong promoting effect on promoting the expression of downstream genes in liver cells.
  • hFIX human coagulation factor IX
  • the reporter gene plasmid carrying only the hF9 proximal promoter, although not expressed at a high level, has a transcription efficiency comparable to that of a plasmid containing an HCR regulatory sequence after the addition of several copies of the 5th site sequence.
  • liver-cell-specific synthetic protein-encoding genes have reference enhancer sequences for screening or synthesis of transcriptionally efficient regulatory elements for use in gene therapy expression frameworks, mediating hepatocyte-specific transcriptional expression. .
  • the present invention provides a mutated ITR sequence (terminal inverted repeat) comprising 106 bases, and the 25th to the 3' wild type ITR sequence SEQ ID NO.: 5
  • the base sequences between the 130 positions are complementary or identical.
  • the wild-type ITR structure contains 145 base pairs, functions in viral replication and mediates the AAV virus that forms a single-stranded DNA core.
  • the wild-type ITR structure is adjusted, and after removing a part of the base sequence at both ends of the 3'-end wild-type ITR sequence, the terminal termination site (TRS) is deleted, and the ITR is deleted.
  • the complementary sequence of the structure is constructed at the 5' end of the expression vector, thereby promoting the formation of a complementary double-stranded structure during AAV replication and promoting transcriptional expression. This structure is further reduced by several base pairs than the engineered ITR structure of Nathwani, so that it can still form complementary double-stranded viral DNA. Based on the above structure, the biological activity and safety of the AAV vector constructed and adjusted were systematically evaluated in HB mice.
  • Hemophilia B belongs to a single-gene hereditary disease and is a sex-linked hereditary hemorrhagic disease caused by a defect in the human coagulation factor IX coding gene (hF9) on chromosome Xq27.1.
  • the incidence rate is about 1/25000.
  • the normal human F9 gene is about 34 kb in length, and the coding sequence cDNA is 1383 bp in length, located in the Xq27.1-q27.2 region of the long arm of the X chromosome, and contains 8 exons and 7 introns. Defects in the human F9 gene result in loss or dysfunction of FIX protein in plasma, which is the underlying cause of hemophilia B.
  • Gene therapy strategy The introduction of normal genes into the normal gene by adeno-associated virus vector AAV has long-term cure potential in hepatocytes. At present, clinical trials and safety results have been obtained in foreign clinical trials. However, there is still room for optimization and improvement in the biological vectors used in gene therapy. Most importantly, there are currently no gene therapy products with similar intellectual property rights in China. Therefore, the present invention combines international advances in progress, conducts research on gene therapy for hemophilia B, develops and optimizes AAV vectors for hemophilia B gene therapy, and confirms its biological activity in preclinical animal models. Safety, which provides a solid foundation for clinical transformation. The development of this vector has filled the gap in the field of gene therapy hemophilia B in China to a certain extent, and its clinical application will lay a solid theoretical and practical foundation for gene therapy for hereditary diseases including hemophilia.
  • the AAV vector for mediating expression of a causative gene in hepatocytes of the present invention and liver-specific regulatory sequences therein are versatile.
  • liver-specific expression can be performed by this vector if the defective gene size is suitable for the AAV vector packaging capacity. Therefore, the AAV vector and the regulatory element involved in the present invention have a very broad application prospect in the field of gene therapy, and provide a favorable research tool for developing more gene therapy strategies in the future.
  • the AAV vector is about 20-25 nm in diameter, and the wild-type AAV virus consists of a protein envelope and a nucleic acid core, and may comprise a DNA fragment of about 4.7 kb in size (Fig. 2A).
  • the engineered viral self-encoding gene can be replaced by a therapeutic gene to form a recombinant AAV viral vector (Fig. 2B).
  • More than 120 kinds of AAV vector capsid proteins have been naturally isolated and identified from human and animal tissues. Tissue affinity assays showed that different AAV vectors could have affinity for a variety of tissues in the human body, while the same tissue could also be infected with AAV vectors of different serotypes (Table 2).
  • AAV2, AAV7, AAV8, AAV9 and AAVrh10 have high affinity, and AAV8 and AAVrh10 have the highest infection efficiency, so they have been applied to blood.
  • the invention also relates to vectors comprising the pSyn regulatory sequences of the invention, as well as host cells genetically engineered using the vectors or pSyn regulatory sequences of the invention.
  • vector refers to bacterial plasmids, phage, yeast plasmids, plant cell viruses, mammalian cell viruses or other vectors well known in the art.
  • any plasmid and vector can be used as long as it can replicate and stabilize in the host.
  • it usually contains an origin of replication, a promoter, a marker gene, and a translational control element.
  • the vector of the invention is an AAV vector.
  • the vector of the present invention can be constructed using methods well known to those skilled in the art. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like.
  • the DNA sequence can be operably linked to a suitable promoter in a vector of the invention to direct mRNA synthesis.
  • the vector of the present invention also includes a ribosome binding site for translation initiation and a transcription terminator.
  • the vectors of the present invention preferably comprise one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and Green fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and Green fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • Vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences, can be used to transform appropriate host cells to enable expression of the protein.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant cell (such as a cell of a crop or a forestry plant).
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a plant cell (such as a cell of a crop or a forestry plant).
  • Representative examples are: Escherichia coli, Streptomyces, Agrobacterium; fungal cells such as yeast; plant cells, animal cells, and the like.
  • the host cell of the invention is a human or non-human mammalian cell, preferably a liver cell.
  • AAV vector of the invention is AAV vector of the invention
  • the AAV vector has a critical delivery transport effect.
  • the sequence expressing the therapeutic gene needs to be transported through the AAV vector in the blood circulation of the body and specifically localized to a specific target organ.
  • Gene therapy The AAV vector of hemophilia B consists essentially of two components: a. therapeutic gene expression framework; b. AAV structural sequence with a mutated ITR structure (Fig. 4).
  • the present invention achieves an improved expression level of normal coagulation function hFIX having a therapeutic effect in gene therapy by optimizing two components.
  • the gene-specifically expressing adeno-associated virus vector (AAV vector) of the gene therapy hemophilia B of the present invention comprises:
  • the expression framework of human FIX includes the hFIX cDNA coding sequence, as well as regulatory sequences such as enhancers/promoters (Fig. 4A).
  • the hFIX coding sequence was codon optimized. The principle of codon optimization is to enhance the transcription of genes by replacing the codons with higher frequency of expression in eukaryotic cells corresponding to the same amino acid by using different codon usage frequencies. Expression efficiency.
  • sequence optimization of the hFIX coding sequence is performed to increase the stability of the mRNA molecule structure and reduce the risk of degradation by endogenous RNase. Sequence optimization is primarily done using OptimumGeneTM analysis.
