WO2022198872A1 - Aav介导的人脂蛋白脂酶肝脏异位表达载体及其用途 - Google Patents

Aav介导的人脂蛋白脂酶肝脏异位表达载体及其用途 Download PDF

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WO2022198872A1
WO2022198872A1 PCT/CN2021/109590 CN2021109590W WO2022198872A1 WO 2022198872 A1 WO2022198872 A1 WO 2022198872A1 CN 2021109590 W CN2021109590 W CN 2021109590W WO 2022198872 A1 WO2022198872 A1 WO 2022198872A1
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lpl
gene
aav
sequence
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吴小兵
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北京锦篮基因科技有限公司
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    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
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    • C12Y301/01034—Lipoprotein lipase (3.1.1.34)
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    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
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Definitions

  • the present invention relates to the field of biotechnology. Specifically, the present invention relates to a recombinant adeno-associated virus vector carrying an optimized human lipoprotein lipase gene expression cassette.
  • a recombinant adeno-associated virus vector carrying an optimized human lipoprotein lipase gene expression cassette By intravenous injection of the recombinant adeno-associated virus vector of the present invention, liver-specific expression of lipoprotein lipase can be obtained, thereby preventing and/or treating severe hypertriglyceridemia or diseases caused by it, such as acute pancreatitis.
  • Lipoprotein lipase is a key enzyme in the hydrolysis of plasma triglyceride (TG), and the dysfunction of this enzyme can make plasma chylomicrons, very low-density lipoproteins and other triglyceride-rich lipoproteins ( Triglyceride-rich lipoprotei (TRL) increased, clinical manifestations were chylomicron/and low-density lipoproteinemia, and plasma triglyceride increased.
  • GPIHBP1 glycosylphosphatidylinositol anchored high density lipoprotein binding protein 1
  • GPIHBP1 is located on the vascular endothelial cells of muscle and adipose tissue, and can bind to LPL from muscle cells and adipocytes from the basal space of the vascular endothelium, and transport it to the luminal surface of capillaries, providing an attachment site for LPL in the vascular lumen , enabling LPL to hydrolyze triglycerides on TRL in plasma.
  • the binding of GPIHBP1 to LPL can also keep LPL structurally stable and have high catalytic activity.
  • GPIHBP1 deficiency prevents LPL from being transported to the capillary lumen to hydrolyze TG on TRL, resulting in severe hypertriglyceridemia (Stephen G Young et al. Cell Metab. 2019;30(1):51-65).
  • Stroes et al. injected two doses of an adeno-associated virus vector carrying the human LPL-S447X gene (1 ⁇ 10 11 vg/kg and 3 ⁇ 10 11 vg/kg) into the legs of patients with LPL gene deficiency intramuscularly, and glycerol was detected 12 weeks later Triglyceride levels, the results showed that compared with before treatment, triglyceride levels were reduced by 27% and 41%, respectively. After 26-36 weeks, active LPL protein was detected in the patient's muscle homogenate without any adverse reactions. (Stroes ES et al, Arterioscler Thromb Vasc Biol. 2018. 28(12): 2303-2304).
  • Gaudet et al. performed gene therapy on 14 patients with LPL gene-deficient HTG-AP by intramuscular injection of AAV1-LPL-S447X.
  • the patients' plasma triglyceride decreased to 40% in 12 weeks, and no hypertriglyceridemia was observed after 2 years of clinical observation.
  • Hypertriglyceridemic acute pancreatitis (HTG-AP) recurrence and serious adverse reactions (Glancy D et al., Gene Ther. 2013.20(4):361-369).
  • the European Medicines Agency (EMA) approved the first gene therapy drug Alipogene Tiparvovec (trade name: Glybera) in Western countries.
  • the symptoms are: patients with LPL gene deficiency who still develop severe or repeated HTG-AP after a strict fat-restricted diet.
  • Glybera expresses LPL by local intramuscular injection of AAV1-LPL-S447X viral vector. Although the concentration of AAV1 virus vector at the injection site is very high, it cannot effectively diffuse to the vascular lumen surface of vascular endothelial cells in the muscle.
  • LPL is only expressed locally at the intramuscular injection site, so although LPL activity is sufficiently high at the site of intramuscular injection, The expression of LPL is limited to the local injection site of the muscle, and there are few functional sites on the surface of vascular endothelial cells that decompose plasma triglycerides.
  • the present invention provides an AAV virus vector carrying an LPL gene expression cassette.
  • the AAV viral vector only retains the two ITR sequences or its variants required for packaging the virus in the wild-type AAV viral genome, and does not contain the protein-coding gene in the wild-type AAV viral genome, which makes the AAV viral vector in the administration.
  • the AAV viral vector of the present invention has high transduction efficiency to the liver after a single intravenous injection, which ensures that the LPL gene expression cassette can efficiently express the LPL protein in the liver, thereby providing a new preventive and therapeutic method. / or gene drugs for the treatment of severe hypertriglyceridemia.
  • the present invention provides an optimized human lipoprotein lipase gene expression cassette, comprising:
  • the optimized human lipoprotein lipase gene expression cassette of the present invention further comprises:
  • TBG enhancer sequence as set forth in SEQ ID NO: 11 or having at least about 90% identity thereto.
  • the optimized human lipoprotein lipase gene expression cassette of the present invention has the nucleotide sequence set forth in SEQ ID NO: 12 or a sequence that is at least about 90% identical to SEQ ID NO: 12 .
  • the expression product of the optimized human lipoprotein lipase gene expression cassette of the present invention is a functional lipoprotein lipase (eg, LPL-S447X), which has LPL activity; in addition, the expression cassette can also inhibit the immune response of the organism.
  • a functional lipoprotein lipase eg, LPL-S447X
  • the expression cassette can also inhibit the immune response of the organism.
  • the present invention provides a viral vector comprising the optimized human lipoprotein lipase gene expression cassette of the present invention.
  • the present invention provides a genetic medicine for the prevention and/or treatment of severe hypertriglyceridemia or a disease caused by it, such as acute pancreatitis, wherein the genetic medicine comprises the optimization of the present invention Viral vector for the expression cassette of the human lipoprotein lipase gene.
  • functional human lipoprotein lipase eg, LPL-S447X
  • the severe hypertriglyceridemia or a disease caused by it, such as acute pancreatitis is induced by LPL gene deficiency and/or GPIHBP1 gene deficiency and/or high-fat diet.
  • the viral vectors of the present invention are recombinant adeno-associated viral vectors, including but not limited to those selected from AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.10, or combinations thereof
  • the recombinant adeno-associated virus vector of the serotype is preferably a recombinant AAV5, AAV3B, AAV8, AAV9 vector.
  • the genomes of the viral vectors of the present invention can self-complement themselves to form double-stranded DNA molecules.
  • the present invention provides the use of the optimized human lipoprotein lipase gene expression cassette of the present invention and the viral vector of the present invention for the preparation of prevention and/or treatment of hypertriglyceridemia or diseases caused by it
  • Gene medicines such as acute pancreatitis, for example, for the preparation of prevention and/or treatment of LPL gene deficiency and/or GPIHBP1 gene deficiency and/or high-fat diet-induced hypertriglyceridemia or diseases caused by it, such as acute pancreatitis of genetic medicines.
  • the genetic preventive and/or therapeutic drug of the present invention is administered by single or multiple intravenous injections, preferably by single intravenous injection.
  • a single administration of the gene drug of the present invention can sustainably reduce the plasma triglyceride concentration in the body, thereby preventing and/or treating hypertriglyceridemia, in particular, preventing and/or treating severe Hypertriglyceridemia, eg, hypertriglyceridemia induced by LPL gene deficiency and/or GPIHBP1 gene deficiency and/or high-fat diet, and diseases caused by hypertriglyceridemia such as acute pancreatitis.
  • hypertriglyceridemia eg, hypertriglyceridemia induced by LPL gene deficiency and/or GPIHBP1 gene deficiency and/or high-fat diet, and diseases caused by hypertriglyceridemia such as acute pancreatitis.
  • the viral vector of the present invention is administered by intravenous injection, preferably by a one-time intravenous injection, which reduces the pain caused by repeated drug administration; the ectopic expression of functional LPL in the liver will not produce obvious toxic effects , but can exert a significant effect on the degradation of triglyceride-rich lipoproteins in circulating blood, thereby greatly improving the preventive and/or therapeutic effect.
  • the genome of the viral vector is a double-stranded AAV vector that can self-complement to form a double-stranded DNA molecule, it takes less time to express functional LPL than a single-stranded AAV vector and can function faster in vivo.
  • the viral vector is an AAV5, AAV3B, AAV8 or AAV9 viral vector
  • high-efficiency infection of liver cells increased expression level
  • liver cells increased expression level
  • the high yield of AAV5, AAV3B, AAV8 or AAV9 viral vectors is conducive to reducing production costs, and is expected to have a better cost-effectiveness advantage after the market.
  • the viral vector of the present invention expands the scope and indications of the use of gene drugs, from severe hypertriglyceridemia caused by LPL gene defect to severe hypertriglyceridemia caused by GPIHBP1 gene defect, and high-fat diet resulting in hypertriglyceridemia, or diseases caused by hypertriglyceridemia such as acute pancreatitis.
  • the present invention realizes the efficient and specific expression of functional LPL gene in the liver from multiple different levels by optimizing the structure of the viral vector, reduces the possible immune response, and improves the viral vector as a gene Efficacy and safety of drugs.
  • FIG. 1A Schematic diagram illustrating the structure of the pscAAV-LP15-EGFP-kan-4x142T vector.
  • the left ITR is the inverted terminal repeat sequence of about 145 bp of AAV virus; the right ITR is the ITR (sometimes also marked as ⁇ ITR) with the D sequence and trs sequence of the AAV virus deleted; LP15 promoter: by human AMBP (X67082.
  • EGFP Enhanced Green fluorescent protein: 4 ⁇ 142-3pT: 4 tandem human miR-142-3p target sequence: BGH polyA: polynucleotide tailing of bovine growth hormone Signal: TBG enhancer: thyroxine-binding globulin enhancer ; Kan: kanamycin resistance gene.
  • Figure 1B Schematic diagram illustrating the structure of the pscAAV-LP15-optLPL-S447X-kan-4x142T vector, where optLPL-S447X is the optimized encoding LPL inserted with the intron sequence shown in SEQ ID NO: 2 - Nucleotide sequence of S447X.
  • Figure 1C A schematic diagram illustrating the structure of the pscAAV-LP15-LPL-S447X-kan-4 ⁇ 142T vector, LPL-S447X in the figure is the encoding LPL-S447X without the insertion of the intron sequence shown in SEQ ID NO: 2 Nucleotide sequence.
  • Figure 2 shows the relative mRNA transcription levels of the LPL-S447X gene after the vectors of Figure 1A (control), Figure 1B, and Figure 1C were introduced into hepatocyte Huh7, respectively.
  • the relative mRNA expression levels were calculated by real-time quantitative RT-qPCR using the ⁇ Ct method. Error bars represent standard deviation.
  • Figure 3 shows the plasma triglyceride levels of Lpl -/- mice before injection of scAAV5-EGFP or scAAV5-optLPL (hereinafter also referred to as AAV5-EGFP, AAV5-optLPL, respectively) and on days 7, 14, 28, and 56 after injection Ester (TG) concentration.
  • AAV5-EGFP scAAV5-EGFP
  • AAV5-optLPL scAAV5-optLPL
  • Lpl -/- mice injected with AAV5-optLPL in three dose groups high dose, 1E13 vector genomes (vg)/kg; intermediate dose, 1E12 vg/kg; low dose, 1E11 vg/kg
  • AAV5-EGFP Blood was collected at different time points (0, 7, 14, 28, 56 days) before and after (1E13vg/kg), and the concentration of TG in plasma was detected.
  • Figure 4 shows the plasma total cholesterol (TC) concentrations in Lpl -/- mice before injection of AAV5-EGFP or AAV5-optLPL and on days 7, 14, 28, 56 after injection.
  • Lpl -/- mice injected with AAV5-optLPL in three dose groups high dose, 1E13vg/kg; middle dose, 1E12vg/kg; low dose, 1E11vg/kg
  • AAV5-EGFP (1E13vg/kg) injection was collected at different time points (0, 7, 14, 28, 56 days) to detect the concentration of TC in plasma.
  • Figure 5 shows the plasma LPL activity on day 14 after injection of AAV5-EGFP or AAV5-optLPL in Lpl -/- mice.
  • Lpl -/- mice injected with AAV5-optLPL in three dose groups (high dose, 1E13vg/kg; middle dose, 1E12vg/kg; low dose, 1E11vg/kg) and after injection of AAV5-EGFP (1E13vg/kg)
  • high dose 1E13vg/kg
  • middle dose 1E12vg/kg
  • low dose 1E11vg/kg
  • AAV5-EGFP 1E13vg/kg
  • Fig. 6 shows that the livers of Lpl -/- mice were harvested on day 56 after injection of AAV5-EGFP or AAV5-optLPL, and the levels of optLPL mRNA in the liver were detected.
  • Lpl -/- mice injected with AAV5-optLPL in three dose groups high dose, 1E13vg/kg; middle dose, 1E12vg/kg; low dose, 1E11vg/kg
  • AAV5-EGFP (1E13vg/kg
  • Figure 7A shows the time-dependent changes in plasma TG in Gpihbp1 -/- mice following intravenous injection of recombinant virus.
  • "ns" means no significant difference.
  • Figure 7B shows the time-dependent changes in plasma cholesterol in Gpihbp1 -/- mice following intravenous injection of recombinant virus.
  • Figure 7C shows that the appearance of peripheral blood plasma of Gpihbp1 -/- mice after intravenous injection of recombinant virus for 2 months is no different from that of wild-type C57 mouse plasma.
  • WT-PBS indicates the group administered PBS to wild-type C57 mice
  • GP-KO PBS indicates the group administered PBS to Gpihbp1 -/- mice
  • GP-KO+AAV5-optLPL indicates the group administered scAAV5- to Gpihbp1 -/- mice optLPL-S447X group.
  • Figure 7D shows the plasma LPL concentration of Gpihbp1 -/- mice after intravenous injection of recombinant virus for 2 months.
  • Figure 7E shows the plasma LPL activity of Gpihbp1 -/- mice 2 months after intravenous injection of recombinant virus.
  • Figure 8A shows the results of HE staining of pancreatic tissue when Gpihbp1 -/- mice were intravenously injected with recombinant virus or PBS for 2 months and then administered a very low dose of cerulein (5 ⁇ g/kg/h*10 times).
  • Cae represents cerulein.
  • Figure 8B shows the plasma amylase levels when Gpihbp1 -/- mice were intravenously injected with recombinant virus or PBS for 2 months, followed by very low doses of cerulein (5 ⁇ g/kg/h*10 times).
  • GP represents Gpihbp1 -/- mice.
  • Figure 8C shows the results of HE staining of pancreatic tissue when Gpihbp1 -/- mice were intravenously injected with recombinant virus or PBS for 2 months and then administered a conventional dose of cerulein (50 ⁇ g/kg/h*10 times).
  • Figure 8D shows that after intravenous injection of recombinant virus or PBS to Gpihbp1 -/- mice for 2 months, and then administered a conventional dose of cerulein (50 ⁇ g/kg/h*10 times) at 0, 6, 12, 24 hours, Changes in plasma amylase activity.
  • WT represents wild-type C57 mice
  • Cae represents cerulein
  • GP represents Gpihbp1 -/- mice
  • AAV5-optLPL represents intravenous injection of 1 ⁇ 10 13 vg/kg scAAV5-optLPL-S447X.
  • Figure 8E shows that after intravenous injection of recombinant virus or PBS to Gpihbp1 -/- mice for 2 months, 0, 6, 12, and 24 hours after administration of conventional dose of cerulein (50 ⁇ g/kg/h*10 times), Changes in plasma lipase activity.
  • the meanings of the abbreviations in the figure are the same as those in FIG. 8D .
  • Figure 8F shows that after intravenous injection of recombinant virus or PBS to Gpihbp1 -/- mice for 2 months, 0, 6, 12, and 24 hours after administration of a conventional dose of cerulein (50 ⁇ g/kg/h*10 times), Changes in plasma TG concentrations.
  • the meanings of the abbreviations in the figure are the same as those in FIG. 8D .
  • Figure 8G shows that after intravenous injection of recombinant virus or PBS to Gpihbp1 -/- mice for 2 months, and then administered a conventional dose of cerulein (50 ⁇ g/kg/h*10 times) at 0, 6, 12, 24 hours, Changes in plasma cholesterol concentrations.
  • cerulein 50 ⁇ g/kg/h*10 times
  • Figure 9A shows that intraorbital intravenous injection of AAV5-optLPL virus to neonatal Gpihbp1 -/- rats 7 days after birth resulted in a significant increase in the 28-day survival rate of Gpihbp1 -/- rats.
  • Figure 9B shows that intraorbital intravenous injection of AAV5-optLPL virus to neonatal Gpihbp1 -/- rats 7 days after birth resulted in a significant decrease in plasma TG in Gpihbp1 -/- rats.
  • Figure 9C shows that secondary injection of AAV8-optLPL virus via tail vein to Gpihbp1 -/- rats 8 weeks after birth resulted in a further decrease in plasma TG.
  • Figure 9D shows that intraorbital intravenous injection of AAV5-optLPL virus to neonatal Gpihbp1 -/- rats 7 days after birth resulted in a significant decrease in plasma cholesterol in Gpihbp1 -/- rats.
  • Figure 9E shows that secondary injection of AAV8-optLPL virus via tail vein to Gpihbp1 -/- rats 8 weeks after birth resulted in a further decrease in plasma cholesterol.
  • Figure 9F shows the appearance of plasma from wild-type SD rats, Gpihbp1 -/- rats in the PBS group, and Gpihbp1 - /- rats in the AAV-optLPL treatment group 9 weeks after the first dose of AAV virus vector intravenously.
  • tubules 1, 2, 3 are plasma from wild-type SD rats; tubules 4, 5, 6 are plasma from Gpihbp1 -/- rats + PBS; tubules 7, 8, 9 are from Gpihbp1 -/- large Murine + 1 x 10 11 vg/kg scAAV8-optLPL-S447X plasma; tubules 10, 11, 12 are plasma from Gpihbp1 -/- rats + 1 x 10 13 vg/kg scAAV8-optLPL-S447X.
  • Figure 9G shows the plasma LPL concentrations from wild-type SD rats, PBS group Gpihbp1 -/- rats, and AAV-optLPL treatment group Gpihbp1 -/- rats 9 weeks after the first dose of AAV virus vector intravenously.
  • AAV-optLPL-treated Gpihbp1 -/- rats LPL concentration was increased in a dose-dependent manner with AAV8-optLPL virus.
