JP2019122371A - Vector plasmid used for gene therapy of hunter syndrome, lentivirus vector system, cell, and cell preparation - Google Patents

Abstract

To provide a novel vector plasmid that performs efficient IDS supply to central nerve and bone, and enables gene therapy of Hunter syndrome.SOLUTION: The present invention provides a vector plasmid composed of CMV hybrid 5'LTR-Ψ-RRE-cPPT-PGK-IDS cDNA-WPRE mut9-3'LTR, a vector plasmid composed of CMV hybrid 5'LTR-Ψ-RRE-cPPT-CD11b-IDS cDNA-WPRE mut9-3'LTR, and a vector plasmid composed of CMV hybrid 5'LTR-Ψ-RRE-cPPT-MND-IDS cDNA-WPRE mut9-3'LTR.SELECTED DRAWING: Figure 2

Description

  The present invention relates to a vector plasmid for use in a lentiviral vector system for gene therapy of Hunter syndrome, a lentiviral vector system comprising the vector plasmid, a cell into which the lentiviral vector system has been introduced, and a cell comprising the cell It relates to a preparation.

  Hunter's syndrome is mucopolysaccharidosis type II (hereinafter sometimes referred to as Mucopolysaccharidosis type II, MPSII), and lacks or decreases mucopolysaccharidolytic enzyme activity, resulting in failure of systemic organs centered on connective tissue. Accumulation of completely degraded mucopolysaccharide is a hereditary metabolic disorder presenting various symptoms. Among mucopolysaccharidosis, it is the only X-chromosome recessive inheritance, which is reported to occur in 1 in 1 to 130,000 male offspring.

  The cause is an abnormality in iduronate-2-sulfatase (hereinafter referred to as iduronate-2-sulfatase: IDS) gene, which is one of lysosomal hydrolases, located in the X short arm (Xq28) of the X chromosome. It is believed that. Genetic abnormalities range from mutations to deletions, and it is thought that genotype diversity reflects phenotype diversity.

  In MPSII, glycosaminoglycans (GAGs) accumulate throughout the body due to IDS deficiency, resulting in central nervous system, bone, heart and other disorders, and severe cases die in their teens. Existing therapies include enzyme replacement therapy (ERT) and hematopoietic stem cell transplantation (HSCT).

  Enzyme replacement therapy is a treatment method in which a defective enzyme is replaced in the body as a drug, and needs to be performed continuously (Non-patent Documents 1 to 6). The earlier the implementation, the more effective, and it is possible to prevent or progress various symptoms by supplementing the enzyme weekly before the symptoms progress. However, it is difficult to treat complete lesions such as bone deformities and valvular heart disease and central nervous symptoms with enzyme replacement therapy alone, and side effects such as allergy and anaphylaxis may occur with administration. Furthermore, enzyme replacement therapy is expensive and expensive, and must be performed once a week for a lifetime, which places a heavy burden on patients.

  Hematopoietic stem cell transplantation is a therapeutic method that can be expected to suppress the progression of symptoms of the brain and heart, and is a therapeutic method to be considered if it is up to about two years before the completion of a lesion (Non-patent documents 7 to 10). When hematopoietic stem cells are engrafted by bone marrow transplantation, enzymes can be produced by themselves, which makes it possible to reduce the frequency of enzyme replacement therapy. However, hematopoietic stem cell transplantation is also not effective in the central nervous system, can not be effective without transplantation treatment under 2 years of age, appropriate donor (bone marrow donor) can not be obtained, Graft Versus Host Disease (GVHD), etc. Have the risk of serious side effects.

  Enzyme replacement therapy, which is the standard treatment for Hunter's syndrome, as described above, greatly affects the patient's QOL because weekly visits are required throughout life. In addition, as described above, hematopoietic stem cell transplantation also has a risk of infection and rejection, and also needs to search for a donor. Therefore, new treatments that reduce the burden on patients are needed. There is gene therapy using a patient's own bone marrow as a new treatment method. This is a method of incorporating a normal gene into the bone marrow of a patient using a viral vector, which improves the enzyme activity more than utilizing the bone marrow of another person, and is expected to have an effect on the head, bones and heart valve. In addition, using their own bone marrow eliminates the risk of infection and rejection, and eliminates the need to look for donors.

