JP7197901B2 - Vector plasmids, lentiviral vector systems, cells and cell preparations used in gene therapy for Hunter syndrome - Google Patents

Description

The present invention provides a vector plasmid used in a lentiviral vector system for gene therapy of Hunter's syndrome, a lentiviral vector system comprising the vector plasmid, a cell introduced with the lentiviral vector system, and a cell containing the cell. Regarding formulations.

Hunter syndrome is mucopolysaccharidosis type II (hereafter sometimes referred to as Mucopolysaccharidosis type II, MPSII), which is caused by deficiency or reduction of mucopolysaccharidase activity, resulting in insufficiency in various organs throughout the body, mainly connective tissue. It is an inherited metabolic disorder that presents various symptoms due to the accumulation of completely degraded mucopolysaccharides. It is the only mucopolysaccharidosis that shows X-chromosome recessive inheritance and is reported to occur in 1 in 110,000 to 130,000 male births.

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

In MPSII, glycosaminoglycans (GAGs) are accumulated throughout the body due to the deficiency of IDS, causing damage to the central nervous system, bones, heart, etc., and severe cases lead to death in the teenage years. Existing treatments include enzyme replacement therapy (ERT) and hematopoietic stem cell transplantation (HSCT).

Enzyme replacement therapy is a therapeutic method in which a deficient enzyme is replenished into the body as a drug, and must be continuously performed (Non-Patent Documents 1 to 6). The sooner it is implemented, the more effective it is. By supplementing enzymes weekly before symptoms progress, it is possible to prevent or suppress the progression of various symptoms. However, it is difficult to treat complete lesions such as bone deformation and valvular heart disease and symptoms of the central nervous system with enzyme replacement therapy alone, and side effects such as allergy and anaphylaxis may occur with administration. Furthermore, the enzyme replacement therapy is expensive and must be performed once a week for the rest of the patient's life, thus placing a heavy burden on the patient.

Hematopoietic stem cell transplantation is a therapeutic method that can be expected to suppress the progression of brain and heart symptoms, and is a therapeutic method that is considered until the age of about 2 years before the lesion is completed (Non-Patent Documents 7 to 10). When bone marrow transplantation is performed and hematopoietic stem cells are engrafted, it becomes possible to produce enzymes on its own, making it possible to reduce the frequency of enzyme replacement therapy. However, hematopoietic stem cell transplantation is not sufficiently effective for the central nervous system, effects cannot be expected unless transplantation is performed before the age of 2 years, appropriate donors (bone marrow donors) cannot be obtained, Graft Versus Host Disease (GVHD), etc. However, there are problems such as the risk of serious side effects.

Enzyme replacement therapy, which is the standard treatment for Hunter syndrome, has a significant impact on the patient's QOL because, as mentioned above, weekly hospital visits are required for the rest of the patient's life. As mentioned above, hematopoietic stem cell transplantation also involves the risk of infection and rejection, and the need to find a donor. Therefore, a new therapeutic method that reduces the burden on patients is eagerly awaited. Gene therapy using the patient's own bone marrow is a new therapeutic method. This is a method of inserting normal genes into the patient's bone marrow using a viral vector. Compared to using other people's bone marrow, enzyme activity is improved, and effects on the head, bones, and heart valves are also expected. Using your own bone marrow also eliminates the risk of infection and rejection, and eliminates the need to find a donor.

The present inventors have found that expression of the IDS gene in hematopoietic stem cells using a (B6/MPS II) viral promoter (MND promoter: Moloney murine leukemia virus LTR/myeloproliferative sarcoma virus enhancer) in model mice cannot be achieved by ERT or HSCT. have succeeded in reducing GAGs in the brain of MPSII model mice (Non-Patent Document 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. O kuyama T, Tanaka A, Suzuki Y, et al: Japan Elaprase Treatment (JET) study: idursulfase enzyme replacement therapy in adult patients with attenuated Hunter syndrome (MucopolysaccharidosisII, MPS II). Mol Genet Metab 2010;99:18-25. 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 disease: Up to 9 years experience with 17 patients. Mol 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 Japanese ministry of health, labor and welfare and the working group of the Japan society for hematopoietic cell transplantation. Pediatr Transplant 2016;20:203-214. Vellodi A, Young E, Cooper A, et al: Long-term follow-up following bone marrow transplantation for Hunter 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 survey in Japan. Mol Genet Metab 2012;107:513-520. Toya Ohashi, Hematopoietic Stem Cell Gene Therapy Corrects 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 that efficiently supply IDS to central nerves and bones in MPS II, have not been realized.