  • the optimized parameters include: (1) gene transcription efficiency regulation parameters, including increasing the preference amino acid codon, reducing or deleting the use of low utilization codon; (2) adjusting the GC content, the applicable interval is 30% to 70%; (3) Modifying the secondary structure of hFIX mRNA, including reducing or deleting implied structures such as hidden cleavage sites, loops or branches, repeat sequences, and applicable stop codon TGA (Fig. 4B).
  • an optimized hFIX sequence was constructed under the driving of the same transcriptional regulatory element, and hemophilia B (HB) mice were injected through the tail vein hypertension.
  • HB hemophilia B mice were injected through the tail vein hypertension.
  • a plasmid carrying the expression framework was introduced into mouse hepatocytes using physical pressure.
  • the codon optimization strategy of the present invention increased the expression level of hFIX (cohFIX) antigen in plasma by about 3 times as compared with the wild type cDNA, and this level has been clinically effective in clinical trials.
  • the hFIX optimized sequence (hFIX.ctrl) is basically the same.
  • the cohFIX expression cassette was transported to the liver tissue of HB mice through the AAV8 vector, and the results of periodic plasma detection revealed that the cohFIX optimized sequence of the present invention (AAV.cohFIX) can stably and efficiently express antigen in mouse hepatocytes. And clotting activity, the transcription efficiency was basically consistent with the positive control (AAV.hFIXctrl), and the results effectively alleviated the bleeding phenotype of the gene-treated mice (Fig. 5B). These results advantageously demonstrate that the sequence optimization strategy of the present invention can effectively increase the transcription efficiency of the hF9 gene and is therefore suitable for expression vectors in gene therapy.
  • the present invention also introduces a point mutation in the coding gene, thereby replacing the arginine at position 338 in the hFIX protein with leucine (hFIX.SIHR338L).
  • This mutation was first discovered in an Italian family.
  • arginine at position 338 in hFIX was replaced by leucine, the result was that the clotting activity was 5-8 times higher than that of normal people, although the level of FIX protein in the patient's blood circulation was comparable to that of normal people. . It has been found that the R338 site is an important part of the binding of factor X to FIX.
  • transcriptional regulatory elements were also screened and optimized. In order to obtain the most efficient transcriptional regulation, different regulatory sequences were combined and compared in parallel with the pLP1 regulatory sequences carried by AAV vectors in clinical trials such as Nathwani.
  • the synthetic pSynthetic (pSyn) regulatory sequence which binds to the regulatory sequences of TTR and Alb, contains binding sites for multiple hepatocyte-specific transcription factors such as HNF-1, HNF-3, HNF-4, and C/EBP. point.
  • pF9 human factor IX regulatory sequence
  • pF9.v1 contains a sequence between -219 and +29 base pairs of the F9 gene
  • pF9.v2 also introduces 5 copies of the enhancer sequence, which effectively enhances pF9 near in vitro cell line assays. The transcription efficiency of the terminal promoter.
  • the ITR structure of the carrier is also adjusted.
  • the control ITR structure (ITR control ) mediates the AAV vector forming the single-stranded DNA core (Fig. 7, sample F), and Nathwani et al. deleted nearly 20 base pairs in the ITR (ITR m1 ) on the AAV vector side, thereby A complementary double-stranded DNA structure was formed during AAV packaging production to promote transcriptional efficiency ( Figure 7, Sample A). Further modifications were made on this basis to delete several base pairs (ITR m2 ), but to retain the ability to form complementary double-stranded viral DNA (Fig. 7, sample BE).
  • a synthetic intron sequence was also added to the hFIX expression framework, and splicing donor and splicing acceptor were added at both ends so as not to affect the coding sequence.
  • the biological activity and safety of the AAV vector constructed by combining the above structural adjustments were systematically evaluated in HB mice.
  • an hFIX expression framework comprising different regulatory elements was constructed and packaged into an AAV8 vector. These vectors enter the HB mice after tail vein injection and mediate liver-specific expression of hFIX protein.
  • the pSyn regulatory sequence has relatively strong liver-specific transcriptional activity, and therefore the in vivo dose increase test was carried out using the AAV.pSyn.cohFIX vector containing the pSyn regulatory sequence, wherein the carrier doses of the high, medium and low concentration groups were injected.
  • HB mice extracted tissues and organs three months after injection of the virus vector for analysis, RNA in liver, spleen, and thymus tissues was extracted and analyzed for hFIX relative RNA expression level of the internal reference gene GAPDH.
  • RNA of human coagulation factor IX was mainly expressed in liver tissues, and the expression level increased as the dose of the vector increased.
  • the level of hFIX RNA expression in the spleen and thymus was at baseline levels compared to liver tissue, consistent with RNA levels in various tissues of HB mice injected with the AAV.GFP vector.
  • the biological distribution of the AAV vector in HB mice was analyzed. According to the results reported in the past, the AAV8 vector should have affinity mainly for mouse liver tissue.
  • the results are shown in Fig. 11.
  • the liver, spleen, thymus, and bone marrow tissues of the gene-treated mice were analyzed and analyzed, and the viral DNA was mainly distributed in the liver tissues, and the distribution concentration was proportional to the dose of the carrier.
  • there was a very small amount of viral DNA distribution in the spleen with an average concentration of about 0.23 copies/cell in the high dose group, which was almost identical to the baseline level in the AAV.GFP group HB mice in other dose groups and other tissues.
  • AAV.pSyn.cohFIX vector in the present application It has the purpose of controlling the specific expression of hFIX in the liver to treat hemophilia B.
  • AAV vector-mediated gene therapy can induce immune response and mild liver damage, accompanied by an increase in liver function.
  • Plasma levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were compared before injection of AAV vector and at 4 and 12 weeks after injection.
  • Fig. 12 compared with the plasma level of HB mice not receiving gene therapy (Week 0), the AST or ALT levels in the plasma of the mice 4 weeks or 12 weeks after the injection of the viral vector were not significant. Sexual increase. This also corresponds to the level of hFIX stably expressed in plasma, indicating that the AAV vector and its expression product did not induce significant liver function damage at these doses.
  • Excessive exogenous coagulation factors can induce a high blood coagulation state in the body.
  • concentration of D-dimer in plasma was measured.
  • the AAV.pSyn vector constructed by the present invention sufficiently functions to specifically express a therapeutic gene of interest in liver tissues.
  • the clotting factor IX can be stably expressed for a long time under the driving of pSyn, thereby restoring the normal coagulation function of the mouse and achieving the purpose of long-term or even permanent healing.