  The present inventors can achieve ERT and HSCT by expressing IDS gene in hematopoietic stem cells using (B6 / MPS II) virus promoter (MND promoter: Moloney murine leukemia virus LTR / myeloproliferative sarcoma virus enhancer) in model mice It succeeded in reducing GAG of the brain of MPSII model mouse which was not (nonpatent literature 11).

Muenzer J, Gucsavas-Calikoglu M, McCandless SE, et al: A phase I / II clinical trial of enzyme replacement therapy in mucopolysaccharidosis II (Hunter syndrome). Mol Genet Metab 2007; 90: 329-337. Muenzer J, Wraith JE, Beck M, et al: A phase II / III clinical study of enzyme replacement therapy with idursulfase in mucopolysaccharidosis II (Hunter syndrome). Genet Med 2006; 8: 465-473. Okuyama T, Tanaka A, Suzuki Y, et al: Japan Eprase Treatment (JET) study: idurulase enzyme replacement therapy in adult patients with attenuation hunters syndrome (Mucopolysaccharidosis II, MPS II). Mol Genet Metab 2010; Muenzer J, Beck M, Eng CM, et al: Long-term, open-labeled extension study of idursulfase in the treatment of Hunter syndrome. Genet Med 2011; 13: 95-101. da Silva EM, Strufaldi MW, Andriolo RB, et al: Enzyme replacement therapy with idursulfase for mucopolysaccharidosis type II (Hunter syndrome). Syst Rev 2016; 2: CD008185. Parini R, Rigoldi, b M, Tedescoet L, et al: Enzymatic replacement therapy for Hunter's disease: Up to 9 years experience with 17 patients. MoI Genet Metab Rep 2015; 3: 65-74. Guffon N, Bertrand Y, Forest I, et al: Bone marrow transplantation in children with Hunter syndrome: Outcome after 7 to 17 years. J Pediatr 2009; 154: 733-737. Kato S, Yabe H, Takakura H, et al: Hematopoietic stem cell transplantation for inborn errors of metabolism: a report from the research committee on transplantation for inborn errors of metabolism of the ministry of health, labour and welfare and the working group of Pediatr Transplant 2016; 20: 203-214. The Japan society for hematopoietic cell transplantation. Vellodi A, Young E, Cooper A, et al: Long-term follow-up following bone marrow transplantation for Hunter's disease. J Inherit Metab Dis 1999; 22: 638-648. Tanaka A, Okuyama T, Suzuki Y, et al: Long-term ef-cacy of hematopoietic stem cell transplantation on brain involvement in patients with mucopolysaccharidosis type II: a nationwide wide survey in Japan. MoI Genet Metab 2012; Toya Ohashi, Hematopoetic Stem Cell Gene Therapy Therapeutics Neuropathic Phenotype in Murine Model of Mucopolysaccharidosis Type II, HUMAN GENE THERAPY 2015; 26: 357-366.

  However, gene therapy methods including vector constructs and gene transfer methods for efficiently supplying IDS to central nerves and bones in MPS II have not been realized.

  The present invention has been made in view of the above problems, and provides a novel vector plasmid capable of efficiently supplying IDS to central nervous system and bone lineage as well as each organ and enabling gene therapy of Hunter syndrome. With the goal.

  The vector plasmid according to the present invention is a vector plasmid consisting of CMV hybrid 5 'LTR-Ψ-RRE-cPPT-PGK-IDS cDNA-WPREmut9-3' LTR, which vector sequence comprises the nucleotide sequence set forth in SEQ ID NO: 1 It is.

  Furthermore, the vector plasmid according to the present invention is a vector plasmid consisting of CMV hybrid 5 'LTR-R-RRE-cPPT-CD11b-IDS cDNA-WPREmut9-3' LTR, which comprises the nucleotide sequence set forth in SEQ ID NO: 2 It is a vector plasmid.

  Furthermore, the vector plasmid according to the present invention is a vector plasmid consisting of CMV hybrid 5 'LTR-R-RRE-cPPT-MND-IDS cDNA-WPREmut9-3' LTR, which comprises the nucleotide sequence set forth in SEQ ID NO: 3 It is a vector plasmid.

  According to the present invention, a novel vector plasmid can be obtained which efficiently supplies IDS to central nervous system and bone lineage, and enables gene therapy of Hunter syndrome.