  • Figure 9H shows the plasma LPL activity from wild-type SD rats, PBS group Gpihbp1 -/- rats, AAV-optLPL treatment group Gpihbp1 -/- rats 9 weeks after the first dose of AAV virus vector intravenously.
  • AAV8-optLPL-treated Gpihbp1 -/- rats LPL activity showed a dose-dependent increase in AAV8-optLPL virus.
  • Figure 10A shows that 14 days after high-fat diet (HFD) feeding, the appearance of the pancreas of Gpihbp1 -/- rats showed obvious encapsulated necrosis.
  • Figure 10B Shows the changes in plasma triglyceride levels in Gpihbp1 -/- rats fed a high-fat diet (HFD) for 14 days.
  • HFD high-fat diet
  • Figure 10C shows changes in plasma cholesterol levels in Gpihbp1 -/- rats fed a high-fat diet (HFD) for 14 days.
  • Figure 10D shows the effect of HE staining on the degree of pancreatic tissue damage in Gpihbp1 -/- rats after 14 days of high-fat diet (HFD) feeding.
  • Figure 10E shows the effect of HE staining on the degree of lung tissue damage in Gpihbp1 -/- rats injected with recombinant AAV virus vector 14 days after high-fat diet (HFD) feeding.
  • Figure 11 Shows plasma TG concentrations before and after high-fat diet feeding in WT hamsters, and after injection of AAV5-EGFP or AAV5-optLPL.
  • 28 days after feeding high-fat diet, and 7, 14, 28 days after injection of AAV5-EGFP or AAV5-optLPL (1E12 vg/kg) 35, 42, 56 from the start of the experiment Day
  • fasted overnight for 12 hours blood was collected, and plasma TG concentration was detected.
  • Figure 12 Shows plasma TC concentrations before and after high-fat diet feeding in WT hamsters, and after injection of AAV5-EGFP or AAV5-optLPL.
  • 28 days after feeding high-fat diet, and 7, 14, 28 days after injection of AAV5-EGFP or AAV5-optLPL (1E12 vg/kg) 35, 42, 56 from the start of the experiment Day
  • fasted overnight for 12 hours blood was collected, and the plasma TC concentration was detected.
  • Figure 13 shows an oral lipid loading test on day 14 after injection of AAV5-EGFP or AAV5-optLPL in WT hamsters. On the 14th day after injection of AAV5-EGFP or AAV5-optLPL (1E12vg/kg), WT hamsters were fasted overnight for 12 hours, and zero-point blood was collected.
  • Figure 14 shows plasma LPL activity at day 28 after injection of AAV5-EGFP or AAV5-optLPL in WT hamsters.
  • AAV5-EGFP or AAV5-optLPL 1E12vg/kg
  • WT hamsters were intravenously injected with heparin, and the plasma after heparin was collected, and the activity of plasma LPL was detected with a kit.
  • Figure 15 shows the detection of optLPL mRNA in the liver of WT hamsters after injection of AAV5-EGFP or AAV5-optLPL on day 30.
  • WT hamsters were harvested on the 30th day (58th day after the start of the experiment) after injection of AAV5-EGFP or AAV5-optLPL (1E12vg/kg), and after perfusion with PBS, the liver tissue was taken, RNA was extracted, and the mRNA of optLPL in the liver was detected by qPCR.
  • the invention discloses a gene medicine for preventing and/or treating severe hypertriglyceridemia, and relates to the design, small-scale preparation and function verification of the gene medicine.
  • lipoprotein lipase is a lipase that lipolysis lipoprotein triglycerides.
  • dietary fats are absorbed, packaged into triglyceride-rich lipoprotein particles, and transported through the organism's circulatory system to appropriate peripheral tissues, which are essential for maintaining the organism's metabolic homeostasis, Defects or dysregulation of these processes can lead to metabolic diseases such as diabetes, obesity and hyperlipidemia.
  • lipolysis of lipoprotein triglycerides requires LPL. As the lipolysis occurs on the luminal surface of capillary endothelial cells, the lipolysis of LPL is partially regulated by the protein GPIHBP1.
  • GPIHBP1 a GPI-anchored protein of capillary endothelial cells, is responsible for the transport of LPL across endothelial cells to the capillary lumen. Without this transport, LPL mislocalizes in the subendothelial interstitial space and fails to reach triglyceride-rich lipoproteins, resulting in severe hypertriglyceridemia.
  • the human LPL gene is located on chromosome 8 p22, about 30kb, consisting of 10 exons and 9 introns, encoding a 475 amino acid protein including a 27 amino acid signal peptide.
  • the LPL gene is mainly expressed in the heart, skeletal muscle and adipose tissue.
  • the messenger RNA encoding LPL is about 3560bp in length, and its signal peptide consists of 27 amino acids.
  • Human mature LPL consists of 448 amino acid residues.
  • the term "functional LPL” refers to a human LPL with full-length wild-type (native) (eg, NCBI protein database (https://www.ncbi.nlm.nih.gov/protein/) accession number NP_000228. 1), variants thereof (e.g., variants with conservative amino acid substitutions), fragments thereof, said variants or fragments providing at least about 10%, at least about 20%, at least about 30%, At least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, or about the same, or greater than 100% of full-length wild-type (natural ) biological activity levels of human LPL.
  • native eg, NCBI protein database (https://www.ncbi.nlm.nih.gov/protein/) accession number NP_000228. 1
  • variants thereof e.g., variants with conservative amino acid substitutions
  • fragments thereof e.g.
  • LPL variants include, but are not limited to, LPL-S447X, which is obtained by removing the last two amino acids from the C-terminus of wild-type (native) human LPL.
  • the term "conservative amino acid substitution” or “conservative amino acid substitution” refers to changing, replacing, or substituting an amino acid with a different amino acid having similar biochemical properties (eg, charge, hydrophobicity, and size), which is a skill in the art known to the personnel.
  • LPL dysfunction results in elevated plasma triglycerides.
  • GPIHBP1 which is closely related to LPL function, also prevents LPL from being transported to the capillary lumen to hydrolyze TG on TRL, resulting in severe hypertriglyceridemia.
  • Acute pancreatits (AP) is a common acute abdomen with a high mortality rate, in which lipoprotein lipase dysfunction and/or GPIHBP1 deficiency are critical for hyperchylomicronemia, hypertriglyceridemia ( Hypertriglyceridemia, HTG) and consequent acute pancreatitis are the most significant.
  • a “signal sequence” is a sequence of amino acids linked to the N-terminal portion of a protein that facilitates secretion of the protein out of the cell.
  • the mature form of the extracellular protein has no signal sequence, which is excised during the secretion process.
  • N-terminal refers to the last amino acid of the N-terminal
  • C-terminal refers to the last amino acid of the C-terminal
  • operably linked means that the specified components are in a relationship that allows them to function in their intended manner.
  • the sequences are aligned for optimal comparison purposes (e.g., between the first and second amino acid sequences or nucleic acid sequences for optimal alignment. Gaps are introduced in one or both or non-homologous sequences can be discarded for comparison purposes).
  • the length of the reference sequences aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60% and even more preferably at least 70%, 80% , 90%, 100% of the reference sequence length.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position.
  • Sequence comparisons and calculation of percent identity between two sequences can be accomplished using mathematical algorithms.
  • the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm (at http://www.gcg.com) is used that has been integrated into the GAP program of the GCG software package available), using the Blossum 62 matrix or the PAM250 matrix and gap weights 16, 14, 12, 10, 8, 6, or 4 and length weights 1, 2, 3, 4, 5, or 6, to determine the distance between two amino acid sequences percent identity.
  • the GAP program in the GCG software package (available at http://www.gcg.com) is used, using the NWSgapdna.CMP matrix and gap weights 40, 50, 60, 70 or 80 and A length weight of 1, 2, 3, 4, 5, or 6 determines the percent identity between two nucleotide sequences.
  • a particularly preferred set of parameters (and one that should be used unless otherwise specified) is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.
  • nucleic acid sequences and protein sequences described herein can be further used as "query sequences" to perform searches against public databases, eg, to identify other family member sequences or related sequences.
  • regulatory sequence refers to a nucleic acid sequence that induces, inhibits or otherwise controls the transcription of a protein of an encoding nucleic acid sequence to which it is operably linked. Regulatory sequences can be, for example, initiation sequences, enhancer sequences, intron sequences, promoter sequences, and the like.
  • exogenous nucleic acid or protein refers to the location of the nucleic acid or protein that is not naturally present in the chromosome or host cell in which it occurs.
  • An exogenous nucleic acid sequence also refers to a sequence derived from and inserted into the same host cell or subject, but present in a non-native state, eg, in a different number of copies, or under the control of different regulatory elements.
  • AAV Addeno-associated virus
  • AAV is a dependent virus that requires helper proteins from other viruses such as adenovirus, herpes simplex virus, human papillomavirus, or cofactors to replicate.
  • AAV2 AAV serotype 2
  • ITRs inverted terminal repeats
  • ORFs open reading frames
  • the ITR is a cis-acting element of the AAV vector genome and plays an important role in the integration, rescue, replication and genome packaging of the AAV virus.
  • the ITR sequence contains a Rep binding site (RBS) and a terminal resolution site (trs), which can be recognized by the Rep protein and generate a nick at the trs.
  • the ITR sequence can also form a unique "T" letter-shaped secondary structure, which plays an important role in the life cycle of AAV viruses.
  • the rest of the AAV2 genome can be divided into two functional regions, the rep gene region and the cap gene region.
  • the rep gene region encodes four Rep proteins, Rep78, Rep68, Rep52 and Rep40.
  • Rep protein plays an important role in the replication, integration, rescue and packaging of AAV virus.
  • Rep78 and Rep68 specifically bind to the terminal melting site trs and GAGY repeat motif in ITR, and initiate the replication process of AAV genome from single-strand to double-strand.
  • the trs and GAGC repeat motifs and/or GAGY repeat motifs in the ITR are the centers of AAV genome replication, so although the ITR sequences are different in various serotypes of AAV viruses, they can all form hairpin structures and have Rep binding. site.
  • There is the p19 promoter at position 19 of the AAV2 genome map which promotes the expression of Rep52 and Rep40, respectively.
  • Rep52 and Rep40 have ATP-dependent DNA helicase activity, but no DNA binding function.
  • the cap gene encodes the capsid proteins VP1, VP2 and VP3 of AAV virus.
  • VP3 has the smallest molecular weight but the largest quantity.
  • the ratio of VP1, VP2 and VP3 in mature AAV particles is approximately 1:1:10.
  • VP1 is required for the formation of infectious AAV;
  • VP2 assists the entry of VP3 into the nucleus;
  • VP3 is the main protein that makes up AAV particles.
  • AAV vector is a highly efficient tool for exogenous gene transfer, that is, AAV vector, which is transformed from wild-type AAV virus with the understanding of the life cycle of AAV virus and its related molecular biological mechanisms. .
  • the modified AAV vector genome only contains the ITR sequence of the AAV virus and the expression cassette carrying the foreign gene to be transported.
  • the Rep and Cap proteins required for AAV virus packaging are provided in trans by other exogenous plasmids, thereby reducing the possible harm caused by the packaging of the rep and cap genes into the AAV vector.
  • the AAV virus itself is not pathogenic, which makes the AAV vector recognized as one of the safest viral vectors.
  • Deleting the D sequence and the trs sequence in the ITR sequence on one side of the AAV virus can also make the genome carried by the packaged recombinant AAV virus vector self-complement to form double strands, which significantly improves the transduction efficiency of the AAV vector in vitro and in vivo (Wang Z et al. , Gene Ther. 2003; 10(26): 2105-2111; McCarty DM et al., Gene Ther. 2003; 10(26): 2112-2118).
  • the packaged virus becomes scAAV (self-complementary AAV) virus, the so-called double-stranded AAV virus. It is different from ssAAV (single-stranded AAV) in which both ITRs are not mutated, that is, conventional AAV viruses.
  • the packaging capacity of the scAAV viral vector is smaller, only half of the packaging capacity of the ssAAV viral vector, about 2.2kb-2.5kb, but the transduction efficiency is higher after infecting cells.
  • AAV virus serotypes There are many AAV virus serotypes, and different serotypes have different tissue infection tropisms, so the application of AAV vectors can transfer foreign genes to specific organs and tissues (Wu Z et al., Mol Ther. 2006; 14(3): 316-327). Some serotype AAV vectors can also cross the blood-brain barrier and introduce exogenous genes into brain neurons, providing the possibility for brain-targeted gene transduction (Samaranch L et al., Hum Gene Ther. 2012; 23 (4 ): 382-389).
  • AAV vectors are stable, showing strong resistance to acids, alkalis and high temperatures (Gruntman AM et al., Hum Gene Ther Methods. 2015; 26(2): 71-76), and it is easy to develop stable high-quality biological products.
  • AAV vector packaging systems mainly include three-plasmid co-transfection system, adenovirus as a helper virus system, Herpes simplex virus type 1 (HSV1) as a helper virus packaging system, and baculovirus-based packaging system.
  • HSV1 Herpes simplex virus type 1
  • baculovirus-based packaging system baculovirus-based packaging system.
  • Each packaging system has its own characteristics, and those skilled in the art can make appropriate selections as required.
  • the three-plasmid transfection packaging system is the most widely used AAV vector packaging system because it does not require helper virus and has high safety. It is also the mainstream production system in the world. A slight disadvantage is that the lack of efficient large-scale transfection method limits the application of the three-plasmid transfection system in the large-scale preparation of AAV vectors.
  • Yuan et al. established a large-scale AAV packaging system using adenovirus as a helper virus (Yuan Z et al., Hum Gene Ther. 2011; 22(5): 613-624).
  • the system has high production efficiency, but the adenovirus in the packaging system is not Finally, the presence of traces in the finished AAV affects the safety of the finished AAV.
  • HSV1 as the packaging system of helper virus is another widely used AAV vector packaging system.
  • Wu Zhijian and Conway et al. proposed an AAV2 vector packaging strategy using HSV1 as a helper virus at the same time (Wu Zhijian, Wu Xiaobing et al., Scientific Bulletin, 1999, 44(5): 506-509; Conway JE et al., Gene Ther. 1999, 6:986-993).
  • Wustner et al. proposed an AAV5 vector packaging strategy using HSV1 as a helper virus (Wustner JT et al., Mol Ther. 2002, 6(4): 510-518).
  • HSV1s carrying the rep/cap gene of AAV and the reversed terminal repeat (ITR)/exogenous gene expression cassette of AAV respectively, and then co-infected with these two recombinant HSV1 viruses Producer cells, packaged to produce AAV virus (Booth MJ et al., Gene Ther. 2004; 11:829-837). Thomas et al. further established a suspension cell system for dual HSV1 virus AAV production (Thomas DL et al., Gene Ther. 2009; 20:861-870), enabling larger-scale AAV virus production.
  • Urabe et al. used three baculoviruses to carry AAV structural genes, non-structural genes and ITR/exogenous gene expression cassettes respectively, and constructed a baculovirus packaging system for AAV vectors. Considering the instability of baculoviruses carrying foreign genes, the number of baculoviruses required in the production system was subsequently reduced, gradually from the initial need of three baculoviruses to the need of two or one baculovirus ( Chen H., Mol Ther. 2008, 16(5): 924-930; Galibert L. et al., J Invertebr Pathol.
  • AAV vectors Due to the above characteristics, AAV vectors have gradually become an exogenous gene transfer tool that is widely used in gene therapy, especially in the gene therapy of genetic diseases. As of August 2016, there are 173 clinical trials of AAV vector-based gene therapy approved in the world (http://www.abedia.com/wiley/vectors.php). More importantly, the AAV vector-based lipoprotein lipase gene therapy drug Glybera was approved by the European Medicines Agency in 2012, becoming the first gene therapy drug approved in the Western world ( S., Mol Ther. 2012; 20(10): 1831-1832); hemophilia B (Kay MA et al., Nat Genet.
  • vector genome refers to the nucleic acid sequences packaged within the rAAV capsid to form the rAAV vector.
  • the vector genome comprises at least the AAV2 5' ITR from 5' to 3', a nucleic acid sequence encoding a functional LPL, and the AAV2 3' ITR. It is also possible to select ITRs from AAVs from different sources other than AAV2.
  • the vector genome may contain regulatory sequences that direct the expression of functional LPL.
  • microRNA refers to a single-stranded non-coding RNA of 18 to 25 nucleotides (nt) in length that is widely present in humans and animals.
  • miRNAs were first discovered in C. elegans in 1993.
  • the lin-4 gene in Caenorhabditis elegans can downregulate the expression of the lin-14 gene, but the encoded product of the lin-4 gene is not a protein, but a small RNA molecule, which indicates that the self-encoded small RNA molecule can regulate the expression of the gene .
  • a variety of similar small RNA molecules have been found in different species and cells, and miRNA began to become the general term for such small RNAs.
  • miRNAs regulate the expression of approximately 60% of human genes (Friedman RC et al., Genome Res. 2009; 19:92-105) and play important roles in a variety of physiological and pathological processes.
  • miRNA genes are typically located in exons, introns, and intergenic regions of the genome (Olena AF et al., J Cell Physiol. 2010; 222:540-545; Kim VN et al., Trends Genet. 2006; 22:165- 173). In cells, miRNAs are produced as described below. First, in the nucleus, the transcription of miRNA genes is initiated by RNA polymerase II or III to generate the initial product pri-microRNA; the pri-microRNA self-folds partial sequence to form a stem-loop structure.
  • the processing complex composed of ribonuclease III Drosha and DGCR8 molecules acts on the pri-microRNA, cutting off the redundant sequence, leaving a stem-loop structure of about 60 nt, that is, the precursor miRNA molecule pre-microRNA.
  • the pre-microRNA enters the cytoplasm from the nucleus, and is processed by Dicer enzyme to remove the ring part in its stem-loop structure and become a double-stranded RNA molecule.
  • double-stranded RNA molecules are bound by protein factors such as AGO2, one of the strands is degraded, and the other strand and the protein factor form an RNA-induced silencing complex (RISC).
  • RISC RNA-induced silencing complex
  • RISC recognizes target sequences in mRNA, reduces mRNA expression levels by degrading mRNA molecules, promoting deadenylation at the 3' end of mRNA molecules, and inhibiting translation, regulating gene expression at the post-transcriptional level (Fabian MR et al., Annu Rev Biochem 2010;79:351-379). Therefore, using a highly expressed miRNA in the cell to insert the target sequence of the miRNA into the 3' UTR (untranslated region) of the exogenous gene can effectively inhibit the expression of the exogenous gene in the introduced cell.
  • miR-142-3p is a miRNA that is highly expressed in hematopoietic stem cell line-derived cells.
  • Immune cells are all differentiated from hematopoietic stem cell lines, so the principle of miRNA inhibition of gene expression is used (Kim VN. Nat Rev Mol Cell Biol. 2005; 6(5): 376-385), the gene carrying the miR-142-3p target sequence Expression is significantly inhibited in immune cells, thereby reducing the probability of the body developing an immune response against the gene expression product (Dismuke DJ et al., Curr Gene Ther. 2013;13(6):434-452).