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Abstract

提供了一种可以驱动增强下游基因在肝脏细胞中的特异性表达的调控序列，该调控序列包含白蛋白和转甲状腺素蛋白的调控序列，可以用于在肝脏细胞中特异性的表达治疗性基因，如凝血因子IX基因。

Description

肝脏特异性转录调控序列及其应用 技术领域
本发明涉及基因工程领域，更具体地涉及一种肝脏特异性转录调控序列及其应用。
背景技术
血友病B(Hemophilia B,HB)属于单基因遗传性疾病，是染色体Xq27.1上的人凝血因子IX编码基因(hF9)缺陷所致的性染色体连锁遗传性出血性疾病，在全球男性中发病率约1/25000。正常的人F9基因全长约34kb，其中编码序列cDNA长度为1383bp，位于X染色体长臂末端Xq27.1-q27.2区域，包含8个外显子和7个内含子。人F9基因缺陷导致血浆中FIX蛋白缺失或功能障碍是导致血友病B的根本病因。目前，血友病患者的主要治疗措施是进行替代治疗(protein replacement therapy,PRT)，包括预防性或治疗性反复输注重组凝血因子IX或血浆制品等。这些治疗措施虽然在一定程度上改善了患者的生存率和生活质量，然而不具备治愈性。与此同时，长期治疗的昂贵费用($100-300,000/年)以及输血或静脉注射引发的感染等并发症都给患者带来诸多弊端。
基因治疗是当前最有潜力治愈血友病的策略之一。通过基因克隆和转移技术，可以有效的把治疗基因转移到基因缺陷细胞以长期稳定地表达功能正常的凝血因子，恢复患者的正常凝血功能，从而达到终身治愈的目的。结合目前血友病基因治疗的基础研究和临床试验进展，发展相对比较成熟的最具有治愈潜力的技术是以腺相关病毒载体(adeno-associated virus，AAV)进行体内基因治疗(In vivo gene therapy)。
2011年和2014年相继发表的世界首例血友病B基因治疗临床试验报道了AAV载体介导基因治疗在10名重症HB患者中取得的显著疗效，部分患者临床症状明显缓解，中断或减少hFIX因子替代品的使用。尽管如此，大多数患者体内的hFIX水平只能达到正常人的1-6％，在高、中、低三个组别中，主要是高剂量组及部分中剂量组的患者取得了较好的临床效应，但相对于>30％的彻底治愈标准还有一定距离。在此临床试验中效果有限的主要原因为：
(1)AAV载体内的表达框架的转录效率还可以进一步提高；
(2)临床试验制剂的生产工艺有待进一步提高，以提高实体病毒颗粒的比 例，降低空壳病毒的污染；
(3)患者机体产生了针对载体包壳蛋白的免疫效应。这种免疫效应虽然可以通过免疫抑制治疗进行控制，但在一定程度上导致了AAV载体被宿主机体清除。
综合以上，进一步改进优化基因治疗血友病B的生物载体并进行临床转化，对于填补国内在此领域的空白有非常重要的意义。
发明内容
本发明的目的在于提供一种肝脏特异性转录调控序列及其应用。
本发明的另一目的在于提供一种包含肝脏特异性转录调控序列的腺相关病毒载体，该腺相关病毒载体可以用于血友病的基因治疗。
在本发明的第一方面，提供了一种分离的肝脏特异性转录调控序列，其特征在于，所述的调控序列包含白蛋白调控序列和转甲状腺素蛋白调控序列。
在另一优选例中，所述的白蛋白调控序列包括转录因子HNF-1和C/EBP的结合位点。
在另一优选例中，所述的转甲状腺素蛋白调控序列包括转录因子HNF-1、HNF-3、HNF-4、和C/EBP的结合位点。
在另一优选例中，所述的转录因子为肝脏特异性转录因子。
在另一优选例中，所述的肝脏特异性转录调控序列为任选自下组的多核苷酸：
(a)核苷酸序列如SEQ ID NO.:1所示的多核苷酸；
(b)核苷酸序列与SEQ ID NO.:1所示序列的同源性≥90％(较佳地≥95％，更佳地≥98％，最佳地≥99％)，且具有肝脏特异性转录活性的多核苷酸；
(c)如SEQ ID NO.:1所示多核苷酸的5’端和/或3’端增加和/或减少1-50个(较佳地1-20，更佳地1-10个，更佳地1-5个)核苷酸，且具有肝脏特异性转录活性的多核苷酸。
在另一优选例中，所述的肝脏特异性转录活性是指驱动下游DNA片段在肝脏细胞中特异性转录的活性。
在另一优选例中，所述的“在肝脏细胞中特异性转录”指在所述肝细胞中的转录活性(或转录量)Z1与在非肝脏细胞(如成纤维细胞)中的转录活性(或转录量)Z2之比(Z1/Z2)≥2，较佳地≥5，更佳地≥10。
在另一优选例中，选自(b)或(c)的多核苷酸保留了SEQ ID NO.:1所示的多核苷酸的肝脏特异性转录活性的至少≥50％，≥60％，≥70％，≥80％，≥90％、≥100％。
在另一优选例中，所述的调控序列为人工重组调控序列。
在另一优选例中，所述的调控序列来源于人或非人哺乳动物。
在本发明的第二方面，提供了一种核酸构建物，其特征在于，所述的构建物含有外源基因或外源DNA片段，以及与所述的外源基因或DNA片段可操作连接的本发明第一方面所述的调控序列。
在另一优选例中，所述的外源基因选自下组：治疗基因、筛选标记基因、抗性基因、抗原蛋白基因、RNAi基因、microRNA基因、或其组合。
在另一优选例中，所述的治疗基因选自下组：人凝血因子IX基因、人凝血因子VIII、人α1-抗胰蛋白酶、人芳基硫酸酯酶B(ARSB)基因、人低密度脂蛋白受体(LDLR)基因、或其组合。
在另一优选例中，所述的凝血因子IX基因包括野生型凝血因子IX基因和突变型凝血因子IX基因。
在另一优选例中，所述的突变型凝血因子IX基因包括(i)密码子优化的凝血因子IX基因，和(ii)点突变凝血因子IX基因，其中所述的点突变凝血因子IX基因编码的多肽在对应于野生型凝血因子IX的第338位精氨酸突变为亮氨酸。
在另一优选例中，所述密码子优化的凝血因子IX基因的序列如SEQ ID NO.:2所示。
在另一优选例中，所述的点突变凝血因子IX基因的序列如SEQ ID NO.:3所示。
在本发明的第三方面，提供了一种载体，其特征在于，所述的载体含有本发明第二方面所述的构建物。
在另一优选例中，所述的载体为质粒、病毒载体。
在另一优选例中，所述的载体为慢病毒载体、腺相关病毒载体。
在另一优选例中，所述的载体为AAV载体，优选地为AAV8载体。
在另一优选例中，所述的载体为AAV8载体，并且所述载体(的一侧)具有本发明第八方面所述的突变的ITR序列。