It is a figure explaining a lentivirus vector system. It is a figure explaining three types of vector plasmids among four types of plasmids contained in the system shown in FIG. FIG. 6 shows the measured values of IDS enzyme activity in serum, cerebellum, cerebrum, heart, liver, spleen after 6 months of gene therapy. FIG. 8 shows the measured values of GAG accumulation in cerebellum, cerebrum, heart, liver, spleen after 6 months of gene therapy.

Hereinafter, embodiments of the present invention will be specifically described with reference to the accompanying drawings, which are for the purpose of facilitating the understanding of the principle of the present invention, and the scope of the present invention is as follows: The present invention is not limited to the embodiment, and other embodiments in which a person skilled in the art appropriately replaces the configuration of the following embodiments are also included in the scope of the present invention.

  The lentiviral vector system according to the present invention has the vector plasmid, the packaging plasmid, the Rev plasmid, and the VSV-G envelope plasmid according to the present invention as described in FIG. The state in which these are integrated to infect cells and integrate into the genome is called a provirus.

  A method of making lentiviral vectors is described. First, the HIV-1 proviral genome is divided into four plasmids: vector, packaging, Rev, and envelope (FIG. 1).

  The packaging plasmid encodes viral genes other than the envelope and supplies in trans the protein for producing viral particles. The modified genes (vif, fpr, vpu, nef) are deleted as the modified gene product is not required for infection of non-dividing cells. Since the modified gene product is not essential but important for HIV-1 replication and is closely related to HIV-1 virulence, deletion increases safety. Moreover, since tat is essential for HIV-1 replication, deleting tat from the packaging plasmid further enhances safety. Since the packaging plasmid does not have a packaging signal (RNA), RNA transcribed from this plasmid is not incorporated into viral particles.

  Rev acts on post-transcriptional RNA, and structural proteins are translated by selectively transporting unspliced mRNA out of the nucleus. In the third generation, the packaging plasmid is a separate plasmid.

  Since the envelope plasmid can only infect CD4 positive cells with the HIV-1 envelope, VSV-G (vesicular stomatitis virus G gylcoprotein) is used to extend the host range. The receptor for VSV-G is considered to be a phospholipid, and by using VSV-G as an envelope, membrane fusion between viral particles and cells occurs independently of the receptor on the cell surface. Therefore, basically, it can be infected regardless of animal species or cytomas. Also, VSV-G is physically strong, and it is possible to easily concentrate virus particles by ultracentrifugation.

  In the vector plasmid, a target gene is integrated between LTRs (repetitive sequences controlling transcription and expression) at both ends containing a packaging signal (Ψ). Furthermore, it has a primer binding site essential for reverse transcription, and RNA transcribed from this plasmid is incorporated into viral particles. Since the HIV-1 LTR promoter activity is very weak in the absence of tat, the internal promoter (FIG. 1 Pro) is used for expression of the incorporated foreign gene. In this embodiment, the internal promoter is a PGK / CD11b / MND promoter and the details are shown in FIG. The performance such as these three types of introduction efficiency is compared and the optimum one is used. The inserted foreign gene (FIG. 1 Purpose gene) to be integrated is the gene IDS cDNA that expresses the MPS II deletion enzyme. RRE (rev responsive element) is necessary for Rev to bind and efficiently transport full-length viral genomic RNA from the nucleus to the cytoplasm. During reverse transcription, the reverse transcription begins with the synthesis of plus strand from the PPT (polypurine tract) sequence immediately upstream of the 3 'LTR, while the lentivirus has the same sequence called cPPT (central polypurine tract) in the central part of the genome. There is one more place where the synthesis is also carried out, so the final double stranded cDNA will have a triple stranded structure of about 100 base pairs called DNA flap at the center. The cPPT sequence has been suggested to affect the efficiency of reverse transcription, and incorporation of the cPPT sequence into a vector increases the efficiency of gene transfer. WPRE (woodchuk hepatitis virus posttranscriptional regulatory element) is incorporated to increase expression efficiency. In other words, WPRE plays a role in active transport of mRNA from nucleus to cytoplasm and stability of mRNA in cytoplasm. By incorporating this sequence into the vector, expression efficiency of titer and transgene can be improved. Go up. In addition, deletion of the enhancer / promoter portion in U3 of the 3 'LTR means that both LTRs of 3' and 3 'in the proviral state have no promoter activity, and transcription of the entire genome from the 5' R region takes place It will not be. As a result, a lentiviral vector corresponding to an HIV-1 proliferative strain or the like deficient strain satisfies the requirements of the position paper "3. The provirus does not have LTR promoter activity and the HIV whole genome is not transcribed", SIN (self inactivating ) Is a vector. In addition, substitution of U3 of 5 'LTR with CMV promoter eliminates tat dependency and makes it possible to delete tat from the packaging plasmid. Although the target gene to be incorporated into the vector plasmid is common to IDS as described above, three types are prepared depending on the difference in internal promoter. That is, CMV hybrid 5 'LTR-Ψ-RRE-cPPT-PGK-IDS cDNA-WPREmut9-3' LTR, comprising the nucleotide sequence of SEQ ID NO: 1 (one or several bases in the nucleotide sequence of SEQ ID NO: 1 Is a vector plasmid containing a substituted, deleted, added, and / or inserted nucleotide sequence), CMV hybrid 5 'LTR-Ψ-RRE-cPPT-CD11b-IDS cDNA-WPREmut9-3' LTR, A vector plasmid comprising a nucleotide sequence set forth in SEQ ID NO: 2 (including a nucleotide sequence in which one or several bases have been substituted, deleted, added and / or inserted in the nucleotide sequence set forth in SEQ ID NO: 2), CMV hybrid 5 'LTR-Ψ-RRE-cPPT-MND-IDS cDNA-WPREmut9-3' LTR, wherein the nucleotide sequence of SEQ ID NO: 3 (one or several bases in the nucleotide sequence of SEQ ID NO: 3 are substituted , Including deletion, addition, and / or inserted base sequences. 2). Among them, the second generation lentivirus vector carrying the same promoter (MND) as the vector plasmid consisting of CMV hybrid 5 'LTR-Ψ-RRE-cPPT-MND-IDS cDNA-WPREmut 9-3' LTR We have succeeded in reducing brain GAG, and it is thought that the vector plasmid described above can efficiently deliver IDS to central nerves and bones.