The present invention has been made in view of these problems, and aims to provide a novel vector plasmid that efficiently supplies IDS not only to each organ but also to the central nervous system and the bone system and enables gene therapy for Hunter's 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 and comprising the nucleotide sequence set forth in SEQ ID NO: 1. is.

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

Further, the vector plasmid according to the present invention is a vector plasmid consisting of CMV hybrid 5'LTR-Ψ-RRE-cPPT-MND-IDS cDNA-WPREmut9-3'LTR and containing the nucleotide sequence set forth in SEQ ID NO:3. vector plasmid.

INDUSTRIAL APPLICABILITY According to the present invention, a novel vector plasmid can be obtained that efficiently supplies IDS to the central nervous system and bone system and enables gene therapy for Hunter's syndrome.

FIG. 2 is a diagram explaining a lentiviral vector system; FIG. 2 is a diagram explaining three types of vector plasmids among the four types of plasmids contained in the system shown in FIG. FIG. 6 shows measured values of IDS enzyme activity in serum, cerebellum, cerebrum, heart, liver, and spleen 6 months after gene therapy. FIG. 6 is a diagram showing measured values of GAG accumulation levels in the cerebellum, cerebrum, heart, liver, and spleen 6 months after gene therapy.

Hereinafter, embodiments of the present invention will be specifically described with reference to the accompanying drawings. The embodiments are intended to facilitate understanding of the principles of the present invention, and the scope of the present invention is as follows. The scope of the present invention is not limited to the embodiments, and other embodiments in which the configurations of the following embodiments are appropriately replaced by those skilled in the art are also included in the scope of the present invention.

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

A method for making a lentiviral vector 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 proteins in trans for making viral particles. Modifier genes (vif, fpr, vpu, nef) are deleted because the modifier gene products are not required for infection of non-dividing cells. Deletion enhances safety because the modifying gene product is critical but not essential for HIV-1 replication and is closely related to HIV-1 virulence. Also, since tat is essential for HIV-1 replication, deletion of tat from the packaging plasmid further increases safety. Since the packaging plasmid does not have a packaging signal (Ψ), RNA transcribed from this plasmid is not incorporated into virus particles.

Rev acts on post-transcriptional RNA, selectively exporting unspliced mRNA out of the nucleus and translating structural proteins. The third generation uses a plasmid separate from the packaging plasmid.

Since the envelope plasmid of HIV-1 can only infect CD4-positive cells, VSV-G (vesicular stomatitis virus G gylcoprotein) is used to expand the host range. The receptor for VSV-G is thought to be a phospholipid, and by using VSV-G as an envelope, membrane fusion between virus particles and cells occurs independently of cell surface receptors. Therefore, it becomes possible to infect basically any animal species or cell tumor. Also, VSV-G is physically strong and virus particles can be easily concentrated by ultracentrifugation.

A vector plasmid has a gene of interest inserted between LTRs (repetitive sequences controlling transcriptional 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 virus particles. Since the LTR promoter activity of HIV-1 is very weak in the absence of tat, an internal promoter (Fig. 1 Pro) is used for the expression of the integrated foreign gene. In this embodiment the internal promoter is the PGK/CD11b/MND promoter and the details are shown in FIG. The performance such as introduction efficiency of these three types is compared and the optimum one is used. The inserted foreign gene (Purpose gene in FIG. 1) to be incorporated is the gene IDS cDNA that expresses the defective enzyme of MPS II. RRE (rev responsive element) is required for Rev binding and efficient transport of full-length viral genomic RNA from the nucleus to the cytoplasm. During reverse transcription, retroviruses start synthesizing positive strands from the PPT (polypurine tract) sequence immediately upstream of the 3'LTR. There is another site, from which synthesis also takes place, so that the final double-stranded cDNA has a triple-stranded structure of about 100 base pairs in the middle called a DNA flap. The cPPT sequence has been suggested to affect the efficiency of reverse transcription, and the incorporation of the cPPT sequence into the vector increases the efficiency of gene transfer. A WPRE (woodchuk hepatitis virus posttranscriptional regulatory element) has been incorporated to increase expression efficiency. In other words, WPRE is believed to play a role in enhancing the active transport of mRNA from the nucleus to the cytoplasm and the stability of the mRNA in the cytoplasm. Go up. In addition, deletion of the enhancer/promoter segment in U3 of the 3'LTR renders both 3'5' LTRs in the proviral state incapable of promoter activity, and transcription of the entire genome from the 5'R region does not occur. There will be no. As a result, the SIN (self-inactivating lentiviral vector) is a lentiviral vector corresponding to HIV-1 proliferative-deficient strains and satisfies the position paper requirement "3. LTR promoter activity in the provirus and the entire HIV genome is not transcribed." ) is considered a vector. Also, by replacing U3 of the 5' LTR with the CMV promoter, tat dependency is eliminated, making it possible to delete tat from the packaging plasmid. Although the target gene to be incorporated in the vector plasmid is common to all IDS as described above, three types are prepared according to the difference in the internal promoter. That is, it consists of CMV hybrid 5'LTR-Ψ-RRE-cPPT-PGK-IDS cDNA-WPREmut9-3'LTR, and the nucleotide sequence set forth in SEQ ID NO: 1 (one or several bases in the nucleotide sequence set forth in SEQ ID NO: 1 is a substituted, deleted, added, and/or inserted nucleotide sequence. A vector plasmid containing the nucleotide sequence of SEQ ID NO: 2 (including a nucleotide sequence in which one or several bases are substituted, deleted, added, and/or inserted in the nucleotide sequence of SEQ ID NO: 2), CMV Consisting of hybrid 5'LTR-Ψ-RRE-cPPT-MND-IDS cDNA-WPREmut9-3'LTR, the base sequence set forth in SEQ ID NO: 3 (one or several bases are substituted in the base sequence set forth in SEQ ID NO: 3) , including deleted, added, and/or inserted nucleotide sequences. Among these, the second-generation lentiviral vector carrying the same promoter (MND) as the vector plasmid consisting of CMV hybrid 5'LTR-Ψ-RRE-cPPT-MND-IDS cDNA-WPREmut9-3'LTR was used to generate MPSII model mice. We have succeeded in reducing GAG in the brain, and it is thought that the vector plasmid described above is capable of efficiently supplying IDS to the central nerves and bones.