在本发明的第四方面，提供了一种宿主细胞，所述的宿主细胞中含有本发明第三方面所述的载体或染色体中整合有外源的本发明第一方面所述的调控序列或本发明第二方面所述的核酸构建物。
在另一优选例中，所述细胞为分离的细胞，和/或所述细胞为基因工程化的细胞。
在另一优选例中，所述细胞为人或非人哺乳动物细胞。
在另一优选例中，所述细胞为肝脏细胞。
在本发明的第五方面，提供了本发明第一方面所述的调控序列、本发明第二方面所述的核酸构建物或本发明第三方面所述的载体的用途，其特征在于，用于制备一制剂或组合物，所述的制剂或组合物用于调控外源基因或外源DNA片段在肝脏细胞或肝脏组织中进行特异性表达。
在本发明的第六方面，提供了本发明第一方面所述的调控序列的用途，其特征在于，用于构建一表达载体，所述的表达载体用于在肝脏细胞或肝脏组织中特异性表达外源基因。
在另一优选例中，所述的外源基因包括结构基因、非结构基因(如编码反义RNA、miRNA或siRNA的非结构基因)。
在本发明的第七方面，提供了一种药物组合物，其特征在于，所述的药物组合物含有本发明第三方面所述的载体和药学上可接受的赋形剂。
在另一优选例中，所述的药物组合物的剂型为口服剂型、或注射剂。
在本发明的第八方面，提供了一种突变的ITR序列(末端反向重复序列)，所述突变的ITR序列含有106个碱基，与3’端野生型ITR序列(SEQ ID NO.:5)的第25位至第130位之间的碱基序列相同或者互补。
在另一优选例中，所述突变的ITR序列删除了野生型ITR序列的对应于SEQ ID NO.:5的第1-24位(5’端)和第131-145位(3’端)。
在另一优选例中，所述突变的ITR序列如SEQ ID NO.:4所示。
在另一优选例中，所述突变的ITR序列为与SEQ ID NO.:4互补的碱基序列。
在本发明的第九方面，提供了一种载体，其特征在于，所述的载体含有本 发明第八方面所述的ITR序列。
在另一优选例中，所述的载体为质粒、病毒载体。
在另一优选例中，所述的载体为慢病毒载体、腺相关病毒载体。
在另一优选例中，所述的载体为AAV载体，优选地为AAV8载体。
在另一优选例中，所述的AAV载体的5’ITR序列为本发明第八方面所述的突变的ITR序列。
在另一优选例中，所述的AAV载体的3’ITR序列为本发明第八方面所述的突变的ITR序列。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了经群体分析在血友病B患者中鉴别诊断的1113种基因突变类型，以及这些突变在人凝血因子9(hF9)基因外显子、内含子和UTR区域的分布。其中，各代码含如下：E，外显子；Int，内含子；UTR，上游调控区域。
图2显示了野生型AAV病毒和AAV载体示意图。
图2A显示了野生型AAV载体是直径为20-25nm的无包膜DNA病毒，内含长约4.7kb的单链DNA基因组。AAV基因组有两组编码基因，rep基因编码病毒复制和组装所需蛋白，cap基因编码3种蛋白以形成60-mer的病毒包壳。AAV载体的外壳蛋白与野生型AAV病毒的一致或非常类似，内含具有转录驱动元件的治疗相关表达基因。AAV载体的分子重量中有74％由蛋白组成，最大DNA的携带量为5kb。
图2B显示了野生型AAV病毒经改造为重组AAV载体，其中病毒自身编码基因被治疗性基因取代。
图3显示了AAV载体介导特异性靶向肝细胞生成凝血因子基因治疗血友病B示意图。
图4显示了AAV载体结构图及hFIX编码序列优化前后比较。
图4A显示了AAV载体结构示意图。
图4B显示了人FIX编码序列优化前后比较，包括优化参数(左)、GC含量(中)、以及cDNA二级结构(右)。
图5显示了序列优化人FIX cDNA有助于提高hFIX在HB小鼠体内的活性和蛋 白水平。
图5A显示了ELISA方法测定hFIX抗原水平的结果。携带野生型hFIX，优化型hFIX(cohFIX)，或优化型hFIX阳性对照(+ve ctrl)表达框架的DNA质粒经高压注射方式进入血友病B小鼠(HB)体内。小鼠血浆于48小时后采集并通过ELISA方法测定hFIX抗原水平。结果以均数±标准差表示。
图5B和图5C分别显示了ELISA方法测定hFIX活性和抗原水平的结果。携带GFP，优化型hFIX(cohFIX)，或优化型hFIX阳性对照(+ve ctrl)表达框架的AAV8载体通过尾静脉注射方式进入HB小鼠。从第一周开始，小鼠血浆每隔四周采集一次，其中hFIX活性和抗原水平分别通过发色底物法和ELISA法进行测定，结果表示为均数±标准差。
图6显示了携带高凝血活性突变hFIX cDNA有助于有效降低AAV载体的生物剂量。表达hFIXR338L突变体的AAV8载体通过尾静脉注射方式进入HB小鼠。从第一周开始，小鼠血浆每隔四周采集一次，其中hFIX活性和抗原水平分别通过发色底物法和ELISA法进行测定，结果表示为均数±标准差。
图7显示了凝胶电泳分析双链AAV载体和单链AAV载体结果。其中ITR m1是含Nathwani等发表的载体中ITR结构，ITR m2内含本项目中改造的ITR突变结构，两者均介导形成含有双链DNA核心的载体(scAAV)。ITR 对照内含对照型ITR结构，经包装形成含单链DNA核心的载体(ssAAV)。AAV载体在碱性胶电泳下可保留其单/双链结构，表现为单链或双链的分子量；而在非变形胶中scAAV的双链结构被破坏，从而全部表现为单链DNA的分子量。
图8显示了肝脏特异性调控元件活性在HB小鼠体内比较。携带不同肝脏特异性调控序列的cohFIX表达框架的AAV载体经尾静脉注射进入血友病B小鼠(HB)体内。从注射后一周开始，小鼠血浆每隔四周采集一次，其中hFIX活性和抗原水平分别通过发色底物法和ELISA法进行测定，结果表示为均数±标准差。
图9显示了携带pSyn.cohFIX表达框架的AAV8载体在HB小鼠体内进行剂量增高试验。AAV载体分别按照高(2×10 12/kg/只)、中(4×10 11/kg/只)、和低(1×10 11/kg/只)三种不同的剂量经尾静脉输入HB小鼠体内(n＝3～6)。AAV.GFP载体(2×10 12/kg/只)被用于作为阴性对照。小鼠于注射后第一周及每隔四周采集血浆一次，hFIX的血浆活性和抗原水平分别通过发色底物法和ELISA法进行测定，结果表示为均数±标准差。