  Next, a method for producing a vector plasmid according to the present invention will be described. The vector plasmid is carried out by a cloning procedure in which the DNA fragment of interest (IDS cDNA) is loaded onto the vector plasmid of interest (pLGT1 plasmid).

  First, in preparation of the insert DNA (preparation of the target DNA fragment IDS cDNA), the restriction enzyme cleavage site of the target DNA fragment is confirmed in advance, and the restriction enzyme used for excision is matched with the cloning site of the vector plasmid to be used next. Select The restriction enzyme site is 5'-MluI ACGCGT / 3'-BamHI GGATCC.

  Next, in preparation of the vector plasmid, the cloning site of the target vector plasmid (pLGT1 plasmid) is cleaved with a restriction enzyme to linearize the vector. Then, the insert DNA and the linearized plasmid are ligated to prepare a vector plasmid according to the present invention.

  The four (packaging, rev, envelope, vector) plasmid systems described above are called third generation lentiviral vectors and have deleted one-third or more of the HIV-1 genome throughout the system, and wild type HIV There is little possibility of producing -1. Since the homologous regions between each plasmid are also minimized, there is almost no possibility that homologous recombination will produce a virus having the ability to grow on its own.

  Next, usage modes of lentiviral vectors are described.

  Co-transfection of 293T cells in which the third generation packaging plasmid, Rev plasmid, and VSV-G envelope plasmid were cultured in large amounts on the different vector plasmids of the three types of promoters constructed, and after 3 days, the supernatant culture fluid was recovered Do. At this point, the culture solution contains virus particles, which are purified by ultracentrifugation and stored at -80 ° C in aliquots. Storage shall be within one year when viral activity is not reduced. The stored vector virus is immediately returned to liquid at 37 ° C and used.

  Furthermore, the cell of the present invention is a cell transformed by introducing the lentiviral vector system of the present invention. The cells into which the lentiviral vector system according to the present invention is introduced are not particularly limited. For example, T cells, NK cells or their precursor cells (hematopoietic stem cells, lymphoid stem cells, etc.) can be used. . The hematopoietic stem cells are CD34 positive cells.

In addition, the cell preparation of the present invention is a cell preparation containing a therapeutically effective amount of cells transformed with the lentiviral vector system of the present invention. The cell preparation can contain, for example, 10 ⁴ to 10 ¹⁰ cells for one administration. In addition, various components such as dimethyl sulfoxide (DMSO) and serum albumin for the purpose of cell protection, antibiotics and the like for the purpose of preventing bacterial contamination, etc. for the purpose of cell activation, proliferation or differentiation induction ( Components such as vitamins, cytokines, growth factors, steroids, etc.) may be contained in the cell preparation.