  • treatment refers to a clinical intervention intended to alter the natural course of disease in an individual being treated. Desired therapeutic effects include, but are not limited to, preventing disease occurrence or recurrence, reducing symptoms, reducing any direct or indirect pathological consequences of disease, reducing the rate of disease progression, improving or alleviating disease state, and relieving or improving prognosis.
  • prevention includes the inhibition of the occurrence or progression of a disease or symptoms of a particular disease.
  • subjects with a family history of high triglycerides are candidates for preventive regimens.
  • prevention refers to the administration of a drug prior to the onset of hypertriglyceridemia, particularly in subjects with LPL gene deficiency and/or GPIHBP1 gene deficiency.
  • the present invention provides a functional human lipoprotein lipase gene expression cassette comprising a nucleotide sequence encoding a functional LPL and a regulatory sequence directing its expression.
  • the expression cassette comprises a nucleotide sequence encoding a functional LPL as described herein and regulatory sequences directing its expression.
  • the nucleotide sequence encoding a functional LPL is at least 70%, 80%, 90% identical to SEQ ID NO: 1, eg, at least 95%, 96%, 97%, 98% identical , 99% or higher identity.
  • the nucleotide sequence encoding a functional LPL is inserted with the intron sequence set forth in SEQ ID NO:2 or inserted with at least about 90% identity to SEQ ID NO:2 (e.g., A nucleotide sequence encoding a functional LPL (eg, LPL-S447X) having at least 95%, 96%, 97%, 98%, 99% or greater identity to intron sequences.
  • SEQ ID NO:2 e.g., A nucleotide sequence encoding a functional LPL (eg, LPL-S447X) having at least 95%, 96%, 97%, 98%, 99% or greater identity to intron sequences.
  • nucleotide sequence encoding a functional LPL is selected from
  • the regulatory sequences directing the expression of functional LPL comprise liver-specific promoters, eg, albumin promoter, hepatitis B virus core promoter, alpha fetoprotein (AFP) promoter, CMV promoter, and the like.
  • the regulatory sequence comprises or is at least about 90% identical to the LP15 promoter sequence set forth in SEQ ID NO: 5 (eg, at least 95%, 96%, 97%, 98%, 99% or more) identity) of the promoter sequence.
  • the use of the LP15 promoter can prevent the CMV promoter from being easily silenced by methylation when the CMV promoter enters the body for expression, improves the expression level of functional LPL, and better achieves the specific expression of liver target organs.
  • the regulatory sequences further comprise sequences that reduce the expression of functional LPL in immune-related cells (eg, antigen-presenting cells), attenuate the immune response elicited by the expression of functional LPL, thereby significantly reducing response to exogenous Probability of an immune response to an LPL protein, such as one or more (eg, 1-8, 2-7, 3, 4, 5, or 6) sequences in tandem with miR-142-3p Complementary target sequences, whereby the expression cassettes of the invention are transcribed to yield untranslated regions (e.g., 5' untranslated regions and/or 3' untranslated regions) of mRNAs encoding functional LPL (e.g., LPL-S447X) It contains human miR-142-3p target sequence, which can effectively inhibit the expression of functional LPL (for example, LPL-S447X) in immune-related cells (such as antigen-presenting cells) and inhibit the immune response.
  • functional LPL for example, LPL-S447X
  • immune-related cells such
  • the regulatory sequence comprises one or more expression enhancers, eg, TBG enhancers, CMV enhancers, and the like.
  • the regulatory sequence comprises or is at least about 90% identical (eg, at least 95%, 96%, 97%, 98%, 99% or more) as set forth in SEQ ID NO: 11 therewith high identity) of the TBG enhancer sequence.
  • the regulatory sequence further comprises a polyadenylation signal (polyA), eg, human growth hormone (hGH) polyadenylation sequence, SV40 polyA, BGH polyA.
  • polyA polyadenylation signal
  • hGH human growth hormone
  • SV40 polyA SV40 polyA
  • BGH polyA BGH polyA
  • the regulatory sequence comprises or is at least about 90% identical (eg, at least 95%, 96%, 97%, 98%, 99% or more) as set forth in SEQ ID NO: 10 therewith high identity) of the BGH polyA sequence.
  • the present invention provides a viral vector, which is an artificial recombinant viral particle, wherein a replication-defective viral genome sequence comprising an expression cassette encoding a functional LPL is packaged in a viral capsid or envelope,
  • the recombinant virus particles are thus unable to produce progeny virions, but retain the ability to infect target cells.
  • the genomic sequence of the viral vector does not contain genes encoding enzymes required for viral replication, therefore, it is considered safe to use viral vectors in gene therapy because the enzymes required for viral replication are absent in the absence of , the replication and infection of progeny virions will not occur.
  • the recombinant viral vector of the present invention may be a recombinant adeno-associated virus (AAV), adenovirus, Bocavirus, AAV/Bocavirus hybrid, herpes simplex virus or lentivirus.
  • AAV adeno-associated virus
  • Bocavirus Bocavirus
  • AAV/Bocavirus hybrid herpes simplex virus or lentivirus.
  • Packaging cell lines for the production of recombinant viral vectors can be prokaryotic or eukaryotic cells (eg, human cells, insect cells, or yeast cells) containing cells by any means (eg, electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, transfection and protoplast fusion) foreign DNA introduced into cells.
  • Packaging cell line cells include, but are not limited to, E. coli cells, yeast cells, human cells, non-human cells, mammalian cells, non-mammalian cells, insect cells, HEK293 cells, hepatocytes, kidney cells, glial cells, or stem cells.
  • target cell refers to a target cell in which it is desired to express functional LPL.
  • target cells include, but are not limited to, hepatocytes, muscle cells.
  • the vector is delivered to target cells in vivo.
  • the viral vector is a recombinant adeno-associated virus (rAAV) vector comprising an AAV capsid and a vector genome packaged therein.
  • the rAAV vector is used to treat LPL gene deficiency and/or GPIHBP1 gene deficiency and/or high-fat diet-induced hypertriglyceridemia or diseases caused by it, such as acute pancreatitis.
  • the vector genome comprises an AAV 5' inverted terminal repeat (ITR) or AAV 5' ⁇ ITR, a nucleic acid sequence encoding a functional LPL, regulatory sequences directing LPL expression in target cells, and an AAV 3'ITR or AAV 3' ⁇ ITR.
  • the ⁇ ITR is the ITR with the D sequence and terminal melting site trs deleted.
  • the ITR is the genetic element responsible for the replication and packaging of the genome during vector production and is the only viral cis-element required for the production of rAAV. ITRs from different sources of AAV can be selected. In one embodiment, the ITR is from an AAV that is different from the capsid of the viral particle.
  • the AAV capsids, ITRs and other AAV components described herein can be readily selected from any AAV, including but not limited to those commonly identified as AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV of serotype AAV8, AAV9, AAVrh.10, or a combination thereof.
  • the AAV capsid is an AAV5 capsid or a variant thereof.
  • the AAV capsid is an AAV3B capsid or a variant thereof.
  • the AAV capsid is an AAV8 capsid or a variant thereof.
  • the AAV capsid is an AAV9 capsid or a variant thereof.
  • the capsid protein is designated by a number or a combination of numbers and letters following the term "AAV" in the rAAV vector name.
  • an rAAV comprising an AAV serotype 5 (AAV5) capsid; and a vector comprising SEQ ID NO: 12 or a sequence at least about 90% identical to SEQ ID NO: 12 Genome.
  • AAV5 AAV serotype 5
  • an rAAV comprising an AAV serotype 3B (AAV3B) capsid; and a vector comprising SEQ ID NO: 12 or a sequence at least about 90% identical to SEQ ID NO: 12 Genome.
  • AAV3B AAV serotype 3B
  • an rAAV comprising an AAV serotype 8 (AAV8) capsid; and a vector comprising SEQ ID NO: 12 or a sequence at least about 90% identical to SEQ ID NO: 12 Genome.
  • an rAAV comprising an AAV serotype 9 (AAV9) capsid; and a vector comprising SEQ ID NO: 12 or a sequence at least about 90% identical to SEQ ID NO: 12 Genome.
  • an AAV “variant” refers to any AAV sequence derived from known AAV sequences, including those AAV sequences with conservative amino acid substitutions, and at least 70%, at least 75% identical to the amino acid or nucleic acid sequence of AAV , at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or greater sequence identity.
  • the AAV capsid includes variants that may contain up to about 10% variation compared to any described or known AAV capsid sequence. That is, the AAV capsid is about 90% to about 99.9% identical, about 95% to about 99% identical or about 97% to about 99% identical to AAV capsids provided herein and/or known in the art About 98% identical.
  • the AAV capsid is at least 95%, 96%, 97%, 98%, 99% or more identical to the AAV capsid variant.
  • any variant protein eg, vp1, vp2, or vp3 can be compared.
  • the recombinant adeno-associated virus (AAV) vectors of the present invention can be produced using known techniques. Such methods involve culturing packaging cells comprising a nucleic acid sequence encoding an AAV capsid; a functional rep gene; an expression cassette as described herein, flanked by an AAV inverted terminal repeat (ITR) or ⁇ ITR; and sufficient helper function to allow packaging of expression cassettes into AAV capsid proteins.
  • AAV adeno-associated virus
  • packaging cells comprising a nucleic acid sequence encoding an AAV capsid; a functional rep gene; an expression cassette as described herein, flanked by an AAV inverted terminal repeat (ITR) or ⁇ ITR; and sufficient helper functions , to allow packaging of the expression cassette into the AAV capsid protein.
  • the host cell is a HEK 293 cell.
  • Suitable methods may include, but are not limited to, baculovirus expression systems or production by yeast.
  • the viral vector of the present invention can express functional LPL for preventing and/or treating severe hypertriglyceridemia ectopic and specific in the liver through intravenous injection.
  • functional LPL is ectopic expression in the liver of adult patients that does not express LPL gene itself, and the expressed functional LPL can achieve hydrolysis of TG in plasma independent of GPIHBP1, Normalizes higher triglyceride levels.
  • the viral vector of the present invention can be used to prevent and/or treat hypertriglyceridemia or diseases caused by it, such as acute pancreatitis.
  • the viral vector of the present invention can be used to prevent and/or treat LPL gene deficiency and/or GPIHBP1 gene deficiency and/or high-fat diet-induced hypertriglyceridemia or diseases caused by it, such as acute pancreas inflammation.
  • an AAV plasmid vector containing the target gene, the target gene expression control element, and the ITR sequence was constructed.
  • the pUC57-optLPL-S447X vector was constructed first.
  • the intron (82 bp in length) of the immunoglobulin heavy chain expression gene in human chromosome 14 as shown in SEQ ID NO: 2 was inserted into the LPL-S447X coding sequence shown in SEQ ID NO: 1.
  • the intron sequence divides the LPL-S447X coding sequence into two parts (referred to as: LPL-S447X CDS part1 and LPL-S447X CDS part2), and after inserting the intron sequence, the LPL-S447X CDS part1-intron is obtained
  • the intron-LPL-S447X CDS part2 sequence, shown in SEQ ID NO: 3 is also referred to herein as "optLPL-S447X” or "optimized LPL-S447X” or simply "optLPL".
  • an intron-uninserted LPL-S447X coding sequence (ie, the nucleotide sequence shown in SEQ ID NO: 1) was also synthesized, hereinafter also referred to as "intron-uninserted LPL-S447X" .
  • the LPL-S447X coding sequence (SEQ ID NO: 1) without intron insertion and the nucleotide sequence (SEQ ID NO: 3) of optLPL-S447X were sent to Nanjing GenScript Biotechnology Co., Ltd. for synthesis, and respectively Clone into pUC57 simple vector (Nanjing GenScript Biotechnology Co., Ltd.) to obtain pUC57-LPL-S447X plasmid vector and pUC57-optLPL-S447X plasmid vector.
  • the pscAAV-LP15-EGFP-kan-4 ⁇ 142T plasmid vector shown in Figure 1A was constructed (hereinafter the vector is also referred to as the pscAAV-LP15-EGFP vector), and its sequence is shown in SEQ ID NO: 4, Include:
  • LP15 promoter the sequence of the LP15 promoter is shown in SEQ ID NO: 5, which consists of the enhancer sequence of human AMBP (X67082.1), the enhancer sequence of human ALB (M12523.1) and the length of 128bp
  • the promoter sequence and the mouse parvovirus (MVM) intron are spliced together;
  • 4x142-3pT 4 tandem human miR-142-3p target sequences, also abbreviated as "4x142-3pT".
  • the sequence of 4 ⁇ 142-3pT is shown in SEQ ID NO:7;
  • BGH polyA the polynucleotide tailing signal of bovine growth hormone, also abbreviated as BGH polyA;
  • thyroxine-binding globulin thyroxine-binding globulin, TBG gene enhancer sequence
  • EGFP which is a nucleotide sequence expressing enhanced green fluorescent protein.
  • the pUC57-optLPL-S447X vector was digested with KpnI and EcoRI to generate two fragments of 2.7 kb and 1507 bp, and the fragment with a length of 1507 bp was recovered, which was the coding sequence of optLPL-S447X.
  • the constructed pscAAV-LP15-EGFP-kan-4 ⁇ 142T vector shown in Figure 1A was digested with KpnI and EcoRI to obtain two fragments of 5403bp and 748bp, and a fragment with a length of 5403bp was recovered.
  • the pUC57-LPL-S447X vector was digested with KpnI and EcoRI, resulting in two fragments of 2.7 kb and 1425 bp, and a fragment of 1425 bp in length was recovered.
  • the pscAAV-LP15-EGFP-kan-4 ⁇ 142T vector shown in Figure 1A was digested with KpnI and EcoRI to obtain two fragments of 5403bp and 748bp, and a fragment with a length of 5403bp was recovered. The obtained fragment of 1425bp in length and the fragment of 5403bp in length obtained by connecting, obtain the carrier shown in Fig.
  • the vector is abbreviated as pscAAV-LP15-LPL-S447X vector.
  • Hepatocytes Huh7 obtained from ATCC were evenly seeded in 24-well plates at 1 ⁇ 10 5 cells/well and incubated overnight in a 37° C. CO 2 incubator to allow cells to adhere.
  • the pscAAV-LP15-optLPL-S447X plasmid and the pscAAV-LP15-LPL-S447X plasmid were transfected into Huh7 cells by lipofection method.
  • the pscAAV-LP15-EGFP plasmid was also transfected into Huh7 cells.
  • the transfection conditions were 1 ⁇ l Lipofectamine 2000: 2 ⁇ g plasmid DNA, the medium was changed 6 h after transfection, and the cells were harvested after culturing in a cell incubator for 48 h. Total cell RNA was extracted, and LPL-S447X mRNA was detected by RT-qPCR. and optLPL-S447X mRNA transcription.
  • the specific process is as follows:
  • LPL-F 5'-TCAACCACAGCTCCAAGACC-3' (SEQ ID NO:8)
  • LPL-R 5'-GTATAGCCGGCGGACACTG-3' (SEQ ID NO:9)
  • the LPL-S447X gene fragment with a length of 173 bp was specifically amplified using LPL-F and LPL-R as primers, using SYBR Green dye-binding method, using SYBR Premix Ex Taq II (Tli RNaseH Plus) reagent (Takara, Dalian, China) , using a fluorescence quantitative PCR instrument (model: ABI 7500fast, ABI) to detect. Please refer to the instruction manual of SYBR Premix Ex Taq II (Tli RNaseH Plus) reagent for the operation process.
  • the HEK293 packaging system was used to co-transfect the three plasmids to package the recombinant AAV virus, and the scAAV5-LP15-opt LPL-S447X recombinant virus was obtained.
  • the pAAV-R2C5 plasmid was constructed.
  • AAV Helper Free System Agilent Technologies, catalog number #240071
  • standard molecular cloning methods were used to encode the capsid protein in the synthetic AAV5 genome (GenBank ID: NC_006152.1).
  • the sequence also known as Cap5 (sequence positions 2207 to 4381 in the genome) replaces the sequence between the HindIII to PmeI restriction sites in the pAAV-RC plasmid (i.e., replaces positions 2013 to 4220 of the pAAV-RC plasmid sequence), the pAAV-R2C5 plasmid was obtained.
  • the pAAV-R2C5 plasmid contains the complete cap gene of AAV5 and the rep gene of AAV2, and provides 4 kinds of Rep proteins (Rep78, Rep68, Rep52 and Rep40) necessary for packaging in the preparation of recombinant AAV5 virus by co-transfection and packaging of three plasmids.
  • AAV5 capsid protein The pAAV-R2C5 plasmid contains the complete cap gene of AAV5 and the rep gene of AAV2, and provides 4 kinds of Rep proteins (Rep78, Rep68, Rep52 and Rep40) necessary for packaging in the preparation of recombinant AAV5 virus by co-transfection and packaging of three plasmids.
  • AAV5 capsid protein The pAAV-R2C5 plasmid contains the complete cap gene of AAV5 and the rep gene of AAV2, and provides 4 kinds of Rep proteins (Rep78, Rep68, Rep52 and Rep40) necessary for packaging in the preparation of recombinant AAV5 virus by co
  • AAV vector plasmid (pscAAV-LP15-optLPL-S447X vector) constructed in Example 1, the helper plasmid (pHelper) (from AAV Helper Free System, Agilent Technologies) and the Rep and Cap protein expression plasmid pAAV-R2C5 of AAV, After mixing the three plasmids at a molar ratio of 1:1:1, HEK293 cells were co-transfected by calcium phosphate method. After 48 hours of transfection, the cells and culture supernatant were harvested, and the cesium chloride density gradient centrifugation method was applied.
  • pHelper from AAV Helper Free System, Agilent Technologies
  • the recombinant AAV virus was isolated and purified to obtain scAAV5-LP15-optLPL-S447X recombinant virus. Since the used plasmid pAAV-R2C5 expresses the AAV5 capsid protein, an AAV5 recombinant virus is obtained.
  • the genomic titer of the AAV5 recombinant virus obtained by the above preparation method was determined by quantitative PCR.
  • the specific process is as follows:
  • LP15-F 5'-AAGTGGCCCTTGGCAGCATCT-3' (SEQ ID NO: 13)
  • LP15-R 5'-GGACAAACGGAGGGAAAATTAGCACT-3' (SEQ ID NO: 14)
  • LP15-F and LP15-R as primers to specifically amplify the LP15 promoter with a length of 229 bp
  • SYBR Green dye binding method 1 ⁇ g/ ⁇ l pscAAV-LP15-optLPL-S447X plasmid and its 10-fold serial dilution were used.
  • the sample was a standard, and the SYBR Premix Ex Taq II (Tli RNaseH Plus) reagent (Takara, Dalian, China) was used to detect the viral genome titer using a fluorescence quantitative PCR instrument (model: ABI 7500fast, ABI).