图10显示了AAV载体剂量增高试验中，各组HB小鼠肝脏、脾和胸腺组织中的RNA表达水平比较。在注射了不同剂量AAV载体后第12周，各组小鼠有2-3只 采集肝、脾、及胸腺组织提取RNA，并逆转录生成cDNA。通过real-time PCR半定量测定hFIX及内参GAPDH的cDNA水平，比较各组织中RNA的水平差异。人凝血因子IX的RNA水平表示为相对RNA拷贝数(2 -Δ(hFIX-GAPDH))。结果显示为个体小鼠数据及均数±标准差。
图11显示了AAV载体在剂量增高试验中各组小鼠组织脏器中的生物性分布。剂量增高试验中各组小鼠2-3只在注射AAV载体后12周采集肝、脾、及胸腺组织提取RNA肝、脾，及胸腺组织中的DNA，通过real-time PCR半定量测定AAV载体DNA在各组织细胞中的浓度。结果显示为均数±标准差。
图12显示了剂量增高试验中各组小鼠肝功能指标测定。各组小鼠2-3只在注射AAV载体前，及注射后第4和12周采集的血浆经过发色底物法测定AST和ALT的浓度，结果显示为均数±标准差。
图13显示了剂量增高试验中各组小鼠高凝血活性指标测定。各组小鼠2-3只在注射AAV载体前，及注射后第4和12周采集的血浆经过ELISA方法检测D二聚体的浓度，结果显示为均数±标准差。
具体实施方式
本发明人经过广泛而深入地研究，首次意外地发现一种肝脏特异性转录调控序列，其可以驱动增强下游基因在肝脏细胞中的特异性表达。实验表明，本发明的调控序列可以增强外源基因(如人凝血因子IX等)在肝脏细胞中的特异性表达，其转录调控效率高(已经在血友病B小鼠模型进行了验证)。本发明的调控序列还可用于在肝脏细胞中特异性地表达其他治疗性基因，对于经肝细胞表达的基因治疗策略都具有非常重要的意义。
pSyn调控序列
如本文所用，术语“调控序列”、“转录调控序列”、“pSyn调控序列”可互换使用，是指一种准确有效起始基因转录功能的核酸序列。本发明的所述的调控序列包括来源于白蛋白、转甲状腺素蛋白的转录调控序列。优选地，所述的调控序列为任选自下组的多核苷酸：
(a)核苷酸序列如SEQ ID NO.:1所示的多核苷酸；
(b)核苷酸序列与SEQ ID NO.:1所示序列的同源性≥90％(较佳地≥95％，更佳地≥98％，最佳地≥99％)，且具有肝脏特异性转录活性的多核苷酸；
(c)如SEQ ID NO.:1所示多核苷酸的5’端和/或3’端增加和/或减少1-50个(较佳地1-20，更佳地1-10个，更佳地1-5个)核苷酸，且具有肝脏特异性转录活性的多核苷酸。
pSyn调控核苷酸序列可以驱动增强下游基因在肝脏细胞中的特异性表达。在本发明中，主要用于增强人凝血因子IX在肝脏细胞中的特异性表达，其转录调控效率和其他肝脏转录调控序列已经在血友病B小鼠模型中进行了比较。除了应用于血友病B的基因治疗外，pSyn调控序列还可用于在肝脏细胞中特异性的表达其他治疗性基因。因此，此段序列对于经肝细胞表达的基因治疗策略都具有非常重要的意义。
在本文中，所述的调控序列包括SEQ ID NO.:1所示调控序列的变体，所述变体可以通过插入或删除调控区域，进行随机或定点突变等来获得。
本发明还包括与本发明的调控序列具有50％或以上(优选60％以上，70％以上，80％以上，更优选90％以上，更优选95％以上，最优选98％以上，如99％)同源性的核酸，所述核酸也具有特异性调控启动植物地上组织表达的功能。“同源性”是指按照位置相同的百分比，两条或多条核酸之间的相似水平(即序列相似性或同一性)。
如本文所用，术语“特异性表达”是指目的基因在特定的时间和/或特定的组织的表达。
如本文所用，“外源的”或“异源的”是指不同来源的两条或多条核酸或蛋白质序列之间的关系。例如，如果启动子与目的基因序列的组合通常不是天然存在的，则启动子对于该目的基因来说是外源的。特定序列对于其所插入的细胞或生物体来说是“外源的”。
本发明的启动子可以被可操作地与外源基因连接，该外源基因相对于启动子而言可以是外源(异源)的。本发明所述的外源基因(也称为目的基因)没有特别的限制，优选的外源基因为经肝细胞表达的用于基因治疗的外源基因，如人凝血因子IX基因。
本发明还提供了包含所述调控序列的核酸构建物和载体。用于制备重组载体的方法是本领域普通技术人员所熟知的。表达载体可以是细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒或其他载体。一种特别优选的载体为AAV8病毒。
本领域普通技术人员可以使用熟知的方法构建含有本发明所述的启动子和/或目的基因序列的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。
pSyn调控序列的构建
为了提高AAV载体介导基因治疗的疗效，对转录调控元件进行了筛选优化。在肝细胞定向基因治疗中，尽管AAV8等病毒载体对肝脏组织具有亲和性并且可以成功感染组织细胞，但也有一定可能感染其他一些非肝脏细胞，结构将产生脱靶表达及相应的不良反应。因此设计筛选的调控元件首要要求是具备肝细胞特异性调控作用。此外，基于双链互补型AAV载体的包装容量只有野生型的一半，为了成功地组装成病毒实体颗粒，调控元件也需要包含尽可能少的碱基数，否则将超过病毒颗粒的包装容量而生成无效产品。转录调控序列主要包含启动子和增强子。启动子负责RNA聚合酶的识别及结合，而增强子包含促进调控转录因子的结合位点，可以增加下游基因的转录效应。
在Nathwani等进行的临床试验中，hFIX序列的调控元件是由人载脂蛋白E肝脏特异性控制区(Human apolipoprotein E-Hepatocyte Control Region,ApoE-HCR)组为增强子，与人α抗胰蛋白酶的启动子组合形成的复合结构序列，并且为了适应AAV的包装容量调整了大小。携带此调控元件的载体属于在肝脏组织中表达效率最强的载体之一，并且成功地在临床试验中体现了一定疗效。然而，这种表达水平只是使患者的表型得到了一定程度缓解，并未完全达到治愈的标准。