  The cell preparation of the present invention is, for example, a cell preparation for the prevention and / or treatment of Hunter syndrome. As used herein, "prevention" includes arresting and delaying the onset of the disease, as well as prevention prior to becoming a disease, as well as preventing the recurrence of the disease after treatment. "Treatment", on the other hand, includes curing the condition, ameliorating the condition and suppressing the progression of the condition.

  As MPSII model mice, IDSs of human hematopoietic stem cell-transplantable NOG mice (NOD / Shi-scid, IL-2RγKO) were knocked out with CRISPR / Cas9 to obtain model mice (NOG / MPS II). Moreover, IDS of B6 mouse | mouth (C57BL / 6 mouse) was knocked out by CRISPR / Cas9, and it was set as model mouse | mouth (B6 / MPS II).

  The MPS II model mice are genetically diagnosed, and 8-week-old hemizygous male mice are pretreated to remove bone marrow from their femurs. After culturing this, a mouse hematopoietic stem cell line which has been separated and extracted by the microbead method etc. is infected with the virus vector purified at MOI = 50, and is transfected. The introduced bone marrow stem cells are administered by tail vein injection after semi-lethal irradiation of another hemizygous male mouse. After that, follow up to 20 weeks of age and analyze enzyme (IDS) activity in circulating blood, enzyme activity in each organ, especially in central nerve, bone, heart, etc., accumulation of substrate (GAG). Conduct behavioral tests to assess central function. These are performed with three types of viral vectors with different promoters, and evaluation is performed between each group.

  The following describes the implementation of gene therapy on B6 / MPS II mice, NOG / MPS II mice. Hematopoietic stem cells of B6 / MPS II mice are infected with lentiviral vector expressing 3 kinds of IDS and lethal dose radiation is transferred to irradiated or anti-c-kit antibody (ACK2) + low dose irradiated B6 / MPS II mice . The reason for using the anti-c-kit antibody is as follows. In this gene therapy, it is necessary to kill the patient's hematopoietic stem cells and to open the bone marrow niche so that the transfected hematopoietic stem cells can be engrafted. Hematopoietic stem cells are present in the bone marrow niche via c-kit etc. By existing in this niche, they do not undergo cell division and maintain the function as stem cells, ie pluripotency and self-replication ability (called dormancy) ). It is resistant to radiation, anticancer drugs, etc. because it does not divide cells. Therefore, in order to kill the patient's hematopoietic stem cells, a large amount of anticancer agent / radiation is required. However, once hematopoietic stem cells leave the niche, they start division and become more sensitive to radiation and anticancer drugs. That is, hematopoietic stem cells are killed even with low doses of radiation. Thus, when anti-c-Kit antibody is administered to the recipient, the recipient's hematopoietic stem cells begin to divide and divide from the niche and become radiosensitive. This allows even low doses of radiation to be sufficient as a pretreatment and avoids a strong pretreatment.

  Human CD34 positive cells are infected with lentiviral vector expressing 3 kinds of IDS and transplanted into NOG / MPS II mice. This is a more clinical treatment as it targets human hematopoietic stem cells. Since pretreatment is an immunodeficient mouse, low dose radiation is sufficient.

  Biochemical evaluation measures IDS activity and GAG at each site such as heart, liver, spleen, central nerve, and bone. The measurement of GAG is performed by a method using liquid chromatography mass spectrometry (LC / MS / MS) which is a modification of the disease specific GAG measurement method (Shimada Y et al. MoI Genet Metab 2016). Specifically, 2-sulfated iduronic acid present at the non-reducing end of GAG extracted and purified from tissue is excised as iduronic acid using recombinant IDS and iduronidase, and quantitative analysis in Multiple Reaction Monitoring (MRM) mode I do.

  In pathological evaluation, accumulation of GAG in nerve cells and the like is performed with a normal light microscope and an electron microscope. With regard to the brain, the number and area of LAMP2-positive cells, GFAP-positive cells, BSI-B4-positive cells, and GM3-positive cells, which appear secondary to inflammatory changes, are examined using immunohistological methods. We also examine SCMAS-positive cells that are said to be associated with autophagy.