  • the AAV vector plasmid pscAAV-LP15-EGFP constructed in Example 1 a helper plasmid (pHelper) (from AAV Helper Free System, Agilent Technologies) and AAV Rep and Cap protein expression plasmid pAAV-R2C5 were used , scAAV5-LP15-EGFP recombinant virus was prepared, and the virus titer was detected.
  • pHelper from AAV Helper Free System, Agilent Technologies
  • Example 3 Intravenous injection of recombinant AAV virus to treat hypertriglyceridemia in LPL gene-deficient mice
  • Conditional LPL knockout mice were introduced from Air Force Military Medical University. Twenty adult Lpl -/- mice, 8-12 weeks old, weighing 18-25 g were used. After the purchase, they were acclimated in the laboratory animal room for one week, then weighed, and blood was collected from the medial canthus with a glass capillary, anticoagulated with heparin, centrifuged at 4°C, 4000 rpm for 10 minutes, and the plasma was separated. Triglyceride (TG) and total cholesterol (TC) were detected using a triglyceride and cholesterol detection kit (purchased from: Zhongsheng Beikong Biotechnology Co., Ltd.).
  • Lpl -/- mice were divided into 4 groups with 5 mice in each group.
  • the administration settings were: one intravenous injection of scAAV5-LP15-optLPL-S447X virus (referred to as AAV5-optLPL virus) in three dose groups (high dose 1E+13vg/kg; middle dose 1E+12vg/kg; low dose 1E+ 11 vg/kg) and a control group with an intravenous injection of scAAV5-LP15-EGFP virus (1E+13 vg/kg) (AAV5-EGFP for short).
  • Example 2 According to the dose of recombinant AAV virus to be injected intravenously and the AAV virus titer detected in Example 2, calculate the required volume of recombinant AAV virus solution injected into each group of mice, and then use sterile PBS to dilute the volume to 100 ⁇ L. Mice in each group were administered the corresponding recombinant AAV virus by tail vein injection.
  • Gpihbp1 -/- mice were purchased from the Jackson Laboratory and housed in the Animal Room of the Department of Comparative Medicine, Eastern Theater General Hospital. 12-week-old adult Gpihbp1 -/- mice were randomly divided into two groups: AAV treatment group and PBS control group, with 15 mice in each group.
  • the AAV treatment group was given tail vein injection of AAV5-optLPL recombinant virus at a dose of 1 ⁇ 10 13 vg/kg.
  • the PBS control group was injected with the same volume of PBS.
  • the plasma LPL concentration of Gpihbp1 -/- mice in the AAV treatment group increased by nearly 10 times compared with the Gpihbp1 -/- mice in the PBS control group after heparin (Fig. 7D), and the plasma LPL activity increased about 2 times. (Fig. 7E).
  • Example 5 Tail vein injection of AAV5-optLPL in Gpihbp1 -/- mice attenuates the susceptibility and severity of cerulein-induced hypertriglyceridemia acute pancreatitis
  • HMG-AP hypertriglyceridemia acute pancreatitis
  • Gpihbp1 -/- mice were treated with tail vein injection of AAV5-optLPL for 2 months in Example 4, and AAV was injected
  • a very low-dose cerulein Cae: 5 ⁇ g/kg/h*10 needles
  • Cae a conventional dose cerulein
  • HE hematoxylin and eosin
  • Example 4 and Example 5 show that Gpihbp1 -/- mice can effectively express and produce LPL protein after injection of AAV5-optLPL virus through the tail vein, increase the concentration and activity of circulating LPL, thereby reducing Gpihbp1 -/-
  • the content of triglycerides in the plasma of mice can eliminate hypertriglyceridemia caused by excessive triglycerides in the blood, reduce the susceptibility and severity of hypertriglyceridemia acute pancreatitis, and achieve the treatment of Objectives of hypertriglyceridemia and its comorbid acute pancreatitis.
  • Example 6 Intravenous injection of AAV-optLPL in Gpihbp1 -/- rats to treat hypertriglyceridemia, reduce the mortality of suckling mice, and alleviate HFD feeding-induced HTG-AP
  • the Gpihbp1 -/- rat animal model was constructed by designing a target on exon 2 of the GPIHBP1 gene and knocking out the 102bp fragment through TALEN gene editing technology. Provided by the team of director Li Weiqin of the center. It was identified as having a severe hyperlipidemia phenotype, with a sharp increase in blood lipid levels from lactation to more than 10,000 mg/dl, spontaneous HTG-AP, and a 28-day survival rate of only 60%. Wild-type SD rats (purchased from Nanjing Qinglongshan Animal Breeding Farm) were used as controls.
  • the surviving adult Gpihbp1 -/- rats gradually increased the blood lipid level and the pancreas inflammation pathology with the increase of rat age.
  • the acute outbreak of HTG-AP and encapsulated necrosis of the pancreas after feeding with high-fat diet (HFD) resulted in a mortality rate of 42.3% after 28 days of HFD feeding.
  • HFD high-fat diet
  • mice were given AAV5-optLPL virus lipid-lowering therapy via orbital intravenous injection 7 days after birth, at a dose of 1 ⁇ 10 13 vg/kg.
  • the mice were injected with the same volume of PBS, observed for 28 days, and the survival curve was recorded.
  • Gpihbp1 -/- rats treated with AAV5-optLPL virus lipid-lowering treatment during lactation increased with age, increased body weight, and the amount of viral vector in the body was relatively insufficient, and the blood lipid level gradually recovered.
  • AAV8-optLPL virus was injected twice through tail vein, and divided into high-dose group (1 ⁇ 10 13 vg/kg) and low-dose group (1 ⁇ 10 11 vg/kg).
  • the secondary injected AAV8-optLPL virus was prepared as follows:
  • the pAAV-R2C8 plasmid was constructed.
  • AAV Helper Free System Agilent Technologies, catalog number #240071
  • standard molecular cloning methods were used to encode the capsid protein in the synthetic AAV8 genome (GenBank ID: NC_006261.1).
  • the sequence also known as Cap8 (sequence positions 2121 to 4337 in the genome) replaces the sequence between the HindIII to PmeI restriction sites in the pAAV-RC plasmid (ie, replaces positions 2013 to 4220 of the pAAV-RC plasmid sequence), the pAAV-R2C8 plasmid was obtained.
  • the pAAV-R2C8 plasmid contains the complete cap gene of AAV8 and the rep gene of AAV2, and provides four Rep proteins (Rep78, Rep68, Rep52 and Rep40) necessary for packaging in the preparation of recombinant AAV8 virus by co-transfection and packaging of three plasmids.
  • AAV8 capsid protein The pAAV-R2C8 plasmid contains the complete cap gene of AAV8 and the rep gene of AAV2, and provides four Rep proteins (Rep78, Rep68, Rep52 and Rep40) necessary for packaging in the preparation of recombinant AAV8 virus by co-transfection and packaging of three plasmids.
  • AAV8 capsid protein The pAAV-R2C8 plasmid contains the complete cap gene of AAV8 and the rep gene of AAV2, and provides four Rep proteins (Rep78, Rep68, Rep52 and Rep40) necessary for packaging in the preparation of recombinant AAV8 virus by co-transfection and packaging of
  • AAV vector plasmid (pscAAV-LP15-optLPL-S447X vector) constructed in Example 1, the helper plasmid (pHelper) (from AAV Helper Free System, Agilent Technologies) and the Rep and Cap protein expression plasmid pAAV-R2C8 of AAV, After mixing these three plasmids at a molar ratio of 1:1:1, HEK293 cells were co-transfected by calcium phosphate method. After 48 hours of transfection, the cells and culture supernatant were harvested, and the cesium chloride density gradient centrifugation method was applied.
  • pHelper from AAV Helper Free System, Agilent Technologies
  • the recombinant AAV8 virus was isolated and purified to obtain scAAV8-LP15-optLPL-S447X recombinant virus (also referred to as "AAV8-optLPL virus"). Since the plasmid pAAV-R2C8 used expresses the AAV8 capsid protein, the AAV8 recombinant virus is obtained. The genome titer of the AAV8 recombinant virus obtained by the above-mentioned preparation method was measured by the quantitative PCR method in the same manner as in Example 2.
  • Spontaneous HTG-AP model was established in Gpihbp1 -/- rats treated with a second injection of AAV8-optLPL virus through the tail vein for 2 weeks and fed a high-fat diet (Research Diet 12492) for 14 days. Blood was collected from the medial canthal vein at different time points (1 day, 3 days, 7 days, 14 days) during HFD feeding to detect the changes of plasma triglyceride (TG) and total cholesterol (CHO). Peripheral blood, pancreatic tissue and lung tissue were collected after 14 days of HFD feeding.
  • TG plasma triglyceride
  • CHO total cholesterol
  • Circulating LPL concentrations and activities were detected in plasma after deheparinization 9 weeks after the first dose of AAV viral vector intravenous injection (2 weeks after the second dose of AAV viral vector intravenous injection), compared with untreated Gpihbp1 -/- rats, AAV- Gpihbp1 -/- rats had a 10-fold increase in LPL concentration (Fig. 9G) and a 2-3-fold increase in LPL activity (Fig. 9H) after optLPL treatment. After centrifugation, the appearance of lipid blood in plasma was significantly reduced, which was close to that of normal rat plasma (Fig. 9F).