为了能够获得更高临床疗效水平的肝脏特异性基因表达，在肝脏特异性基因启动子数据库(The Liver Specific Gene Promoter Database)中进行了检索(http://rulai.cshl.org/LSPD/index.html)。在肝细胞高效特异表达的蛋白中，白蛋白(Albumin)是其中最主要成员之一。由此可见，白蛋白的增强子和启动子具备较多的肝脏特异性调控元素，从而与肝脏细胞中的转录调控系统联系密切。白蛋白的启动子包括转录起始位点至上游217bp之间区域，其序列中包含有不同转录因子的结合位点(表5)，例如肝脏特异性转录因子HNF-1(Hepatocyte nuclear factor 1)(LF-B1,PAF,AFP1)、C/EBP、CTF/NF1、C/EBP和DBP等。而在人白蛋白基因5’端的上游区域至少存在三个增强子，包括启动子上游约-265bp，-1.7kb，以及-6kb区域。这些富含AT和GC区域均属于转录因子，如HNF-1等的主要结合位点，这种蛋白-DNA相互作用对于下游基因 的转录具有明显促进的作用。
表5.白蛋白和甲胎蛋白基因的启动子和增强子中富含AT和GT序列的区域
Figure PCTCN2018114844-appb-000001
转甲状腺素蛋白(transthyretin，TTR)，又称前白蛋白(prealbumin)，也是主要在肝细胞内合成生产的蛋白之一。小鼠TTR基因的启动子长度包含从转录起始位点至5’上游202个碱基对的区域，其中从-108至-202区域属于TTR在肝脏细胞中特异性转录表达必需区域。此外，在5’上游介于-1.86kb至-1.96kb之间有约100bp的DNA片段具有很强的增强子作用，在肝癌细胞株中可以促进近端启动子驱动下的表达效率增强5～10倍。经鉴定，在TTR的启动子和增强子区域包含至少有4种不同转录因子的结合位点，如HNF-1、HNF-3、HNF-4、以及C/EBP等。这些因子大部分属于肝脏特异性转录因子，对于促进下游基因在肝细胞内的表达具有较强的促进作用。
人凝血因子IX(hFIX)的主要合成部位也位于肝脏细胞，其编码基因hF9的启动子定位于-219至+29碱基对之间，属于TATA序列缺乏、转录效率较弱的启动子。然而，经鉴定发现在hF9启动序列中包含有5个顺式调控元件。这些元件中可以与肝脏内含量丰富的转录因子，如C/EBPα、NF1、AR、DBP或HNF-4等，结合从而促进hF9转录。在所有元件中，以5号位点的调控序列(-219至-199)的转录促进作用最强。只携带hF9近端启动子的报告基因质粒虽然表达水平不高，但在加入了数个拷贝的5号位点序列以后，其转录效率可以与包含了HCR调控序列的质粒大致相当32。这些数据目前只是在体外细胞系试验中得出了结论，尚未进行体内试验验证。因此，为了获得生理性表达水平的凝血因子IX，值得尝试应用这些生理性调控序列构建表达框架，以用于在肝细胞内特异性表达hFIX的基因治疗载体中。
以上这些经肝脏细胞特异性合成蛋白的编码基因均有可以借鉴的增强子 序列以进行筛选或合成转录效率高的调控元件以用于基因治疗中的表达框架，介导肝细胞特异性的转录表达。
突变的ITR序列
本发明提供了一种突变的ITR序列(末端反向重复序列)，所述突变的ITR序列含有106个碱基，与3’端野生型ITR序列SEQ ID NO.:5的第25位至第130位之间的碱基序列互补或者相同。
具体地，野生型ITR结构含有145个碱基对，在病毒复制中发挥作用并介导形成单链DNA核心的AAV病毒。本发明对野生型ITR结构进行了调整，在3’端野生型ITR序列的两端各去除部分碱基序列后，删除了其中的末端终止位点(terminal resolution site，TRS)，并将此ITR结构的互补序列构建于表达载体的5’端，从而促进在AAV复制过程中形成互补双链结构，促进转录表达。此结构比Nathwani等改造的ITR结构进一步缩减了数个碱基对，使其仍然可以形成互补双链病毒DNA。综合以上结构，调整而构建的AAV载体的生物活性和安全性在HB小鼠体内进行了系统评估。
人凝血因子IX基因
血友病B(Hemophilia B,HB)属于单基因遗传性疾病，是染色体Xq27.1上的人凝血因子IX编码基因(hF9)缺陷所致的性染色体连锁遗传性出血性疾病，在全球男性中发病率约1/25000。正常的人F9基因全长约34kb，其中编码序列cDNA长度为1383bp，位于X染色体长臂末端Xq27.1-q27.2区域，包含8个外显子和7个内含子。人F9基因缺陷导致血浆中FIX蛋白缺失或功能障碍是导致血友病B的根本病因。经群体分析3721名HB患者及其家系成员，总共鉴定诊断出1113种特异性突变(图1)。在这些突变中，约73％为点突变，呈单个或多个广泛地分布于外显子内(700/812)。大约50％突变致使hFIX蛋白表达障碍或功能缺陷，从而诱发严重的临床表型(表1)。
表1.血友病B患者突变类型和疾病严重程度的相关分析
突变类型/疾病严重程度 总数 重度 中度 轻度 其他
错义突变 558 218 98 100 142
无义突变 77 58 2 1 16
移码突变 130 102 20 3 3
剪切位点突变 83 41 18 6 18
大片段突变(＞50bp) 10 9 1 0 0
小片段突变(＜50bp) 15 11 3 0 1
同义突变 7 0 0 3 4
启动子 18 3 4 5 6
3′UTR 4 0 1 1 2
总数 902 442(49％) 147(16％) 119(13％) 194(22％)
目前中国血友病的发病率为5-10/10万，约15％属于血友病B患者，重症患者占其中一半左右。以目前的医疗经济状况，绝大部分重度HB患者无法承担预防性替代治疗所需的昂贵费用。
基因治疗策略通过腺相关病毒载体AAV导入正常基因在肝细胞内表达凝血因子具有长期治愈的潜能，目前国外的临床试验中初步取得了一定的临床疗效及安全性结果。然而，基因治疗中使用的生物载体还存在一定的优化改进空间。最重要的是，目前国内尚无具有同类知识产权的基因治疗产品。因此，本发明结合国际前沿进展，开展了基因治疗血友病B的研究，开发优化了用于血友病B基因治疗的AAV载体，并且在临床前期的动物模型中证实了其生物学活性和安全性，从而为其临床转化提供了坚实的基础。这个载体的开发在一定程度上填补了我国目前在基因治疗血友病B领域的空白，其临床应用将为基因治疗包括血友病在内的遗传性疾病奠定坚实的理论和实践基础。
此外，本发明中的用于介导在肝细胞中表达致病基因的AAV载体及其中的肝脏特异性调控序列具有通用性。对于其他类型的单基因缺陷性遗传性疾病，如果缺陷基因尺寸适用于AAV载体包装容量，均可通过此载体进行肝脏特异性表达。因此，本发明中涉及的AAV载体及调控元件理论上在基因治疗领域具有非常广泛的适用前景，为未来发展更多的基因治疗策略提供了有利的研究工具。
腺相关病毒载体