(1) Preparation of Vector Plasmid A basic vector of lentiviral vector plasmid was prepared by the following procedure. Based on pLV-SIN-CMV Neo vector (Takara Bio Inc.), the hybrid LTR is created by replacing the U3 promoter region of 5 'HIV-1 LTR with the CMV promoter, reducing Gag residual sequence downstream of packaging signal, internal Remove the promoter PCMMIE to the neomycin resistance gene, change it to a mut9 sequence (X protein frameshift by removing one base) to suppress the expression of X protein contained in the WPRE sequence, and cHS4 core insulator in the 3 'SIN LTR region A lentiviral vector plasmid pLGT1 carrying a sequence (forward direction) was constructed. A vector plasmid was prepared by the cloning procedure in which the IDS cDNA fragment was loaded onto the vector plasmid pLGT1.

  First, in preparation of insert DNA (preparation of IDS cDNA fragment), restriction enzyme cleavage sites of the target DNA fragment were confirmed in advance, and restriction enzymes used for excision were selected according to the cloning site of the vector plasmid to be used next. The restriction enzyme site was 5'-MluI ACGCGT / 3'-BamHI GGATCC.

  A reaction solution was prepared by mixing substrate DNA (≦ 1 μg), Hind III (1 μl), BamH I (1 μl), 10 × K Buffer (2 μl) and sterile distilled water (20 μl). The reaction was allowed to proceed at 37 ° C. for 1 hour, loading buffer was added to half (about 10 μl) of the reaction solution, agarose gel electrophoresis was performed, and cleavage was confirmed. The gel containing the band of the target DNA fragment was excised. The DNA was purified from the gel using NucleoSpin Gel and PCR Clean-up to make an insert DNA solution. The PCR amplification of insert DNA was performed using the primer which 5 'side added the restriction enzyme cutting site used for ligation with a vector.

  Next, in preparation of the vector plasmid, the cloning site of the target vector plasmid (pLGT1 plasmid) was digested with a restriction enzyme to linearize the vector. A reaction solution was prepared by mixing vector plasmid DNA (≦ 1 μg), Hind III (1 μl), BamH I (1 μl), 10 × K Buffer (2 μl) and sterile distilled water (20 μl). The reaction was allowed to proceed at 37 ° C. for 1 hour, and DNA was purified from the reaction solution using NucleoSpin Gel and PCR Clean-up. The recovered solution was used as a plasmid DNA solution.

  Then, 100 fmol of insert DNA solution, 50 ng (25 fmol) of plasmid DNA solution, 7.5 μl of Ligation Mix, and 15 μl of sterile distilled water were mixed using DNA Ligation Kit, and reacted at 16 ° C. for 30 minutes. Thereby, the insert DNA and the linearized plasmid were ligated to produce a vector plasmid according to the present invention.

(2) Virus Packaging In lentivirus packaging, in addition to the packaging plasmids pCAG-HIVgp and VSVG / REV plasmids pCMV-VSV-G-RSV-Rev, the SIN vector plasmid pLGT1 plasmid prepared above was used.

Cells were seeded in 10cm diameter of Poly-L-Lysine treated dishes of subconfluent 293T cells 5 × 10 ^6/10 ml DMEM medium / dish (5-6-fold dilution). The cells were cultured at 37 ° C., 5% or 10% CO ₂ incubator for about 24 hours to reach 70-80% confluence.

  In a 1.5 ml eppendorf tube or a 5 ml polystyrene tube (Falcon 2058), 500 μl of HBSS / one dish was prepared. The following plasmid DNA was added to each dish and mixed by vortexing or pipetting.

Packaging plasmid (pCAG-HIVgp) 3 μg
VSV-G, Rev plasmid (pCMV-VSV-G-RSV-Rev) 3 μg
SIN vector plasmid 6 μg
24 μl of PEI (1 mg / ml) was added to each dish, mixed by vortexing or pipetting, and left at room temperature for 15 to 20 minutes. The mixture was mixed by pipetting several times with a pipetman (P1000), and then little by little was uniformly spread on a dish, and cultured for 12-16 hours at 37 ° C., 5% or 10% CO ₂ incubator.

The culture supernatant was aspirated and replaced with 7.5 ml (37 ° C.) of DMEM medium (+10 μM Forskolin). The cells were cultured at 37 ° C., 5% or 10% CO ₂ incubator for about 48 hours.