  • Gpihbp1 -/- rats treated with AAV5-optLPL and AAV8-optLPL were fed HFD, the blood lipid level increased rapidly after 3 days of stress, and the plasma triglyceride level in the low-dose AAV8-optLPL treatment group increased for 3 days As high as more than 5000 mg/dl, and then gradually decreased to about 2000 mg/dl (Fig. 10B); plasma cholesterol level increased slightly at first and then gradually decreased (Fig. 10C); pancreatic histopathological HE showed inflammatory cell infiltration and mild pancreatic necrosis formed, significantly reducing the severity of HTG-AP.
  • the plasma triglyceride level was slightly increased to 1000 mg/dl; the plasma cholesterol level was maintained at a low level; the appearance of the pancreatic tissue was basically normal, and the pathological HE showed no inflammatory cell infiltration and no pancreatic necrosis formation.
  • the occurrence of HTG-AP (Fig. 10D).
  • Example 7 Intravenous injection of recombinant AAV virus to treat hamster hyperlipidemia model induced by high-fat diet
  • a high-fat feed with a cholesterol content of 0.5% and a lard content of 20% was ordered from Beijing Botai Hongda Biotechnology Co., Ltd.
  • the high-fat diet was started after the hamsters were acclimated for one week (day 0 of the experiment).
  • the high-fat diet was fed for 4 weeks (the 28th day of the experiment), blood was taken after an overnight fast for 12 hours, and plasma TG and TC were measured. , TC no significant difference as the principle), 6 in each group.
  • mice One group of mice was given a jugular vein injection of scAAV5-EGFP (referred to as AAV5-EGFP), and the other group of mice was given a jugular vein injection of scAAV5-optLPL-S447X (referred to as AAV5-optLPL).
  • AAV5-EGFP a jugular vein injection of scAAV5-optLPL-S447X
  • the injection volume was calculated, and the volume was adjusted to 300 ⁇ L with sterile PBS before injection.
  • blood On the 7th, 14th, and 28th days after injection, blood was collected by overnight fasting, centrifuged to obtain plasma, and plasma TG and TC were detected by kits.
  • Oral lipid loading test was performed on the 14th day after administration, that is, after overnight fasting, blood was collected (recorded as 0-point blood), and then the hamsters were given 10 mL/kg of olive oil by gavage. Blood was collected at 2, 4, and 8 hours, and plasma TG was measured. The results showed that the postprandial clearance of TG in the hamsters injected with AAV5-optLPL was significantly accelerated (Fig. 13).
  • the samples were taken on the 30th day after administration (ie, the 58th day of the experiment), and the mRNA expression level of the exogenous LPL gene in the liver was detected, and the mRNA expression of the exogenous LPL gene was detected in the AAV5-optLPL group ( FIG. 15 ).

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Abstract

提供了一种优化的人脂蛋白脂酶基因表达框以及包含所述优化的人脂蛋白脂酶基因表达框的重组腺相关病毒载体。所述基因表达框主要包含：LP15启动子（SEQ ID NO:5）、插入有内含子（SEQ ID NO:2）的脂蛋白脂酶LPL-S447X（SEQ ID NO:3）、串联的miR-142-3p靶序列（SEQ ID NO:7）。所述病毒载体作为基因药物，通过静脉注射，在肝脏特异性表达人脂蛋白脂酶，可预防和/或治疗严重高甘油三酯血症或由其引起的疾病如急性胰腺炎。

Description

AAV介导的人脂蛋白脂酶肝脏异位表达载体及其用途 技术领域
本发明涉及生物技术领域。具体地，本发明涉及一种携带优化的人脂蛋白脂酶基因表达框的重组腺相关病毒载体。通过静脉注射本发明的重组腺相关病毒载体，能够获得脂蛋白脂酶的肝脏特异性表达，由此预防和/或治疗严重高甘油三酯血症或由其引起的疾病如急性胰腺炎。
背景技术
脂蛋白脂酶(lipoprotein lipase,LPL)是水解血浆甘油三酯(triglyceride，TG)的关键酶,该酶功能障碍可使血浆乳糜微粒、极低密度脂蛋白等富含甘油三酯的脂蛋白(triglyceride-rich lipoprotei，TRL)增高，临床表现为乳糜微粒/及低密度脂蛋白血症,血浆甘油三酯升高。
与LPL密切相关的是糖基磷脂酰肌醇锚定的高密度脂蛋白结合蛋白1(glycosylphosphatidylinositol anchored high density lipoprotein binding protein 1，GPIHBP1)。GPIHBP1位于肌肉和脂肪组织的血管内皮细胞上，能够从血管内皮的基底间隙与来自肌肉细胞和脂肪细胞的LPL结合，并将其转运至毛细血管腔面，在血管腔内为LPL提供附着位点，使得LPL能够水解血浆中TRL上的甘油三酯。GPIHBP1与LPL的结合还能够使LPL保持结构稳定和较高的催化活性。GPIHBP1缺陷会使得LPL无法转运至毛细血管腔发挥水解TRL上TG的作用，造成严重的高甘油三酯血症(Stephen G Young等人，Cell Metab.2019；30(1):51-65)。
现有技术中报导了通过肌肉注射重组AAV1病毒载体(所述AAV1是肌肉靶向性高的腺相关病毒载体)介导的人LPL-S447X表达能够有效治疗LPL基因缺陷小鼠，具有良好的安全性，可长期(约1年)降低血浆甘油三酯，呈剂量依赖性(Ross CJ等人，Hum Gene Ther.2012；15(9):906-919)。Carpentier等人发现肌肉注射AAV1-LPL-S447X可降低LPL基因缺陷患者餐后乳糜微粒水平(Carpentier AC等人，J Clin Endocrinol Metab.97(5):1635-1644)。Stroes等人向LPL基因缺陷患者腿部肌肉注射两种剂量的携带人LPL-S447X基因的腺相关病毒载体(1×10 11vg/kg和3×10 11vg/kg)，12周后检测甘油三酯水平,结果表明，与治疗前相比较，甘油三酯水平分别降低27％、41％，26-36周后,在患者肌肉匀浆中检测到有活性的LPL蛋白，无任何不良反应出现(Stroes ES等人，Arterioscler Thromb Vasc Biol.2018.28(12):2303-2304)。Gaudet等对14例LPL基因缺陷型HTG-AP患者通过肌肉注射施用AAV1-LPL-S447X进行基因治疗，患者血浆甘油三酯在12周下降到40％，经过2年临床观察无高甘油三酯血症性胰腺炎(hypertriglyceridemic acute pancreatitis,HTG-AP)复发和严重不良反应(Glancy D等人，Gene Ther.2013.20(4):361-369)。鉴于前期临床研究结果证实了该基因治疗方法的安全性和有效性，2012年，欧洲药品局(EMA)批准了西方国家第一个基因治疗药物Alipogene Tiparvovec(商品名：Glybera)，该药的适应症为：严格限制脂肪饮食后仍然发生严重或反复HTG-AP的LPL基因缺陷患者。
Glybera是通过肌肉局部注射AAV1-LPL-S447X病毒载体来表达LPL。虽然AAV1病毒载体在注射部位浓度很高，但不能有效扩散至肌肉中血管内皮细胞的血管腔面，需要在患者大腿 股四头肌注射二百多个点，不仅需要的AAV1病毒载体的量非常大，而且造成了患者不便依从；此外，肌肉转导AAV1-LPL-S447X对降低体内甘油三酯作用有限：LPL只是在肌肉注射局部表达，因此尽管在肌肉的注射位置处LPL活性足够高，但LPL表达仅限于肌肉的局部注射部位，在分解血浆甘油三酯的血管内皮细胞表面的功能部位很少，其降低血浆甘油三酯的疗效有限，长期观察发现难以将血浆甘油三酯降至11.3mmol/L以下。因此Glybera在上市4年后并没有真正用于治疗临床病人，而是在4年后由生产厂家宣布退出了市场。
所以本领域仍需要预防和/或治疗高甘油三酯血症的备选基因疗法，并且通过多方优化，将这种备选基因疗法达到单次或多次注射后，能够长期表达功能性LPL、且表达的功能性LPL可有效扩散至患者的能够有效分解血浆甘油三酯的组织或器官。
发明概述
本发明提供了一种携带LPL基因表达框的AAV病毒载体。所述AAV病毒载体仅保留了野生型AAV病毒基因组中包装病毒所需要的两个ITR序列或其变体，不含有野生型AAV病毒基因组中的蛋白编码基因，这使得所述AAV病毒载体在施用于患者后的免疫原性低；此外，AAV病毒载体通常以不整合的染色体外遗传物质形式实现携带外源基因读框的持续稳定表达，避免了导入生物体的外源基因随机整合而带来的安全性问题。
本发明的AAV病毒载体在单次静脉注射后，对肝脏具有较高的转导效率，保证了LPL基因表达框能够在肝脏内高效地表达LPL蛋白，由此，提供了一种新的预防和/或治疗严重高TG血症的基因药物。
在第一方面，本发明提供了一种优化的人脂蛋白脂酶基因表达框，其包含：
(1)如SEQ ID NO:5所示的启动子序列或者与其具有至少约90％同一性的启动子序列；
(2)插入有SEQ ID NO:2所示的内含子序列或者插入有与SEQ ID NO:2具有至少约90％同一性的内含子序列的编码功能性脂蛋白脂酶(例如，LPL-S447X)的核苷酸序列，例如，
(i)如SEQ ID NO:3所示的优化的编码LPL-S447X的核苷酸序列；
(ii)与如SEQ ID NO:3所示的核苷酸序列互补的核苷酸序列；
(iii)与(i)或(ii)的核苷酸序列编码相同的LPL-S447X，但因遗传密码的简并性而与(i)或(ii)的核苷酸序列不同的核苷酸序列；或
(iv)与(i)或(ii)或(iii)所述核苷酸序列具有至少70％同一性的序列；和
(3)至少一个(例如，2－8个)SEQ ID NO:6所示的人miR-142-3p靶序列，例如，具有4个串联的人miR-142-3p靶序列，例如，SEQ ID NO:7所示的人miR-142-3p靶序列。
在一个实施方案中，本发明的优化的人脂蛋白脂酶基因表达框还包含：
(4)如SEQ ID NO:10所示的或者与其具有至少约90％同一性的BGH polyA序列；和/或
(5)如SEQ ID NO:11所示的或者与其具有至少约90％同一性的TBG增强子序列。
在一个具体实施方案中，本发明的优化的人脂蛋白脂酶基因表达框具有如SEQ ID NO:12所示的核苷酸序列或与SEQ ID NO:12具有至少约90％同一性的序列。
本发明的优化的人脂蛋白脂酶基因表达框的表达产物为功能性脂蛋白脂酶(例如，LPL-S447X)，具有LPL活性；此外，所述表达框还能够抑制生物体的免疫反应。
在第二方面，本发明提供了一种病毒载体，其包含本发明的优化的人脂蛋白脂酶基因表达框。
在第三方面，本发明提供了一种用于预防和/或治疗严重高甘油三酯血症或其引起的疾病如急性胰腺炎的基因药物，其中所述基因药物是包含本发明的优化的人脂蛋白脂酶基因表达框的病毒载体。通过静脉注射本发明的所述基因药物，能够在肝脏特异性表达功能性人脂蛋白脂酶(例如，LPL-S447X)，发挥预防和/或治疗严重高甘油三酯血症或其引起的疾病如急性胰腺炎的目的。在一些实施方案中，所述严重高甘油三酯血症或其引起的疾病如急性胰腺炎是LPL基因缺陷和/或GPIHBP1基因缺陷和/或高脂饮食诱发的。
在一些实施方案中，本发明的病毒载体是重组腺相关病毒载体，包括但不限于选自AAV1、AAV2、AAV3B、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAVrh.10或它们的组合的血清型的重组腺相关病毒载体，优选地为重组AAV5、AAV3B、AAV8、AAV9载体。
在一些实施方案中，本发明的病毒载体的基因组可以自我互补形成双链DNA分子。
在第四方面，本发明提供了本发明的优化的人脂蛋白脂酶基因表达框和本发明的病毒载体的用途，用于制备预防和/或治疗高甘油三酯血症或其引起的疾病如急性胰腺炎的基因药物，例如，用于制备预防和/或治疗LPL基因缺陷和/或GPIHBP1基因缺陷和/或高脂饮食诱发的高甘油三酯血症或其引起的疾病如急性胰腺炎的基因药物。
在一些实施方案中，本发明的基因预防和/或治疗药物的给药方式为单次或多次静脉注射，优选地为单次静脉注射。
在一些实施方案中，本发明的基因药物的一次给药可持续降低体内的血浆甘油三脂浓度，由此预防和/或治疗高甘油三酯血症，特别地，预防和/或治疗严重的高甘油三酯血症，例如，由LPL基因缺陷和/或GPIHBP1基因缺陷和/或高脂饮食诱发的高甘油三酯血症，以及由高甘油三酯引起的疾病如急性胰腺炎。
发明的技术效果
本发明的病毒载体通过静脉注射方式给药，优选地，通过一次性静脉注射方式给药，减少了反复施用药物所带给患者的痛苦；在肝脏异位表达功能性LPL不会产生明显毒性作用，却可对循环血液中富含甘油三酯脂蛋白的降解发挥明显效应，由此大大提高了预防和/或治疗效果。当病毒载体的基因组是可以自我互补形成双链DNA分子的双链AAV载体时，较单链AAV载体表达功能性LPL所需的时间更少，可以在体内更快地发挥作用。
进一步地，当病毒载体是AAV5、AAV3B、AAV8或AAV9病毒载体时，可以实现对肝脏细胞的高效感染(提高表达水平)，提高了药物有效性。而且，AAV5、AAV3B、AAV8或AAV9病毒载体的产量高，利于降低生产成本，预期上市后有更好的性价比优势。
本发明的病毒载体扩大了基因药物使用的范围和适应症，从LPL基因缺陷引起的严重高甘油三酯血症，扩大至GPIHBP1基因缺陷引起的严重高甘油三酯血症、以及由高脂饮食导致的高甘油三酯血症、或由高甘油三酯血症引起的疾病如急性胰腺炎。
因此，本发明通过对病毒载体结构的优化，从多个不同的层次共同实现了功能性LPL基因在肝脏的高效、特异性表达，同时降低了有可能引起的免疫反应，提高了病毒载体作为基因 药物的有效性和安全性。
附图简述
结合以下附图一起阅读时，将更好地理解以下详细描述的本发明的优选实施方案。出于说明本发明的目的，图中显示了目前优选的实施方案。然而，应当理解本发明不限于图中所示实施方案的精确安排和手段。
图1A：例示了pscAAV-LP15-EGFP-kan-4×142T载体的结构示意图。左侧ITR为AAV病毒的约145bp的反向末端重复序列；右侧ITR为删除了AAV病毒的D序列和trs序列的ITR(有时也标记为ΔITR)；LP15启动子：由人AMBP(X67082.1)的增强子序列、人ALB(M12523.1)的增强子序列和长度为128bp的启动子序列以及小鼠微小病毒(MVM)内含子(大小为67bp)拼接而成；EGFP：增强型绿色荧光蛋白：4×142-3pT：4个串联的人miR-142-3p靶序列：BGH polyA：牛生长激素的多聚核苷酸加尾信号：TBG增强子：甲状腺素结合球蛋白增强子；Kan：卡那霉素抗性基因。
图1B：例示了pscAAV-LP15-optLPL-S447X-kan-4×142T载体的结构示意图，图中的optLPL-S447X是插入有SEQ ID NO:2所示的内含子序列的经优化的编码LPL-S447X的核苷酸序列。
图1C：例示了pscAAV-LP15-LPL-S447X-kan-4×142T载体的结构示意图，图中的LPL-S447X是未插入SEQ ID NO:2所示的内含子序列的编码LPL-S447X的核苷酸序列。
图2：显示了图1A(对照)、图1B、图1C的载体分别导入肝细胞Huh7后，LPL-S447X基因的相对mRNA转录水平。以GAPDH作为内参，通过实时定量RT-qPCR，用ΔΔCt法计算mRNA相对表达水平。误差棒表示标准差。
图3：显示了Lpl -/-小鼠注射scAAV5-EGFP或scAAV5-optLPL(以下也分别简称为AAV5-EGFP、AAV5-optLPL)前，注射后第7，14，28，56天，血浆甘油三酯(TG)浓度。Lpl -/-小鼠注射AAV5-optLPL的高中低3个剂量组(高剂量，1E13个载体基因组(vg)/kg；中间剂量，1E12vg/kg；低剂量，1E11vg/kg)和注射AAV5-EGFP(1E13vg/kg)前后不同时间点(0，7，14，28，56天)采血，检测血浆中的TG浓度。
图4：显示了Lpl -/-小鼠注射AAV5-EGFP或AAV5-optLPL前，注射后第7，14，28，56天，血浆总胆固醇(TC)浓度。Lpl -/-小鼠注射AAV5-optLPL的高中低3个剂量组(高剂量，1E13vg/kg；中间剂量，1E12vg/kg；低剂量，1E11vg/kg)和注射AAV5-EGFP(1E13vg/kg)前后不同时间点(0，7，14，28，56天)采血，检测血浆中的TC浓度。
图5：显示了Lpl -/-小鼠注射AAV5-EGFP或AAV5-optLPL后第14天血浆LPL活性。Lpl -/-小鼠注射AAV5-optLPL的高中低3个剂量组(高剂量，1E13vg/kg；中间剂量，1E12vg/kg；低剂量，1E11vg/kg)和注射AAV5-EGFP(1E13vg/kg)后第14天，静脉注射肝素，取肝素后血浆，检测血浆LPL活性。
图6：显示了Lpl -/-小鼠注射AAV5-EGFP或AAV5-optLPL后第56天取肝脏，并检测肝脏optLPL mRNA水平。Lpl -/-小鼠注射AAV5-optLPL的高中低3个剂量组(高剂量，1E13vg/kg；中间剂量，1E12vg/kg；低剂量，1E11vg/kg)和注射AAV5-EGFP(1E13vg/kg)后第56天，PBS 灌流后，取肝组织，提取RNA，通过qPCR检测肝脏中optLPL的mRNA。
图7A：显示了Gpihbp1 -/-小鼠静脉注射重组病毒后的血浆TG随时间的变化。图中，“n.s.”表示无显著性差异。
图7B：显示了Gpihbp1 -/-小鼠静脉注射重组病毒后的血浆胆固醇随时间的变化。