AAV载体直径约20-25nm，野生型AAV病毒由蛋白包壳和核酸核心组成，可以包含大小约4.7kb的DNA片段(图2A)。经改造后病毒自身编码基因可以被治疗性基因所取代，从而构成重组AAV病毒载体(图2B)。迄今从人和动物组织中 自然分离鉴定的AAV载体包壳蛋白(capsid protein)有120余种。组织亲和性检测结果显示不同的AAV载体可以对人体多种组织具有亲和性，与此同时同一种组织也可以被不同血清型的AAV载体感染(表2)。对于血友病基因治疗主要的靶向器官肝脏(图3)，AAV2、AAV7、AAV8、AAV9以及AAVrh10均有较高的亲和性，其中AAV8及AAVrh10的感染效率最高，因此已经被应用于血友病基因治疗的临床试验。
表2.不同血清型AAV的组织嗜亲性
Figure PCTCN2018114844-appb-000002
本发明的载体
本发明也涉及包含本发明的pSyn调控序列的载体，以及利用本发明的载体或pSyn调控序列经基因工程产生的宿主细胞。
术语“载体”指本领域熟知的细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒或其他载体。总之，只要能在宿主体内复制和稳定，任何质粒和载体都可以用。对于载体而言，通常含有复制起点、启动子、标记基因和翻译控制元件。优选的，本发明载体为AAV载体。
可以利用本领域技术人员熟知的方法构建本发明的载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的DNA序列可有效连接到本发明载体中的适当启动子上，以指导mRNA合成。本发明载体还包括翻译起始用的核糖体结合位点和转录终止子。
此外，本发明载体优选地包含一个或多个选择性标记基因，以提供用于选择转化的宿主细胞的表型性状，如真核细胞培养用的二氢叶酸还原酶、新霉素抗性以及绿色荧光蛋白(GFP)，或用于大肠杆菌的四环素或氨苄青霉素抗性。
包含上述的适当DNA序列以及适当启动子或者控制序列的载体，可以用于 转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如植物细胞(如农作物和林业植物的细胞)。代表性例子有：大肠杆菌，链霉菌属、农杆菌；真菌细胞如酵母；植物细胞、动物细胞等。优选的，本发明的宿主细胞为人或非人哺乳动物细胞，较佳地为肝脏细胞
本领域一般技术人员都清楚如何选择适当的载体、启动子、增强子和宿主细胞。
本发明的AAV载体
在基因治疗血友病B的策略中，AAV载体具有关键性的传递运输作用。表达治病基因的序列需要经过AAV载体才能在机体血液循环中运输并且特异性定位于特定的靶器官。基因治疗血友病B的AAV载体主要包含由两部分组成：a.治病基因表达框架；b.具有突变ITR结构的AAV结构序列(图4)。本发明通过优化两个组成部分从而达到提高基因治疗中具有治疗性作用的正常凝血功能hFIX的表达水平。具体地，本发明的基因治疗血友病B的肝脏特异性表达腺相关病毒载体(AAV载体)包含：
1.肝脏特异性转录调控序列，pSynthetic(简称pSyn)；
2.序列优化的人凝血因子IX编码序列；
3.优化改良的ITR结构(突变的ITR序列)。
本发明的主要优点包括：
(a)在肝脏组织中特异性高效驱动下游基因的转录表达；
(b)具有长期稳定的在肝脏组织中驱动下游基因表达的活性；
(c)具有较好的安全性，在非肝脏组织中基本不表达，不干扰其他非治病基因的表达水平。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
实施例1
序列优化人F9编码基因
人FIX的表达框架包括hFIX cDNA编码序列，以及增强子/启动子等调控序列(图4A)。首先，对hFIX编码序列进行了密码子优化。密码子优化的原理是通过评估不同密码子使用频率的差异，将同一个氨基酸所对应的在真核细胞中表达频率较高的密码子替换相对使用频率较低的密码子，从而增强基因的转录表达效率。
此外，对hFIX编码序列进行序列优化，以提高mRNA分子结构的稳定程度，降低其被内源性RNase降解的风险。序列优化主要利用OptimumGeneTM分析完成。其中优化的参数包括：(1)基因转录效率调控参数，包括增加偏好性氨基酸密码子，降低或删除利用率低下密码子的使用；(2)调整GC含量，适用区间为30％～70％；(3)修改hFIX的mRNA二级结构，包括减少或删除隐含的剪切位点、环状或分枝等不稳定结构、重复序列、以及适用终止密码子TGA等(图4B)。
为了验证本发明的优化策略是否可以有效的提高hF9基因的表达效率，在同样的转录调控元件的驱动下构建了经优化的hFIX序列，通过尾静脉高压注射血友病B(HB)小鼠，利用物理压力将携带表达框架的质粒导入小鼠肝细胞中。
结果如图5A所示，与野生型cDNA比较，本发明的密码子优化策略使血浆中的hFIX(cohFIX)抗原表达水平提高了约3倍，这种水平已经与临床试验中取得了明显临床疗效的hFIX优化序列(hFIX.ctrl)基本一致。
随后，将携带cohFIX表达框架通过AAV8载体转运至HB小鼠的肝脏组织，血浆定期检测的结果发现，本发明的cohFIX优化序列(AAV.cohFIX)可以在小鼠肝细胞中稳定高效的进行表达抗原和凝血活性，其转录效率与阳性对照(AAV.hFIXctrl)基本一致，其结果有效的缓解了基因治疗小鼠的出血表型(图5B)。这些结果有利地证明了本发明的序列优化策略能够有效的提高hF9基因的转录效率，因此适用于基因治疗中的表达载体。
除了有效的优化cDNA序列，本发明也在编码基因中引入了一个点突变，从而使hFIX蛋白中第338位的精氨酸替换为亮氨酸(hFIX.SIHR338L)。这个突变最初发现于一个意大利家系，当hFIX中第338位的精氨酸被亮氨酸取代，结果导致尽管患者血循环中FIX蛋白水平和正常人相当，但凝血活性达到正常人的5-8倍。经研究发现R338位点是凝血因子X与FIX结合的重要部位，被亮氨酸取代后可以增强两者之间的结合效率，从而明显增强FIX的凝血活性。此外，携带此突变的FIX蛋白的抗纤溶效应也增强。因此，将此突变位点引入cohFIX序列，经AAV载体在HB小鼠肝细胞中表达，结果显示hFIXR338L突变体的表达可以 有效的降低AAV制剂的剂量，在低剂量1×10 11vg/kg基础上即可在小鼠体内产生～100％正常人血浆水平的凝血因子活性和抗原，较野生型AAV载体降低了数倍(图6)。
实施例2
构建并筛选肝脏特异性调控序列