  The virus-containing culture supernatant was recovered, and suspended cells and the like were removed through a 0.45 μm filter. When not concentrated, the filter filtrate was dispensed as it is and stored at -80 ° C. When concentrating, transfer the virus-containing culture supernatant through a 0.45 μm filter to Amicon Ultra-0.5 (100 K), and add up to the volume (500 μl) of the filter cup with HBSS to obtain 14,000 x g (angle) 10 ° C at 4 ° C. -Centrifuge for 30 minutes and discard the filtrate in the lower tube.

  The filter cup was placed in a new tube in the reverse direction, and centrifuged at 1,000 × g, 4 ° C. for 2 minutes, and the virus solution was collected in the new tube below. After adding 100 μl of HBSS (<4 μl / dish) to the filter cup and washing the membrane with pipetting several times, it was collected in the lower tube.

  In order to recover the virus solution remaining in the filter cup, the filter cup was again set in the reverse direction and centrifuged at 1,000 × g, 4 ° C. for 2 minutes to recover all virus solutions in the lower tube. The recovered virus solution was made up to 10 ml with PBS (−), and ultracentrifuged at 50,000 × g (19,400 rpm for BECKMAN SW28) at 4 ° C. for 2 hours. The supernatant was decanted or aspirated and suspended in 1 ml PBS using a pipetman (P200). Transfer to a 50 ml centrifuge tube and vortex for 3 hours.

  Transfer Vortex suspension to 1.5 ml eppendorf tube, centrifuge at 5000 rpm for 1 min to remove debris as pellet, pipet the supernatant into screw capped tube and store at -80 ° C, 1 It was decided to use it within the year.

(3) Ex Vivo Gene Therapy Ex vivo Gene Therapy was performed by the following method using the three types of lentiviral vectors described above. First, MPSII model mice were genetically diagnosed, and 8-week-old hemizygous male mice were pretreated to remove bone marrow from their femurs. After culturing this, a mouse hematopoietic stem cell line isolated and extracted by the microbead method or the like was infected with the virus vector purified at MOI = 50, and was transfected. The introduced bone marrow stem cells were administered by tail vein injection after semi-lethal irradiation was applied to another hemizygous male mouse. After that, they were followed up to 20 weeks of age and analyzed for enzyme (IDS) activity in circulating blood, enzyme activity in each organ, especially in central nerve, bone, heart, etc., accumulation of substrate (GAG). These were performed with three types of viral vectors different in promoter, and evaluation was performed between each group.

  As shown in FIG. 3, IDS enzyme activity after 6 months of gene therapy was significantly different in the group treated with the vector carrying the MND promoter in serum, cerebellum, cerebrum, cerebrum, heart, liver and spleen. Showed a high enzyme activity. In addition, after 6 months in this treatment group, serum, liver, spleen and heart showed 10 times or more higher enzyme activity than the normal group.

  As shown in FIG. 4, the substrate (GAG) accumulation amount after 6 months of gene therapy is the normal group in cerebellum, cerebrum, heart, liver, spleen among the three groups treated with the vector carrying the MND promoter And it improved without a significant difference. In the group treated with the vector carrying PGK promoter and CD11b promoter, significant difference was observed in the cerebrum and cerebellum from the normal group.

  From the above results, it was found that the vector carrying the MND promoter has the highest transduction efficiency including the central nervous system.

  It can be used for gene therapy of Hunter syndrome.

  Sequence number 1-3: vector

Claims (7)

  CMV hybrid 5'LTR-Ψ-RRE-cPPT-PGK-IDS cDNA-WPREmut9-3'LTR A vector plasmid comprising the nucleotide sequence set forth in SEQ ID NO: 1.   CMV hybrid 5'LTR-Ψ-RRE-cPPT-CD11b-IDS cDNA-WPREmut9-3'LTR A vector plasmid comprising the nucleotide sequence set forth in SEQ ID NO: 2.   CMV hybrid 5'LTR-Ψ-RRE-cPPT-MND-IDS cDNA-WPREmut9-3'LTR A vector plasmid comprising the nucleotide sequence set forth in SEQ ID NO: 3.   A lentiviral vector system comprising the vector plasmid according to claim 1, 2 or 3.   A cell transformed with the lentiviral vector system according to claim 4 introduced.   A cell preparation comprising a therapeutically effective amount of the cell according to claim 5.   A cell preparation for preventing and / or treating Hunter syndrome, which comprises a therapeutically effective amount of the cell according to claim 5.