图7C：显示了Gpihbp1 -/-小鼠静脉注射重组病毒2个月后，小鼠外周血血浆外观与野生型C57小鼠血浆无差别。图中WT-PBS表示对野生型C57小鼠施用PBS组；GP-KO PBS表示对Gpihbp1 -/-小鼠施用PBS组；GP-KO+AAV5-optLPL表示对Gpihbp1 -/-小鼠施用scAAV5-optLPL-S447X组。
图7D：显示了Gpihbp1 -/-小鼠静脉注射重组病毒2个月后的血浆LPL浓度。
图7E：显示了Gpihbp1 -/-小鼠静脉注射重组病毒2个月后的血浆LPL活性。
图8A：显示了对Gpihbp1 -/-小鼠静脉注射重组病毒或PBS 2个月后，再施用极低剂量雨蛙素(5μg/kg/h*10次)时，胰腺组织的HE染色结果。图中，Cae表示雨蛙素。
图8B：显示了对Gpihbp1 -/-小鼠静脉注射重组病毒或PBS 2个月后，再施用极低剂量雨蛙素(5μg/kg/h*10次)时，血浆的淀粉酶水平。图中，GP表示Gpihbp1 -/-小鼠。
图8C：显示了对Gpihbp1 -/-小鼠静脉注射重组病毒或PBS 2个月后，再施用常规剂量雨蛙素(50μg/kg/h*10次)时，胰腺组织的HE染色结果。
图8D：显示了对Gpihbp1 -/-小鼠静脉注射重组病毒或PBS 2个月后，再施用常规剂量雨蛙素(50μg/kg/h*10次)后第0、6、12、24小时，血浆淀粉酶活性的变化。图中，WT表示野生型C57小鼠；Cae表示雨蛙素；GP表示Gpihbp1 -/-小鼠，AAV5-optLPL表示静脉注射1×10 13vg/kgscAAV5-optLPL-S447X。
图8E：显示了对Gpihbp1 -/-小鼠静脉注射重组病毒或PBS 2个月后，再施用常规剂量雨蛙素(50μg/kg/h*10次)后第0、6、12、24小时，血浆脂肪酶活性的变化。图中各缩写的含义同图8D。
图8F：显示了对Gpihbp1 -/-小鼠静脉注射重组病毒或PBS 2个月后，再施用常规剂量雨蛙素(50μg/kg/h*10次)后第0、6、12、24小时，血浆TG浓度的变化。图中各缩写的含义同图8D。
图8G：显示了对Gpihbp1 -/-小鼠静脉注射重组病毒或PBS 2个月后，再施用常规剂量雨蛙素(50μg/kg/h*10次)后第0、6、12、24小时，血浆胆固醇浓度的变化。图中各缩写的含义同图8D。
图9A：显示了对新生Gpihbp1 -/-大鼠出生后7天经眶静脉注射AAV5-optLPL病毒导致了Gpihbp1 -/-大鼠的28天生存率显著提高。
图9B：显示了对新生Gpihbp1 -/-大鼠出生后7天经眶静脉注射AAV5-optLPL病毒导致了Gpihbp1 -/-大鼠的血浆TG显著下降。
图9C：显示了对Gpihbp1 -/-大鼠出生后8周经尾静脉二次注射AAV8-optLPL病毒导致了血浆TG的进一步下降。
图9D：显示了对新生Gpihbp1 -/-大鼠出生后7天经眶静脉注射AAV5-optLPL病毒导致了Gpihbp1 -/-大鼠的血浆胆固醇显著下降。
图9E：显示了对Gpihbp1 -/-大鼠出生后8周经尾静脉二次注射AAV8-optLPL病毒导致了血浆胆固醇的进一步下降。
图9F：显示了第一针AAV病毒载体静脉注射后9周，来自野生型SD大鼠、PBS组Gpihbp1 -/-大鼠、AAV-optLPL治疗组Gpihbp1 -/-大鼠的血浆外观。图9F小管1、2、3是来自野生型SD大鼠的血浆；小管4、5、6是来自Gpihbp1 -/-大鼠+PBS的血浆；小管7、8、9是来自Gpihbp1 -/-大鼠+1×10 11vg/kgscAAV8-optLPL-S447X的血浆；小管10、11、12是来自Gpihbp1 -/-大鼠+1×10 13vg/kgscAAV8-optLPL-S447X的血浆。
图9G：显示了第一针AAV病毒载体静脉注射后9周，来自野生型SD大鼠、PBS组Gpihbp1 -/-大鼠、AAV-optLPL治疗组Gpihbp1 -/-大鼠的血浆LPL浓度。在AAV-optLPL治疗组Gpihbp1 -/-大鼠中，LPL浓度呈AAV8-optLPL病毒的剂量依赖性升高。
图9H：显示了第一针AAV病毒载体静脉注射后9周，来自野生型SD大鼠、PBS组Gpihbp1 -/-大鼠、AAV-optLPL治疗组Gpihbp1 -/-大鼠的血浆LPL活性。在AAV8-optLPL治疗组Gpihbp1 -/-大鼠中，LPL活性呈AAV8-optLPL病毒的剂量依赖性升高。
图10A：显示了高脂饲料(HFD)喂饲后14天，Gpihbp1 -/-大鼠胰腺外观呈现明显包裹性坏死。
图10B：显示了用高脂饲料(HFD)喂饲的14天期间，Gpihbp1 -/-大鼠血浆甘油三脂水平的变化。
图10C：显示了用高脂饲料(HFD)喂饲的14天期间，Gpihbp1 -/-大鼠血浆胆固醇水平的变化。
图10D：显示了高脂饲料(HFD)喂饲后14天，HE染色观察注射重组AAV病毒载体对Gpihbp1 -/-大鼠的胰腺组织损伤程度的影响。
图10E：显示了高脂饲料(HFD)喂饲后14天，HE染色观察注射重组AAV病毒载体对Gpihbp1 -/-大鼠的肺组织损伤程度的影响。
图11：显示了WT仓鼠高脂饲料喂饲前后，以及注射AAV5-EGFP或AAV5-optLPL后的血浆TG浓度。在喂饲高脂饲料前，喂饲高脂饲料后第28天，以及注射AAV5-EGFP或AAV5-optLPL(1E12vg/kg)后第7，14，28天(实验开始的第35，42，56天)，过夜禁食12小时，取血，检测血浆TG浓度。
图12：显示了WT仓鼠高脂饲料喂饲前后，以及注射AAV5-EGFP或AAV5-optLPL后血浆TC浓度。在喂饲高脂饲料前，喂饲高脂饲料后第28天，以及注射AAV5-EGFP或AAV5-optLPL(1E12vg/kg)后第7，14，28天(实验开始的第35，42，56天)，过夜禁食12小时，取血，检测血浆TC浓度。
图13：显示了WT仓鼠注射AAV5-EGFP或AAV5-optLPL后第14天口服脂质负荷试验。WT仓鼠注射AAV5-EGFP或AAV5-optLPL(1E12vg/kg)后第14天，过夜禁食12小时，取零点血，橄榄油灌胃后，检测1，2，4，8小时的血浆TG浓度。
图14：显示了WT仓鼠注射AAV5-EGFP或AAV5-optLPL后第28天血浆LPL活性。WT仓鼠注射AAV5-EGFP或AAV5-optLPL(1E12vg/kg)后第28天，静脉注射肝素，取肝素后血浆，用试剂盒检测血浆LPL活性。
图15：显示了WT仓鼠注射AAV5-EGFP或AAV5-optLPL第30天取材后，肝脏optLPL mRNA检测。WT仓鼠注射AAV5-EGFP或AAV5-optLPL(1E12vg/kg)后第30天(实验开始后第58天)取材，PBS灌流后，取肝组织，提RNA，通过qPCR检测肝脏中optLPL的mRNA。
发明详述
本发明公开了一种预防和/或治疗严重高甘油三酯血症的基因药物，涉及该基因药物的设计、小量制备及功能验证。
除非下文中另外定义，否则本说明书中所用的全部技术与科学术语具有如本发明所属领域的普通技术人员通常理解的相同含义。本文所提及的全部出版物、专利申请、专利和其他参考文献通过引用的方式完整地并入。此外，本文中所述的材料、方法和例子仅是说明性的并且不意在是限制性的。本发明的其他特征、目的和优点将从本说明书及附图并且从后附的权利要求书中显而易见。
I.定义
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数字数值大5％的上限的范围内的数字数值。
如本文中所用，术语“包含”或“包括”意指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。
术语“脂蛋白脂酶(lipoprotein lipase,LPL)”是一种对脂蛋白甘油三酯进行脂解的脂肪酶。在肠道中，膳食脂肪被吸收、包装成富含甘油三酯的脂蛋白颗粒，并通过生物体的循环系统输送至合适的外周组织，这对于维持生物体的代谢稳态是必不可少的，这些过程的缺陷或失调会导致代谢疾病，例如，糖尿病、肥胖症和高脂血症。脂蛋白甘油三酯的脂解需要LPL。当所述脂解发生在毛细血管内皮细胞的腔表面(luminal surface)时，LPL的脂解作用部分地受到蛋白质GPIHBP1的调节。GPIHBP1是毛细血管内皮细胞的一种GPI锚定蛋白，负责将LPL跨过内皮细胞转运至毛细血管腔。没有这种转运，LPL会错误地定位在内皮下胞间间隙(interstitial space)，无法到达富含甘油三酯的脂蛋白处，从而导致严重的高甘油三酯血症。
人类LPL基因位于第8号染色体p22上，约30kb，由10个外显子和9个内含子组成，编码包含27个氨基酸信号肽在内的475个氨基酸蛋白。LPL基因主要在心脏、骨骼肌和脂肪组织中表达。编码LPL的信使RNA长约3560bp，其信号肽由27个氨基酸组成。人的成熟LPL由448个氨基酸残基组成。
如本文中所用，术语“功能性LPL”是指具有全长野生型(天然)人LPL(如NCBI protein数据库(https://www.ncbi.nlm.nih.gov/protein/)登录号NP_000228.1所示)的氨基酸序列的酶、其变体(例如，具有保守氨基酸置换的变体)、其片段，所述变体或片段提供至少约10％、至少约20％、至少约30％、至少约40％、至少约50％、至少约60％、至少约70％、至少约75％、至少约80％、至少约90％、或大约相同、或大于100％的全长野生型(天然)人LPL的生物活性水平。
LPL变体的示例包括但不限于LPL-S447X，其是通过去掉野生型(天然)人LPL的C端最后两个氨基酸而获得。
如本文中所用，术语“保守氨基酸置换”或“保守氨基酸取代”是指将氨基酸改变、置换或取代成具有相似生物化学性质(例如电荷、疏水性和大小)的不同氨基酸，这是本领域技术人员已知的。
存在多种通过常规方法来测量LPL表达和活性水平的测定法。参见，例如本说明书实施例3。
LPL功能障碍会导致血浆甘油三酯升高。与LPL功能密切相关的GPIHBP1的缺陷也会使得LPL无法转运至毛细血管腔发挥水解TRL上TG的作用，造成严重的高甘油三酯血症。急性胰腺炎(acute pancreatits,AP)是一种常见的急腹症，死亡率极高，其中，脂蛋白脂酶功能障碍和/或GPIHBP1缺陷对高乳糜微粒血症、高甘油三酯血症(hypertriglyceridemia，HTG)及随之发生的急性胰腺炎的影响最为显著。
“信号序列”是连接至蛋白质的N-端部分的氨基酸的序列，其促进蛋白质分泌至细胞外。细胞外蛋白质的成熟形式没有信号序列，其在分泌过程期间被切除。
术语“N端”指N端的最末氨基酸，术语“C端”指C端的最末氨基酸。
术语“有效连接”意指指定的各组分处于一种允许它们以预期的方式起作用的关系。
如下进行序列之间序列同一性的计算。
为确定两个氨基酸序列或两个核酸序列的同一性百分数，将所述序列出于最佳比较目的比对(例如，可以为了最佳比对而在第一和第二氨基酸序列或核酸序列之一或二者中引入空位或可以为比较目的而抛弃非同源序列)。在一个优选实施方案中，为比较目的，所比对的参考序列的长度是至少30％、优选地至少40％、更优选地至少50％、60％和甚至更优选地至少70％、80％、90％、100％的参考序列长度。随后比较在对应氨基酸位置或核苷酸位置处的氨基酸残基或核苷酸。当第一序列中的位置由第二序列中对应位置处的相同氨基酸残基或核苷酸占据时，则所述分子在这个位置处是相同的。
可以利用数学算法实现两个序列间的序列比较和同一性百分数的计算。在一个优选实施方案中，使用已经集成至GCG软件包的GAP程序中的Needlema和Wunsch((1970)J.Mol.Biol.48:444-453)算法(在http://www.gcg.com可获得)，使用Blossum 62矩阵或PAM250矩阵和空位权重16、14、12、10、8、6或4和长度权重1、2、3、4、5或6，确定两个氨基酸序列之间的同一性百分数。在又一个优选的实施方案中，使用GCG软件包中的GAP程序(在http://www.gcg.com可获得)，使用NWSgapdna.CMP矩阵和空位权重40、50、60、70或80和长度权重1、2、3、4、5或6，确定两个核苷酸序列之间的同一性百分数。特别优选的参数集合(和除非另外说明否则应当使用的一个参数集合)是采用空位罚分12、空位延伸罚分4和移码空位罚分5的Blossum 62评分矩阵。
还可以使用PAM120加权余数表、空位长度罚分12，空位罚分4)，利用已经并入ALIGN程序(2.0版)的E.Meyers和W.Miller算法,((1989)CABIOS,4:11-17)确定两个氨基酸序列或核苷酸序列之间的同一性百分数。
额外地或备选地，可以进一步使用本文所述的核酸序列和蛋白质序列作为“查询序列”以针对公共数据库执行检索，以例如鉴定其他家族成员序列或相关序列。
术语“调控序列”或“表达控制序列”是指这样的核酸序列，其诱导、抑制或以其它方式控制与之有效连接的编码核酸序列的蛋白质转录。调控序列可以是例如起始序列、增强子序列、内含子序列和启动子序列等。
描述核酸或蛋白质时所用的术语“外源的”是指核酸或蛋白质不是天然存在于其存在的染色体或宿主细胞的位置。外源核酸序列也指衍生自并插入相同宿主细胞或受试者但以非天然状态存在的序列，例如，不同的拷贝数，或受不同调控元件的控制。
如本文中所用，术语“腺相关病毒(Adeno-associated virus，AAV)”因在腺病毒制品中发现而得名。AAV是微小病毒科(Parvovirus)成员，包含多种血清型，其基因组为单链DNA。
AAV是依赖性病毒，需要其它病毒如腺病毒、单纯疱疹病毒、人乳头瘤病毒、或辅助因素提供辅助功能蛋白才能复制。
最早分离到的AAV病毒是血清型2型AAV(AAV2)。AAV2基因组长约4.7kb，基因组两端为长度145bp的“反向末端重复序列”(inverted terminal repeat,ITR)，呈回文-发卡结构。基因组中有两个大开放阅读框(ORF)，分别编码rep和cap基因。已将AAV2的全长基因组克隆至大肠杆菌质粒中(Samulski RJ等人，Proc Natl Acad Sci USA.1982；79:2077-2081.Laughlin CA等人，Gene.1983；23:65-73)。
ITR是AAV载体基因组的顺式作用元件，在AAV病毒的整合、拯救、复制和基因组包装中发挥重要作用。ITR序列中包含Rep蛋白结合位点(Rep binding site，RBS)和末端解链位点trs(terminal resolution site)，能够被Rep蛋白结合识别并在trs处产生切口。ITR序列还可形成独特的“T”字母型二级结构，在AAV病毒的生活周期中发挥重要作用。
AAV2基因组其余部分可分为2个功能区，rep基因区和cap基因区。
rep基因区编码Rep78、Rep68、Rep52和Rep40四种Rep蛋白。Rep蛋白对于AAV病毒的复制、整合、拯救和包装都具有重要作用。其中Rep78和Rep68与ITR中的末端解链位点trs和GAGY重复基序特异性结合，启动AAV基因组由单链向双链的复制过程。ITR中trs和GAGC重复基序和/或GAGY重复基序是AAV基因组复制的中心，因此虽然在各种血清型的AAV病毒中ITR序列都不尽相同，但是都能形成发卡结构和存在Rep结合位点。在AAV2基因组图谱位置19处有p19启动子，启动分别表达Rep52和Rep40。Rep52和Rep40具有ATP依赖的DNA解旋酶活性，但没有结合DNA的功能。
cap基因编码AAV病毒的衣壳蛋白VP1、VP2和VP3。其中，VP3分子量最小，但数量最多，在成熟的AAV颗粒中VP1、VP2、VP3的比例大致为1:1:10。VP1是形成有感染性的AAV所必需的；VP2协助VP3进入细胞核；VP3是组成AAV颗粒的主要蛋白。
如本文中所用，术语“AAV载体”是人们随着对AAV病毒生活周期及其相关分子生物学机制的了解，将野生型AAV病毒改造成的一种高效的外源基因转移工具，即AAV载体。改造后的AAV载体基因组中只包含AAV病毒的ITR序列和携带待转运的外源基因表达框。AAV病毒包装需要的Rep和Cap蛋白通过其他外源质粒反式提供，由此降低了rep和cap基因包装入AAV载体可能带来的危害。进一步地，AAV病毒本身不具有致病性，这使得AAV载体成为公认的最安全的病毒载体之一。删除AAV病毒的一侧ITR序列中的D序列和trs序列还能够使包装得到的重组AAV病毒载体所携带基因组自我互补，形成双链，显著提高AAV载体 的体内外转导效率(Wang Z等人，Gene Ther.2003；10(26):2105-2111；McCarty DM等人，Gene Ther.2003；10(26):2112-2118)。包装得到的病毒成为scAAV(self-complementary AAV)病毒，即所谓的双链AAV病毒。它不同于双侧ITR均未突变的ssAAV(single-stranded AAV)，即传统的AAV病毒。
scAAV病毒载体的包装容量更小，仅为ssAAV病毒载体包装容量的一半，约为2.2kb-2.5kb，但感染细胞后转导效率更高。
AAV病毒血清型众多，不同的血清型具有不同的组织感染嗜性，因此应用AAV载体能够将外源基因转运至特定的器官和组织(Wu Z等人，Mol Ther.2006；14(3):316-327)。某些血清型AAV载体还可穿越血脑屏障，将外源基因导入至大脑神经元中，为靶向大脑的基因转导提供了可能(Samaranch L等人，Hum Gene Ther.2012；23(4):382-389)。
此外，AAV载体的理化性质稳定，对酸、碱和高温体现出较强的耐受性(Gruntman AM等人，Hum Gene Ther Methods.2015；26(2):71-76)，容易开发出稳定性较高的生物制品。
现有技术中对AAV载体具有相对成熟的包装系统，这便于规模化生产AAV载体。
目前常用的AAV载体包装系统主要包括三质粒共转染系统、腺病毒作为辅助病毒的系统、单纯疱疹病毒(Herpes simplex virus type 1，HSV1)作为辅助病毒的包装系统、以及基于杆状病毒的包装系统。每种包装系统都各具特点，本领域技术人员可以根据需要做出合适的选择。
三质粒转染包装系统因无需辅助病毒，安全性高，是应用最为广泛的AAV载体包装系统，也是目前国际上主流的生产系统。略显不足的是，高效大规模转染方法的缺失限制了三质粒转染系统在AAV载体大规模制备中的应用。
Yuan等建立以腺病毒为辅助病毒的AAV大规模包装系统(Yuan Z等人，Hum Gene Ther.2011；22(5):613-624)，该系统生产效率高，但包装系统中腺病毒在最后AAV成品中的痕量存在，影响了AAV成品的安全性。
HSV1作为辅助病毒的包装系统是另一类应用较为广泛的AAV载体包装系统。伍志坚和Conway等几乎同时在国际上提出了以HSV1为辅助病毒的AAV2载体包装策略(伍志坚，吴小兵等，科学通报，1999，44(5)：506-509；Conway JE等人，Gene Ther.1999，6：986-993)。随后Wustner等提出了以HSV1为辅助病毒的AAV5载体包装策略(Wustner JT等人，Mol Ther.2002，6(4)：510-518)。在此基础上，Booth等利用两个HSV1分别携带AAV的rep/cap基因和AAV的反向末端序列(Inverted terminal repeat，ITR)/外源基因表达框，然后用这两个重组HSV1病毒共同感染生产细胞，包装产生AAV病毒(Booth MJ等人，Gene Ther.2004；11:829-837)。Thomas等进一步建立双HSV1病毒AAV生产的悬浮细胞系统(Thomas DL等人，Gene Ther.2009；20:861-870)，使更大规模的AAV病毒生产成为可能。
Urabe等利用三个杆状病毒分别携带AAV的结构基因、非结构基因和ITR/外源基因表达框，构建了AAV载体的杆状病毒包装系统。考虑到杆状病毒携带外源基因的不稳定性，随后减少了生产系统中所需杆状病毒的个数，逐渐从最开始的需要三个杆状病毒到需要两个或一个杆状病毒(Chen H.，Mol Ther.2008，16(5)：924-930；Galibert L.等人，J Invertebr Pathol.2011；107Suppl:S80-93)以及一个杆状病毒组合一株诱导细胞株策略(Mietzsch M等人，Hum Gene Ther.2014；25:212-222，Mietzsch M等人，Hum Gene Ther.2015；26(10):688-697)。
由于上述特点，AAV载体逐渐成为一种广泛应用于基因治疗，特别是遗传病的基因治疗的外源基因转运工具。截至2016年8月，世界上批准的基于AAV载体的基因治疗临床试验方案有173项(http://www.abedia.com/wiley/vectors.php)。更为重要的是，基于AAV载体的脂蛋白脂酶基因治疗药物Glybera已于2012年被欧洲药监局批准上市，成为西方世界批准的第一个基因治疗药物(
Figure PCTCN2021109590-appb-000001