为了提高AAV载体介导基因治疗的疗效，除了对hF9基因进行优化外，还对转录调控元件进行了筛选优化。为了获得最有效率的转录调控，将不同的调控序列进行组合，并与Nathwani等临床试验中AAV载体携带的pLP1调控序列进行了平行比较。
1.人工合成的pSynthetic(pSyn)调控序列，结合了TTR和Alb的调控序列，包含HNF-1、HNF-3、HNF-4、以及C/EBP等多个肝细胞特异性转录因子的结合位点。
2.人凝血因子IX调控序列(pF9)有长短两种不同的版本。其中pF9.v1包含了F9基因-219至+29碱基对之间的序列，而pF9.v2还引入了5个拷贝的增强子序列，该序列在体外细胞系试验中有效的提高了pF9近端启动子的转录效率。
除此之外，还对载体的ITR结构进行了调整。对照型ITR结构(ITR 对照)介导形成单链DNA核心的AAV载体(图7，样本F)，Nathwani等删除了AAV载体一侧的ITR(ITR m1)中近20余个碱基对，从而在AAV包装生产的过程中形成了互补双链DNA结构，促进转录效率(图7，样本A)。在此基础上进一步改造删减了几个碱基对(ITR m2)，但使其保留形成互补双链病毒DNA的能力(图7，样本B-E)。还在hFIX的表达框架中加入了一段人工合成的内含子序列，并且在两端加上splicing donor和splicing acceptor，从而不影响编码序列。综合以上结构调整而构建的AAV载体的生物活性和安全性在HB小鼠体内进行了系统评估。
为了比较确定本发明设计构建的肝脏特异性调控元件是否具有高效稳定的转录驱动效率，构建了包含不同调控元件的hFIX表达框架并且包装成AAV8载体。这些载体经过尾静脉注射后进入HB小鼠体内，并且介导肝脏特异性表达hFIX蛋白。
结果如图8所示，经过活性测定及ELISA分析，显示在近四个月的追踪观察中，注射了内含pSyn调控元件的AAV载体的HB小鼠组血浆中的hFIX抗原和活性水平最高。与LP1调控元件组相比较，pTTR和pF9组的HB小鼠血浆中hFIX的蛋白 和活性水平相对较弱，提示其转录调控效率相对较低。
实施例3
pSyn调控序列的体内剂量增高试验
根据以上结果显示pSyn调控序列有相对较强的肝脏特异性转录活性，因此利用包含pSyn调控序列的AAV.pSyn.cohFIX载体进行了体内剂量增高试验，其中高、中、低浓度组注射的载体剂量分别与Nathwani等设计的临床试验中应用的剂量想对应。
结果显示，血浆中的hFIX抗原和活性从注射载体后一周就开始升高，到第四周达到高峰，并且在4个月的观察周期中维持在相对稳定的水平。此外，血浆中的凝血因子活性和抗原水平随着剂量的增高成正比上升，中剂量组的小鼠血浆中hFIX的活性和浓度可以达到正常人水平的100％左右，而高剂量组已经在正常人水平的600～900％之间(图9)。这些结果证明了AAV.pSyn.cohFIX载体可以有效地在HB小鼠体内长期稳定的表达并发挥其凝血作用。
实施例4
pSyn调控序列的组织特异性试验
为了明确AAV载体介导表达hFIX的组织特异性，部分HB小鼠在注射病毒载体三个月后提取了组织器官进行分析，肝、脾、和胸腺组织中的RNA被提取出来并且分析了hFIX相对内参基因GAPDH的RNA表达水平。
结果如图10所示，人凝血因子IX的RNA主要在肝脏组织中表达，并且表达水平随着载体剂量的升高而增加。与肝脏组织相比，脾脏和胸腺中hFIX RNA表达水平处于基线水平，与注射了AAV.GFP载体的HB小鼠各组织中的RNA水平一致。这些结果说明AAV.pSyn.hFIX载体介导的RNA表达具有肝脏组织特异性。
实施例5
AAV.pSyn.cohFIX载体的生物性分布
随后，分析了AAV载体在HB小鼠体内的生物性分布。根据以往报道的结果，AAV8载体应该主要对小鼠肝脏组织具有亲和性。
结果如图11所示，经分析检测基因治疗小鼠的肝、脾、胸腺、及骨髓组织，病毒DNA主要分布在肝脏组织中，而且分布浓度和载体剂量成正比。此外在脾脏中也有极少量的病毒DNA分布，在高剂量组的平均浓度约0.23拷贝/细胞，在 其他剂量组及其他组织中均与AAV.GFP组HB小鼠中的基线水平差不多一致。结合这些数据以及RNA表达水平的分析结果，说明AAV8结合肝脏特异性调控序列的基因传递策略可以有效的保证目的治病基因在肝脏组织中的特异性表达，本申请中的AAV.pSyn.cohFIX载体具有控制在肝脏内特异性表达hFIX以治疗血友病B的目的。
实施例6
肝功能指标测定
在既往的临床试验中显示AAV载体介导的基因治疗可以诱发机体免疫反应及轻度的肝脏损害，伴随着肝功能指标的上升。比较了AAV载体注射前以及注射后第4、12周的血浆谷草转氨酶(AST)和谷丙转氨酶(ALT)的水平。
结果如图12所示，和未接受基因治疗的HB小鼠血浆内的水平相比(Week 0)，在注射病毒载体后4周或12周的小鼠血浆内的AST或ALT水平都没有显著性升高。这也与血浆内稳定表达的hFIX水平相对应，说明在这些剂量下AAV载体及其表达产物没有诱发明显的肝功能损害。
实施例7
小鼠高凝血活性指标测定
过量的外源性凝血因子可以诱发机体的高凝血状态。为了检测不同剂量AAV载体表达的hFIX是否会产生高凝血风险，检测了血浆中的D二聚体浓度。
结果如图13所示，和基因治疗前水平以及AAV.GFP对照组水平相比，不同剂量下的AAV载体表达凝血因子没有引发过度的凝血反应，证明了在这些剂量下HB小鼠体内不存在高凝血风险。
综合以上数据，本发明构建的AAV.pSyn载体充分具备在肝脏组织中特异性表达目的治病基因的功能。在血友病基因治疗中，在pSyn驱动下可以长期稳定地表达凝血因子IX，从而恢复小鼠的正常凝血功能，达到长期甚至永久治愈的目的。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
Figure PCTCN2018114844-appb-000003

Claims (2)

  1. 一种载体，其特征在于，所述的载体含有权利要求7所述的构建物。
  2. 权利要求1所述的调控序列、权利要求7所述的构建物或权利要求9所述的载体的用途，其特征在于，用于制备一制剂或组合物，所述的制剂或组合物用于调控外源基因或外源DNA片段在肝脏细胞或肝脏组织中进行特异性表达。
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