S.，Mol Ther.2012；20(10):1831-1832)；血友病B(Kay MA等人，Nat Genet.2000；24(3):257-261)和先天性黑蒙症(RPE65基因突变引起)(Jacobson SG等人，Arch Ophthalmol.2012；130(1):9-24)的AAV载体基因治疗药物均取得不错的临床试验效果，预期在不久的将来会上市销售，造福广大患者。
术语“载体基因组(vg)”是指包装在rAAV衣壳内形成rAAV载体的核酸序列。在一个实施方案中，载体基因组至少包含5’至3’的AAV2 5’ITR、编码功能性LPL的核酸序列和AAV23’ITR。也可以选择来自除AAV2以外的不同来源AAV的ITR。此外，载体基因组可以包含指导功能性LPL表达的调控序列。
如本文中所用，术语“miRNA(microRNA)”是指广泛存在于人类和动物体内的长度为18至25个核苷酸(nucleotide，nt)的单链非编码RNA。
1993年miRNA首先在秀丽隐杆线虫(C.elegans)中发现。秀丽隐杆线虫中lin-4基因能够下调lin-14基因的表达，但lin-4基因的编码产物不是蛋白质，而是一种小RNA分子，这表明自身编码的小RNA分子能够调节基因的表达。随后，多种类似的小RNA分子在不同的物种和细胞中相继发现，miRNA开始成为该类小RNA的统称。
miRNA调节人类大约60％基因的表达(Friedman RC等人，Genome Res.2009；19:92-105)，在多种生理和病理过程中发挥重要作用。
miRNA基因通常位于基因组的外显子、内含子和基因间区中(Olena AF等人，J Cell Physiol.2010；222:540-545；Kim VN等人，Trends Genet.2006；22:165-173)。在细胞内，miRNA的产生过程如下所述。首先，在细胞核中，miRNA基因由RNA聚合酶II或III启动转录产生初始产物pri-microRNA；pri-microRNA自我折叠部分序列形成茎环结构。随后，由核糖核酸酶III Drosha和DGCR8分子组成的加工复合体作用于pri-microRNA，切去多余序列，留下60nt左右的茎环结构，即前体miRNA分子pre-microRNA。然后，在转运蛋白Exportin-5的协助下，pre-microRNA从细胞核进入细胞质中，经Dicer酶加工去掉其茎环结构中的环形部分，变为双链RNA分子。最后，双链RNA分子被AGO2等蛋白因子结合，其中一条链发生降解，另一条链和蛋白因子形成RNA诱导的沉默复合物(RNA induced silencing complex，RISC)。RISC识别mRNA中的靶序列，通过降解mRNA分子、促进mRNA分子3’端去腺苷化和抑制翻译来降低mRNA的表达水平，在转录后水平调节基因的表达(Fabian MR等人，Annu Rev Biochem.2010；79:351-379)。因此，利用细胞内高表达的miRNA，在外源基因的3’UTR(untranslated region)插入该miRNA的靶序列，能够有效地抑制外源基因在导入细胞中的表达。
miR-142-3p是一种miRNA，其在造血干细胞系来源细胞中高表达。
免疫细胞均分化来源于造血干细胞系，因此利用miRNA抑制基因表达的原理(Kim VN.Nat Rev Mol Cell Biol.2005；6(5):376-385)，携带miR-142-3p靶序列的基因表达会在免疫细胞中受到明显抑制，从而降低机体产生针对基因表达产物免疫反应的概率(Dismuke DJ等人，Curr  Gene Ther.2013；13(6):434-452)。
术语“治疗”指意欲改变正在接受治疗的个体中疾病之天然过程的临床介入。想要的治疗效果包括但不限于防止疾病出现或复发、减轻症状、减小疾病的任何直接或间接病理学后果、降低病情进展速率、改善或缓和疾病状态，以及缓解或改善预后。
用于本文时，“预防”包括对疾病或特定疾病的症状的发生或发展的抑制。在一些实施方式中，具有高甘油三脂家族病史的受试者是预防性方案的候选。通常，术语“预防”是指在高甘油三脂血症发生前，特别是在具有LPL基因缺陷和/或GPIHBP1基因缺陷的受试者中于高甘油三脂血症发生前的药物施用。
II.表达框
在一个方面，本发明提供了一种功能性人脂蛋白脂酶基因表达框，其包含编码功能性LPL的核苷酸序列和指导其表达的调控序列。
在一个实施方案中，表达框包含如本文所述的编码功能性LPL的核苷酸序列和指导其表达的调控序列。在一个实施方案中，编码功能性LPL的核苷酸序列与SEQ ID NO:1具有至少70％、80％、90％的同一性，例如，具有至少95％、96％、97％、98％、99％或更高的同一性。又在一个实施方案中，编码功能性LPL的核苷酸序列是插入有SEQ ID NO:2所示的内含子序列或者插入有与SEQ ID NO:2具有至少约90％同一性(例如，具有至少95％、96％、97％、98％、99％或更高的同一性)的内含子序列的编码功能性LPL(例如，LPL-S447X)的核苷酸序列。
在一些实施方案中，编码功能性LPL的核苷酸序列选自
(i)如SEQ ID NO:1或SEQ ID NO:3所示的编码功能性LPL的核苷酸序列；
(ii)与如SEQ ID NO:1或SEQ ID NO:3所示的核苷酸序列互补的核苷酸序列；
(iii)与(i)或(ii)的核苷酸序列编码相同的功能性LPL，但因遗传密码的简并性而与(i)或(ii)的核苷酸序列不同的核苷酸序列；或
(iv)与(i)或(ii)或(iii)所述核苷酸序列具有至少70％、80％、90％同一性(例如，具有至少95％、96％、97％、98％、99％或更高同一性)的序列。
在一个实施方案中，指导功能性LPL表达的调控序列包含肝特异性启动子，例如，白蛋白启动子、乙肝病毒核心启动子、α甲胎蛋白(AFP)启动子、CMV启动子等。优选地，调控序列包含如SEQ ID NO:5所示的LP15启动子序列或者与其具有至少约90％同一性(例如，具有至少95％、96％、97％、98％、99％或更高同一性)的启动子序列。与使用CMV启动子相比较，使用所述LP15启动子能够避免CMV启动子进入体内表达易被甲基化沉默，提高功能性LPL的表达水平，更好地实现肝脏靶器官的特异性表达。
在一个实施方案中，调控序列进一步包含可降低功能性LPL在免疫相关细胞(如抗原呈递细胞)中的表达，减弱功能性LPL表达带来的免疫反应的序列，由此显著降低针对外源性LPL蛋白发生免疫反应的概率，所述序列为例如一个或多个(例如，1－8个、2－7个、3个、4个、5个或6个)串联的与miR-142-3p互补的靶序列，由此，本发明的表达框转录得到编码功能性LPL(例如，LPL-S447X)的mRNA的非翻译区(例如，5’端非翻译区和/或3’端非翻译区中含有人miR-142-3p靶序列，能够有效地抑制功能性LPL(例如，LPL-S447X)在免疫相关细胞(如抗原呈递细胞)中的表达，抑制免疫反应。
在一个实施方案中，调控序列包含一个或多个表达增强子，例如，TBG增强子、CMV增强子等。在一个具体实施方案中，调控序列包含如SEQ ID NO:11所示的或者与其具有至少约90％同一性(例如，与其具有至少95％、96％、97％、98％、99％或更高同一性)的TBG增强子序列。
在一个实施方案中，调控序列进一步包含聚腺苷酸化信号(polyA)，例如，人生长激素(hGH)聚腺苷酸化序列、SV40polyA、BGH polyA。在一个具体实施方案中，调控序列包含如SEQ ID NO:10所示的或者与其具有至少约90％同一性(例如，与其具有至少95％、96％、97％、98％、99％或更高同一性)的BGH polyA序列。
III.病毒载体
又在一个方面，本发明提供了一种病毒载体，其为人造的重组病毒颗粒，其中，包含编码功能性LPL的表达框的复制缺陷型病毒基因组序列被包装在病毒衣壳或包膜中，从而所述重组病毒颗粒不能产生子代病毒体，但保留了感染靶细胞的能力。
在一个实施方案中，病毒载体的基因组序列不包含编码病毒复制所需的酶的基因，因此，认为在基因疗法中使用病毒载体是安全的，因为在不存在病毒复制所需的酶的情况下，子代病毒体的复制和感染是不会发生的。
本发明的重组病毒载体可以是重组的腺相关病毒(AAV)、腺病毒、博卡病毒(Bocavirus)、AAV/博卡病毒杂交体、单纯疱疹病毒或慢病毒。
生产重组病毒载体(例如，重组AAV载体)的包装细胞系可以是原核细胞或真核细胞(例如，人类细胞、昆虫细胞或酵母细胞)，其含有通过任何方式(例如电穿孔、磷酸钙沉淀、显微注射、转化、病毒感染、转染和原生质体融合)引入细胞中的外源DNA。包装细胞系细胞包括但不限于大肠杆菌细胞、酵母细胞、人类细胞、非人类细胞、哺乳动物细胞、非哺乳动物细胞、昆虫细胞、HEK293细胞、肝细胞、肾细胞、神经胶质细胞或干细胞。
如本文所用，术语“靶细胞”是指其中期望表达功能性LPL的靶细胞。靶细胞的示例包括但不限于肝细胞、肌细胞。在一些实施方案中，将载体体内递送至靶细胞。
在一个优选的实施方案中，病毒载体是重组腺相关病毒(rAAV)载体，其包含AAV衣壳和包装在其中的载体基因组。rAAV载体用于治疗LPL基因缺陷和/或GPIHBP1基因缺陷和/或高脂饮食诱发的高甘油三酯血症或其引起的疾病如急性胰腺炎。载体基因组包含AAV 5’反向末端重复序列(ITR)或AAV 5’ΔITR、编码功能性LPL的核酸序列、指导LPL在靶细胞中表达的调控序列、以及AAV 3’ITR或AAV 3’ΔITR。所述ΔITR是删除了D序列和末端解链位点trs的ITR。ITR是在载体生产期间负责基因组的复制和包装的遗传元件，并且是产生rAAV所需的唯一病毒顺式元件。可以选择来自不同来源AAV的ITR。在一个实施方案中，ITR来自与病毒颗粒的衣壳不同的AAV。
除非另有说明，否则本文描述的AAV衣壳、ITR和其它AAV组分可以容易地从任何AAV中选择，包括但不限于通常被鉴定为AAV1、AAV2、AAV3B、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAVrh.10或它们的组合的血清型的AAV。在一个实施方案中，AAV衣壳是AAV5衣壳或其变体。在一个实施方案中，AAV衣壳是AAV3B衣壳或其变体。在一个实施方案中，AAV衣壳是AAV8衣壳或其变体。在一个实施方案中，AAV衣壳是AAV9衣壳或 其变体。在某些实施方案中，衣壳蛋白由rAAV载体名称中术语“AAV”之后的数字或数字和字母的组合来指定。
在一个实施方案中，提供一种这样的rAAV，其包含AAV血清型5(AAV5)衣壳；和包含SEQ ID NO:12或与SEQ ID NO:12具有至少约90％同一性的序列的载体基因组。
在一个实施方案中，提供一种这样的rAAV，其包含AAV血清型3B(AAV3B)衣壳；和包含SEQ ID NO:12或与SEQ ID NO:12具有至少约90％同一性的序列的载体基因组。
在一个实施方案中，提供一种这样的rAAV，其包含AAV血清型8(AAV8)衣壳；和包含SEQ ID NO:12或与SEQ ID NO:12具有至少约90％同一性的序列的载体基因组。
在一个实施方案中，提供一种这样的rAAV，其包含AAV血清型9(AAV9)衣壳；和包含SEQ ID NO:12或与SEQ ID NO:12具有至少约90％同一性的序列的载体基因组。
如本文中所用的，AAV“变体”是指衍生自已知的AAV序列的任何AAV序列，包括具有保守氨基酸置换的那些AAV序列，以及与AAV的氨基酸或核酸序列具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少97％、至少99％或更大的序列同一性的序列。在另一个实施方案中，AAV衣壳包括可以包含与任何描述或已知的AAV衣壳序列相比高达约10％的变异的变体。即，AAV衣壳与本文提供的和/或本领域已知的AAV衣壳具有约90％的同一性至约99.9％的同一性，约95％至约99％的同一性或约97％至约98％的同一性。在一个实施方案中，AAV衣壳与AAV衣壳变体具有至少95％、96％、97％、98％、99％或更高的同一性。当确定AAV衣壳的百分同一性时，可以对任何变体蛋白(例如vp1、vp2或vp3)进行比较。
IV.病毒载体的制备方法
本发明的重组腺相关病毒(AAV)载体可以使用已知的技术产生。此类方法涉及培养包装细胞，其包含编码AAV衣壳的核酸序列；功能性rep基因；如本文描述的表达框，其侧接有AAV反向末端重复序列(ITR)或ΔITR；和足够的辅助功能，以允许将表达框包装到AAV衣壳蛋白中。
本文还提供了包装细胞，其包含编码AAV衣壳的核酸序列；功能性rep基因；如本文描述的表达框，其侧接有AAV反向末端重复序列(ITR)或ΔITR；和足够的辅助功能，以允许将表达框包装到AAV衣壳蛋白中。在一个实施方案中，宿主细胞是HEK 293细胞。
可以利用本领域已知的产生rAAV的其它方法。合适的方法可以包括但不限于杆状病毒表达系统或通过酵母生产。
V.病毒载体的用途
本发明的病毒载体作为一种基因药物，能够通过静脉注射，在肝脏异位地、特异性表达预防和/或治疗严重高甘油三酯血症的功能性LPL。
通过静脉注射本发明的病毒载体，在成年患者的机体本身不表达LPL基因的肝脏中异位表达功能性LPL，且所表达的功能性LPL不依赖于GPIHBP1即可实现对血浆中TG的水解，使较高的甘油三酯水平趋于正常。
本发明的病毒载体可以用于预防和/或治疗高甘油三酯血症或其引起的疾病如急性胰腺炎。在一些实施方案中，本发明的病毒载体可以用于预防和/或治疗LPL基因缺陷和/或 GPIHBP1基因缺陷和/或高脂饮食诱发的高甘油三酯血症或其引起的疾病如急性胰腺炎。
实施例
下面对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明的一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。如无特殊说明，实施例中涉及的各种反应试剂均可以通过商业渠道购买得到。
实施例1.AAV质粒载体的构建
在本实施例中构建了包含目的基因和目的基因表达调控元件、以及ITR序列的AAV质粒载体。
首先构建了pUC57-optLPL-S447X载体。
具体而言，登录NCBI protein数据库(https://www.ncbi.nlm.nih.gov/protein/)，搜索得到人LPL蛋白质氨基酸序列(NP_000228.1)(448aa)，去掉C端最后两个氨基酸即为LPL-S447X的氨基酸序列(446aa)，将LPL-S447X对应的编码序列进行人源密码子优化，并增加一个终止密码子，获得编码功能性LPL的如SEQ ID NO:1所示的核苷酸序列。
将人14号染色体中免疫球蛋白重链表达基因的如SEQ ID NO:2所示的内含子(长度为82bp)插入SEQ ID NO:1所示的LPL-S447X编码序列。该内含子序列将LPL-S447X编码序列分为两部分(分别称为：LPL-S447X CDS part1和LPL-S447X CDS part2)，插入该内含子序列后，获得了LPL-S447X CDS part1-内含子-LPL-S447X CDS part2序列，如SEQ ID NO:3所示，文中也称为“optLPL-S447X”或“优化的LPL-S447X”或简称为“optLPL”。
作为对照，也合成了未插入内含子的LPL-S447X编码序列(即，SEQ ID NO:1所示的核苷酸序列)，下文中也称为“未插入内含子的LPL-S447X”。
将未插入内含子的LPL-S447X编码序列(SEQ ID NO:1)和optLPL-S447X的核苷酸序列(SEQ ID NO:3)均送至南京金斯瑞生物科技有限公司合成，并分别克隆入pUC57 simple载体(南京金斯瑞生物科技有限公司)，得到pUC57-LPL-S447X质粒载体和pUC57-optLPL-S447X质粒载体。
另外，构建了图1A所示的pscAAV-LP15-EGFP-kan-4×142T质粒载体(下文中也将该载体简称为pscAAV-LP15-EGFP载体)，其序列如SEQ ID NO:4所示，包含：
i)LP15启动子，所述LP15启动子的序列如SEQ ID NO:5所示，由人AMBP(X67082.1)的增强子序列、人ALB(M12523.1)的增强子序列和长度为128bp的启动子序列以及小鼠微小病毒(MVM)内含子(大小为67bp)拼接而成；
ii)4个串联的人miR-142-3p靶序列，也缩写为“4×142-3pT”。4×142-3pT的序列如SEQ ID NO:7所示；
iii)牛生长激素的多聚核苷酸加尾信号，也缩写为BGH polyA；
iv)人甲状腺素结合球蛋白(thyroxine-binding globulin，TBG)基因增强子序列；
v)EGFP，所述EGFP是表达增强型绿色荧光蛋白的核苷酸序列。
然后，用KpnI和EcoRI双酶切消化pUC57-optLPL-S447X载体，产生2.7kb和1507bp两个片段，回收长度为1507bp的片段，为optLPL-S447X编码序列。用KpnI和EcoRI双酶切消化构建的图1A所示的pscAAV-LP15-EGFP-kan-4×142T载体，得到5403bp和748bp两个片段，回收长度为5403bp的片段。连接所获得的长度为1507bp的片段和长度为5403bp的片段，得到图1B所示的pscAAV-LP15-optLPL-S447X-kan-4×142T载体，经SmaI酶切(产生3149bp/1356bp/1275bp片段)鉴定该载体，下文中也将该载体简称为pscAAV-LP15-optLPL-S447X载体。
类似地，用KpnI和EcoRI双酶切消化pUC57-LPL-S447X载体，产生2.7kb和1425bp两个片段，回收长度为1425bp的片段。另外，用KpnI和EcoRI双酶切消化图1A所示的pscAAV-LP15-EGFP-kan-4×142T载体，得到5403bp和748bp两个片段，回收长度为5403bp的片段。连接所获得的长度为1425bp的片段和长度为5403bp的片段，得到图1C所示的载体，经SmaI酶切(产生5636bp/2487bp/2476bp/144bp/133bp片段)鉴定该载体，下文中也将该载体简称为pscAAV-LP15-LPL-S447X载体。
将肝细胞Huh7(获自ATCC)均匀接种在24孔板中，1×10 5个细胞/孔，37℃ CO 2培养箱中温育过夜使细胞贴壁。使用脂质体转染法分别将pscAAV-LP15-optLPL-S447X质粒和pscAAV-LP15-LPL-S447X质粒转染入Huh7细胞中，作为对照，同样地将pscAAV-LP15-EGFP质粒转染入Huh7细胞中，转染条件均为1μl Lipofectamine 2000:2μg质粒DNA，转染后6h换液，放入细胞培养箱中继续培养48h后收获细胞，提取细胞总RNA，通过RT-qPCR分别检测LPL-S447X mRNA和optLPL-S447X mRNA的转录。具体过程如下：
在LPL-S447X基因上设计两条引物LPL-F和LPL-R：
LPL-F：5’-TCAACCACAGCTCCAAGACC-3’(SEQ ID NO:8)
LPL-R：5’-GTATAGCCGGCGGACACTG-3’(SEQ ID NO:9)
以LPL-F和LPL-R为引物特异性地扩增LPL-S447X基因长度为173bp片段，采用SYBR Green染料结合法，应用SYBR Premix Ex Taq II(Tli RNaseH Plus)试剂(Takara，大连，中国)，使用荧光定量PCR仪(型号：ABI 7500fast，ABI)检测。操作过程参见SYBR Premix Ex Taq II(Tli RNaseH Plus)试剂说明书。
检测结果如图2所示，显示了插入SEQ ID NO:2所示的内含子后，optLPL-S447X mRNA的转录效率显著提高，是未插入内含子的LPL-S447X mRNA的近10倍。
实施例2.重组AAV病毒的制备和检定
在本实施例中采用三质粒共转染HEK293包装系统包装重组AAV病毒，得到了scAAV5-LP15-opt LPL-S447X重组病毒。
首先，构建了pAAV-R2C5质粒。以AAV Helper Free System(Agilent Technologies，目录号#240071)中的pAAV-RC质粒为基本骨架，采用标准的分子克隆方法，用合成的AAV5基因组(GenBank ID：NC_006152.1)中的衣壳蛋白编码序列(也称为Cap5)(基因组中第2207至4381位序列)替换pAAV-RC质粒中HindIII至PmeI限制性酶切位点之间的序列(即，替换pAAV-RC质粒的第2013至4220位序列)，获得了pAAV-R2C5质粒。所述pAAV-R2C5质粒包含完整的 AAV5的cap基因和AAV2的rep基因，在三质粒共转染包装制备重组AAV5病毒中提供包装所必须的4种Rep蛋白(Rep78、Rep68、Rep52和Rep40)和AAV5衣壳蛋白。
然后，取实施例1构建的AAV载体质粒(pscAAV-LP15-optLPL-S447X载体)、辅助质粒(pHelper)(来自AAV Helper Free System，Agilent Technologies)和AAV的Rep及Cap蛋白表达质粒pAAV-R2C5，将这三种质粒按照1:1:1的摩尔比混匀后，采用磷酸钙方法共转染HEK293细胞，转染48小时后，收获细胞和培养上清液，应用氯化铯密度梯度离心法分离纯化重组AAV病毒，得到scAAV5-LP15-optLPL-S447X重组病毒。由于使用的质粒pAAV-R2C5表达了AAV5衣壳蛋白，由此，获得的是AAV5重组病毒。
采用定量PCR方法测定通过上述制备方法得到的AAV5重组病毒的基因组滴度。具体过程如下：
在LP15启动子中设计两条引物LP15-F和LP15-R：
LP15-F：5’-AAGTGGCCCTTGGCAGCATCT-3’(SEQ ID NO:13)
LP15-R：5’-GGACAAACGGAGGGAAATTAGCACT-3’(SEQ ID NO:14)
以LP15-F和LP15-R为引物特异性地扩增LP15启动子长度为229bp片段，采用SYBR Green染料结合法，以1μg/μl的pscAAV-LP15-optLPL-S447X质粒及其10倍梯度稀释的样品为标准品，应用SYBR Premix Ex Taq II(Tli RNaseH Plus)试剂(Takara，大连，中国)，使用荧光定量PCR仪(型号：ABI 7500fast，ABI)检测病毒基因组滴度。操作过程参见SYBR Premix Ex Taq II(Tli RNaseH Plus)试剂说明书。病毒的处理方法参见文献(Ulrich-Peter R.等人，J Virol Methods.2002；106:81-88)，获得的AAV5重组病毒滴度约为6E+12vg/ml。
类似地，通过三质粒法，使用实施例1构建的AAV载体质粒pscAAV-LP15-EGFP、辅助质粒(pHelper)(来自AAV Helper Free System，Agilent Technologies)和AAV的Rep及Cap蛋白表达质粒pAAV-R2C5，制备了scAAV5-LP15-EGFP重组病毒，并检测了病毒滴度。
实施例3.静脉注射重组AAV病毒治疗LPL基因缺陷小鼠的高甘油三酯血症
从空军军医大学引进条件性LPL基因敲除小鼠。采用20只成年Lpl -/-小鼠，年龄为8-12周，体重在18-25g。购买后在实验室动物房适应一周，然后称重，用玻璃毛细管进行内眦静脉采血，肝素抗凝，4℃，4000rpm，离心10分钟，分离血浆。使用甘油三酯和胆固醇检测试剂盒(购自：中生北控生物科技股份有限公司)检测甘油三脂(TG)和总胆固醇(TC)。
根据检测的甘油三脂(TG)和总胆固醇(TC)结果将Lpl -/-小鼠分为4组，每组5只小鼠。施用设置为：一次静脉注射scAAV5-LP15-optLPL-S447X病毒(简称AAV5-optLPL病毒)的高中低3个剂量组(高剂量1E+13vg/kg；中间剂量1E+12vg/kg；低剂量1E+11vg/kg)和一次静脉注射scAAV5-LP15-EGFP病毒(1E+13vg/kg)(简称AAV5-EGFP)的对照组。
根据待静脉注射的重组AAV病毒剂量和实施例2检测的AAV病毒滴度，计算出所需的对每组小鼠注射的重组AAV病毒液的体积，然后用无菌PBS定容至100μL，将各组小鼠分别通过尾静脉注射给予相应的重组AAV病毒。
在重组AAV病毒静脉注射后的第7、14、28、56天，分别在将动物过夜禁食12小时后，内眦静脉采血，测定血浆TG、TC。结果显示，静脉注射scAAV5-LP15-optLPL-S447X病毒后， 血浆中的TG和TC均显著性降低，特别是TG的下降幅度更加明显(图3，图4)。
另外，在静脉注射重组AAV病毒后第14天，按照100U/kg的剂量通过尾静脉注射肝素，注射10分钟后取血，离心分离得到血浆，用该肝素后血浆作为酶源，使用LPL试剂盒(Abcam)，测量LPL活性。结果表明：静脉注射AAV5-optLPL病毒的三个组的血浆均具有LPL活性，且该LPL活性随着注射的病毒载体剂量的加大而增加，而AAV5-EGFP病毒组没有检测到血浆LPL活性(图5)。
在重组AAV病毒静脉注射后第56天，处死小鼠，取肝脏，检测外源性LPL基因的mRNA表达水平。在施与scAAV5-optLPL-S447X病毒的小鼠组中检测到了optLPL-S447X mRNA,并且optLPL-S447X mRNA水平呈剂量依赖性增加(图6)。
实施例4 Gpihbp1 -/-小鼠尾静脉注射AAV5-optLPL治疗高甘油三酯血症
Gpihbp1 -/-小鼠购自Jackson Laboratory，饲养于东部战区总医院比较医学科动物房。12周龄成年Gpihbp1 -/-小鼠随机分为AAV治疗组和PBS对照组两组，每组15只。AAV治疗组给予尾静脉注射AAV5-optLPL重组病毒，注射剂量为1×10 13vg/kg。PBS对照组给予注射同等体积PBS。在注射后不同的时间点(0天、7天、14天、28天和2月)内眦静脉取血检测血浆甘油三酯(TG)和总胆固醇(CHO)含量变化，注射治疗后2月，取肝素后血浆检测循环LPL浓度及活性。
从图7A和图7B的结果可知，Gpihbp1 -/-小鼠AAV治疗组和PBS对照组相比，治疗前0天基线血浆甘油三酯和总胆固醇水平无显著差别，均呈现重度高甘油三酯血症。经尾静脉注射AAV5-optLPL重组病毒后7天，治疗组Gpihbp1 -/-小鼠血浆甘油三脂水平即明显降低，接近正常值水平。降脂效果维持至治疗后2个月，长期有效，离心后的小鼠外周血血浆外观与野生型C57小鼠(购自北京维通利华实验动物技术有限公司)血浆无差别(图7C)。
治疗两个月后，AAV治疗组Gpihbp1 -/-小鼠肝素后血浆LPL浓度相较于PBS对照组Gpihbp1 -/-小鼠升高接近10倍(图7D)，血浆LPL活性升高约2倍(图7E)。
实施例5.Gpihbp1 -/-小鼠尾静脉注射AAV5-optLPL减轻施用雨蛙素诱导的高甘油三酯血症急性胰腺炎的易感性与严重程度
对实施例4的Gpihbp1 -/-小鼠尾静脉注射AAV5-optLPL治疗2月后，进一步对所述Gpihbp1 -/-小鼠评估了施用雨蛙素后的影响。
施用雨蛙素诱导建立高甘油三酯血症急性胰腺炎(HTG-AP)模型的实验方案：在实施例4中对Gpihbp1 -/-小鼠尾静脉注射AAV5-optLPL治疗2月后，将注射AAV治疗组再随机分为极低剂量雨蛙素(Cae:5μg/kg/h*10针)组(n＝6)，常规剂量雨蛙素(Cae:50μg/kg/h*10针)组(n＝6)和未造模(腹腔注射同等体积PBS)组(n＝3)，其中所述10针是10次腹腔注射，每小时注射一次。PBS对照组同样随机分为极低剂量雨蛙素(Cae:5μg/kg/h*10针)组(n＝6)，常规剂量雨蛙素(Cae:50μg/kg/h*10针)组(n＝6)和未造模(腹腔注射同等体积PBS)组(n＝3)。造模后6、12、24h，取材小鼠外周血，进行血浆生化淀粉酶检测；留取胰腺组织，显微拍摄胰腺组织切片的苏木精和伊红(HE)染色照片，结果如下。
当对野生型C57小鼠施用极低剂量雨蛙素(5μg/kg/h*10次)时，不会诱发急性胰腺炎。对于Gpihbp1 -/-小鼠未治疗的PBS对照组由于重度高脂血症，当给予极低剂量雨蛙素(5μg/kg/h*10次)时，从图8A、图8B的结果可知，增加了AP的易感性，胰腺病理呈现轻度水肿、炎症细胞浸润的胰腺炎症表现，同时血浆生化淀粉酶水平增高；而经尾静脉注射AAV5-optLPL治疗后的Gpihbp1 -/-小鼠，血脂水平接近正常，AP易感性降低，胰腺病理无炎症表现，血浆生化淀粉酶水平轻度升高。
另外，从图8C－图8G的结果可知，给予常规剂量雨蛙素(50μg/kg/h*10次)诱导AP模型，未治疗的PBS对照组Gpihbp1 -/-小鼠呈现重度HTG-AP表现，胰腺组织水肿、大量炎症细胞浸润并伴有胰腺组坏死，血浆淀粉酶、脂肪酶水平增高。而经尾静脉注射AAV5-optLPL降脂治疗后的Gpihbp1 -/-小鼠，血脂水平接近正常，HTG-AP严重程度显著降低，胰腺病理仅表现为轻度水肿，血浆生化淀粉酶、脂肪酶水平相较于对照组显著下降。
实施例4和实施例5的结果表明，Gpihbp1 -/-小鼠经尾静脉注射AAV5-optLPL病毒后，能够有效地表达产生LPL蛋白，增加循环LPL浓度和活性，由此，降低Gpihbp1 -/-小鼠血浆中甘油三酯的含量，消除因血液中甘油三酯过高带来的高甘油三酯血症，减轻高甘油三酯血症性急性胰腺炎的易感性及严重程度，从而达到治疗高甘油三酯血症及其合并症急性胰腺炎的目的。
实施例6.Gpihbp1 -/-大鼠静脉注射AAV-optLPL治疗高甘油三酯血症，降低乳鼠死亡率，减轻HFD喂饲诱导的HTG-AP
Gpihbp1 -/-大鼠动物模型是通过TALEN基因编辑技术，在GPIHBP1基因外显子2上设计靶点，敲除102bp片段构建而成，由北京大学医学部刘国庆教授团队和东部战区总医院重症胰腺炎中心李维勤主任团队提供。经鉴定具有重度高脂血症表型，并从哺乳期血脂水平急剧增高达10000mg/dl以上，自发HTG-AP，28天存活率仅为60％。使用野生型SD大鼠(购自南京青龙山动物繁殖场)用作对照。
存活的成年Gpihbp1 -/-大鼠随着鼠龄增加，血脂水平逐渐增加，胰腺炎症病理逐渐加重。给予高脂饲料(HFD)喂饲后急性暴发HTG-AP，胰腺包裹性坏死，HFD喂饲后28天死亡率达42.3％。
为挽救Gpihbp1 -/-大鼠哺乳期高死亡率，给予新生乳鼠出生后7天经眶静脉注射AAV5-optLPL病毒降脂治疗，剂量1×10 13vg/kg，对照组Gpihbp1 -/-乳鼠注射同等体积PBS，观察28天，记录生存曲线。在注射后不同的时间点(7天、14天、21天、28天、35天、42天、49天、56天)内眦静脉取血检测血浆甘油三酯(TG)和总胆固醇(CHO)含量变化。哺乳期经AAV5-optLPL病毒降脂治疗的Gpihbp1 -/-大鼠随着鼠龄增长，体重增加，体内病毒载体量相对不足，血脂水平逐渐复升，于8周龄成年时对所述大鼠经尾静脉二次注射AAV8-optLPL病毒，分为高剂量组(1×10 13vg/kg)和低剂量组(1×10 11vg/kg)。所述二次注射的AAV8-optLPL病毒是如下制备的：
首先，构建了pAAV-R2C8质粒。以AAV Helper Free System(Agilent Technologies，目录号#240071)中的pAAV-RC质粒为基本骨架，采用标准的分子克隆方法，用合成的AAV8基因组(GenBank ID：NC_006261.1)中的衣壳蛋白编码序列(也称为Cap8)(基因组中第2121至4337 位序列)替换pAAV-RC质粒中HindIII至PmeI限制性酶切位点之间的序列(即，替换pAAV-RC质粒的第2013至4220位序列)，获得了pAAV-R2C8质粒。所述pAAV-R2C8质粒包含完整的AAV8的cap基因和AAV2的rep基因，在三质粒共转染包装制备重组AAV8病毒中提供包装所必须的4种Rep蛋白(Rep78、Rep68、Rep52和Rep40)和AAV8衣壳蛋白。
然后，取实施例1构建的AAV载体质粒(pscAAV-LP15-optLPL-S447X载体)、辅助质粒(pHelper)(来自AAV Helper Free System，Agilent Technologies)和AAV的Rep及Cap蛋白表达质粒pAAV-R2C8，将这三种质粒按照1:1:1的摩尔比混匀后，采用磷酸钙方法共转染HEK293细胞，转染48小时后，收获细胞和培养上清液，应用氯化铯密度梯度离心法分离纯化重组AAV8病毒，得到scAAV8-LP15-optLPL-S447X重组病毒(也简称为“AAV8-optLPL病毒”)。由于使用的质粒pAAV-R2C8表达了AAV8衣壳蛋白，由此，获得的是AAV8重组病毒。与实施例2同样地通过定量PCR方法测定通过上述制备方法得到的AAV8重组病毒的基因组滴度。
对Gpihbp1 -/-大鼠经尾静脉二次注射AAV8-optLPL病毒治疗后2周，喂饲高脂饲料(Research Diet 12492)14天建立自发HTG-AP模型。喂饲HFD期间的不同时间点(1天、3天、7天、14天)内眦静脉取血检测血浆甘油三酯(TG)和总胆固醇(CHO)含量变化。喂饲HFD14天后取材外周血、胰腺组织和肺组织。
从图9A-9E的结果可知，和未治疗Gpihbp1 -/-哺乳期乳鼠相比，新生Gpihbp1 -/-乳鼠出生后7天经眶静脉注射AAV5-optLPL病毒降脂治疗后，显著提高Gpihbp1 -/-大鼠28天生存率至80％，血浆生化甘油三酯和总胆固醇水平显著下降，TG水平维持在800-1000mg/dl。8周成年后，经尾静脉二次注射AAV8-optLPL病毒，血脂水平进一步下降至正常水平，并且与注射的AAV8-optLPL病毒载体呈剂量依赖效应。
第一针AAV病毒载体静脉注射后9周(第二针AAV病毒载体静脉注射后2周)去肝素后血浆检测循环中LPL浓度和活性，相较于未治疗Gpihbp1 -/-大鼠，AAV-optLPL治疗后Gpihbp1 -/-大鼠LPL浓度升高10倍(图9G)和LPL活性升高2-3倍(图9H)。离心后血浆外观脂血明显减轻，接近正常大鼠血浆(图9F)。
喂饲HFD饲料后，未治疗Gpihbp1 -/-大鼠血脂水平急剧升高，最高值达18000mg/dl(图10B)。喂饲HFD饲料后14天胰腺外观呈现明显包裹性坏死(图10A)，胰腺组织HE染色见大片状胰腺组织坏死，大量炎症细胞浸润，呈现纤维化表现，自发HTG-AP严重(图10D)，同时全身炎症反应，肺组织病理表现为急性肺损伤(图10E)，多脏器功能障碍。
对AAV5-optLPL和AAV8-optLPL治疗后的Gpihbp1 -/-大鼠喂饲HFD，血脂水平3天应激性升高后，迅速下降，低剂量AAV8-optLPL治疗组血浆甘油三脂水平3天升高至大于5000mg/dl，随后逐渐降至约2000mg/dl(图10B)；血浆胆固醇水平起初略有升高，随后逐渐降低(图10C)；胰腺组织病理HE表现炎症细胞浸润，轻度胰腺坏死形成，显著减轻了HTG-AP的严重程度。高剂量AAV8-optLPL治疗组血浆甘油三脂水平轻度升高至1000mg/dl；血浆胆固醇水平维持在较低水平；胰腺组织外观基本正常，病理HE表现无炎症细胞浸润，无胰腺坏死形成，预防了HTG-AP的发生(图10D)。
以上结果表明，Gpihbp1 -/-大鼠经静脉注射AAV-optLPL病毒治疗后，能够有效地表达产生LPL蛋白，降低大鼠血浆中甘油三酯的含量，降低Gpihbp1 -/-大鼠哺乳期因重度高脂血症诱 发HTG-AP导致的高死亡率，同时预防和减轻HFD喂饲诱导的HTG-AP发病，从而达到治疗高甘油三酯血症及其合并症急性胰腺炎的目的。
实施例7.静脉注射重组AAV病毒治疗高脂饲料喂饲诱发的仓鼠高脂血症模型
从伊维沃生物科技公司购买12只雄性WT仓鼠，年龄在2-3月龄。仓鼠适应性饲养一周后称重。过夜禁食后，用玻璃毛细管进行内眦静脉采血，肝素抗凝，4度，4000rpm，离心10分钟，分离得到血浆。使用甘油三酯和胆固醇检测试剂盒(中生北控生物科技股份有限公司)检测甘油三脂(TG)和总胆固醇(TC)。
从北京博泰宏达生物科技公司订制胆固醇含量为0.5％且猪油含量为20％的高脂饲料。在仓鼠适应性饲养一周后开始喂饲该高脂饲料(实验的第0天)。在喂饲该高脂饲料4周(实验的第28天)时，过夜禁食12小时后取血，测量血浆TG、TC,根据血脂结果将仓鼠分为两组(以两组之间血浆TG、TC无明显差异为原则)，每组6只。一组小鼠给予颈静脉注射scAAV5-EGFP(简称AAV5-EGFP)，另一组小鼠给予一次颈静脉注射scAAV5-optLPL-S447X(简称AAV5-optLPL)。按照1E+12vg/kg的注射剂量，根据相应的AAV滴度，计算出注射容积，用无菌PBS定容至300μL后给予注射。在注射后第7、14、28天，过夜禁食取血，离心得到血浆，使用试剂盒检测血浆TG，TC。
结果显示，喂饲高脂饲料4周后，仓鼠血浆TG明显升高，TC也有轻微升高。在注射重组AAV病毒后第14天，发现与注射AAV5-EGFP组相比，AAV5-optLPL组血浆TG明显降低。在第28天，AAV5-optLPL组的血浆TG仍然较低(图11，12)。
给药后第14天进行口服脂质负荷试验，即，过夜禁食后，取血(记录为0点血)，然后按照10mL/kg的剂量给予仓鼠橄榄油灌胃，灌胃后第1、2、4、8小时取血，测量血浆TG，结果显示注射AAV5-optLPL组的仓鼠餐后TG的清除明显加快(图13)。
给药后第28天取完测血脂用的禁食血后，给动物喂饲高脂饲料，4小时后再按照500U/kg的剂量腹腔注射肝素，注射20分钟后取血，离心分离得到血浆，用肝素后血浆作为酶源，使用LPL试剂盒(Abcam)，测量LPL活性。结果显示AAV5-optLPL组的LPL活性明显高于AAV5-EGFP组(图14)。
在给药后第30天(即，实验的第58天)取材，检测肝脏外源性LPL基因的mRNA表达水平，在AAV5-optLPL组检测到了外源性LPL基因的mRNA表达(图15)。
以上描述了本发明的示例性实施方案。本领域技术人员可以借鉴本文内容，适当改进工艺参数来实施本申请的发明。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明的范围内。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明的技术。

Claims (10)

  1. 一种优化的人脂蛋白脂酶基因表达框，其包含：
    (1)如SEQ ID NO:5所示的启动子序列或者与其具有至少约90％同一性的启动子序列；
    (2)插入有SEQ ID NO:2所示的内含子序列或者插入有与SEQ ID NO:2具有至少约90％同一性的内含子序列的编码功能性脂蛋白脂酶(例如，LPL-S447X)的核苷酸序列，例如，
    (i)如SEQ ID NO:3所示的优化的编码LPL-S447X的核苷酸序列；
    (ii)与如SEQ ID NO:3所示的核苷酸序列互补的核苷酸序列；
    (iii)与(i)或(ii)的核苷酸序列编码相同的LPL-S447X，但因遗传密码的简并性而与(i)或(ii)的核苷酸序列不同的核苷酸序列；或
    (iv)与(i)或(ii)或(iii)所述核苷酸序列具有至少70％同一性的序列；和
    (3)至少一个(例如，2－8个)SEQ ID NO:6所示的人miR-142-3p靶序列，例如，具有4个串联的人miR-142-3p靶序列，例如，SEQ ID NO:7所示的人miR-142-3p靶序列。
  2. 根据权利要求1所述的优化的人脂蛋白脂酶基因表达框，其还包含：
    (4)如SEQ ID NO:10所示的或者与其具有至少约90％同一性的BGH polyA序列；和/或
    (5)如SEQ ID NO:11所示的或者与其具有至少约90％同一性的TBG增强子序列。
  3. 根据权利要求2所述的基因表达框，其碱基全序列如SEQ ID NO:12所示或与SEQ ID NO:12具有至少约90％同一性，特征在于，表达框表达产物为LPL-S447X。
  4. 一种病毒载体，其特征在于，包含权利要求1－3中任一项所述的优化的人脂蛋白脂酶基因表达框。
  5. 一种用于预防和/或治疗严重高甘油三酯血症或其引起的疾病如急性胰腺炎的基因药物，特征在于，
    (1)该基因药物是一种病毒载体；
    (2)该病毒载体包含权利要求1－3中任一项所述的优化的人脂蛋白脂酶基因表达框；和
    (3)将该基因药物静脉注射，通过肝脏特异性表达人脂蛋白脂酶(例如，LPL-S447X)来达到预防和/或治疗严重高甘油三酯血症或其引起的疾病如急性胰腺炎的目的，
    例如，所述严重高甘油三酯血症或其引起的疾病如急性胰腺炎是LPL基因缺陷和/或GPIHBP1基因缺陷和/或高脂饮食诱发的。
  6. 根据权利要求4所述的病毒载体或根据权利要求5所述的基因药物，其特征在于，
    (1)所述病毒载体是重组腺相关病毒载体；和/或
    (2)所述病毒载体的基因组可自我互补形成双链DNA分子。
  7. 根据权利要求6所述的病毒载体或基因药物，其特征在于，所述重组腺相关病毒载体是选自AAV1、AAV2、AAV3B、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAVrh.10或它们的组合的血清型的重组腺相关病毒载体，优选的血清型为AAV5、AAV3B、AAV8、AAV9。
  8. 根据权利要求1－3中任一项所述的优化的人脂蛋白脂酶基因表达框和根据权利要求4、6和7中任一项所述的病毒载体的用途，用于制备预防和/或治疗高甘油三酯血症或其引起的疾病如急性胰腺炎的基因药物，例如，用于制备预防和/或治疗LPL基因缺陷和/或GPIHBP1基因缺陷和/或高脂饮食诱发的高甘油三酯血症或其引起的疾病如急性胰腺炎的基因药物。
  9. 根据权利要求8所述的用途，其特征在于，所述基因药物的给药方式为静脉注射，所述高甘油三酯血症是LPL基因缺陷和/或GPIHBP1基因缺陷和/或高脂饮食引起的高甘油三酯血症。
  10. 根据权利要求8或权利要求9所述的用途，其特征在于，所述基因药物的一次给药可持续降低体内的血浆TG浓度，由此预防和/或治疗高甘油三酯血症，特别地，预防和/或治疗严重的高甘油三酯血症。
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