Next, a method for constructing the vector plasmid according to the present invention will be described. A vector plasmid is produced by a cloning operation in which a full- length DNA ( IDS cDNA) is loaded onto a target vector plasmid (pLGT1 plasmid).

Next, in preparing the vector plasmid, the cloning site of the desired 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 the vector plasmid according to the present invention.

The four (packaging, rev, envelope, vector) plasmid systems described above are called third-generation lentiviral vectors. -1 is very unlikely to be produced. Since the homologous regions between each plasmid are also minimized, there is almost no possibility of generating self-replicating viruses by homologous recombination.

Next, the mode of use of the lentiviral vector will be described.

The three constructed vector plasmids with different promoters were co-transfected with the third-generation packaging plasmid, Rev plasmid, and VSV-G envelope plasmid into mass-cultured 293T cells, and after 3 days, the supernatant culture was collected. do. At this point, the culture medium contains virus particles, which are purified by ultracentrifugation and aliquoted and stored at -80°C. Storage should be within 1 year when viral activity does not decrease. Stored vector viruses are immediately reconstituted at 37°C for use.

Also, the cells according to the present invention are cells transformed by introduction of the lentiviral vector system according to the present invention. Cells into which the lentiviral vector system according to the present invention is introduced are not particularly limited, but for example, T cells, NK cells or their progenitor cells (hematopoietic stem cells, lymphoid stem cells, etc.) can be used. . 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. Cell preparations can contain, for example, 10 ⁴ to 10 ¹⁰ cells for a single administration. In addition, dimethyl sulfoxide (DMSO) and serum albumin are used to protect cells, and antibiotics are used to prevent bacterial contamination. components such as vitamins, cytokines, growth factors, steroids, etc.) may be contained in the cell preparation.

A cell preparation according to the present invention is, for example, a cell preparation for prevention and/or treatment of Hunter's syndrome. As used herein, "prevention" includes suppressing and delaying the onset of disease, and includes not only prevention before the onset of disease, but also prevention against recurrence of disease after treatment. On the other hand, "treatment" includes curing symptoms, ameliorating symptoms, and suppressing progression of symptoms.

The MPSII model mice were NOG mice (NOD/Shi-scid, IL-2RγKO) that can be transplanted with human hematopoietic stem cells. In addition, IDS of B6 mouse (C57BL/6 mouse) was knocked out by CRISPR/Cas9 and used as a model mouse (B6/MPS II).

MPSII model mice are genetically diagnosed, 8-week-old hemizygous male mice are pretreated, and bone marrow is removed from their femurs. After culturing, mouse hematopoietic stem cell lines separated and extracted by the microbead method or the like are infected with a purified viral vector at MOI=50 to introduce genes. The transduced bone marrow stem cells are administered by tail vein injection after sublethal irradiation to another hemizygous male mouse. After that, we will follow up until 20 weeks old or more, and analyze the enzyme (IDS) activity in the circulating blood, the enzyme activity in each organ, especially the central nerve, bone, heart, etc., and the substrate (GAG) accumulation. Behavioral tests are also performed to assess central function. These tests are performed using three types of viral vectors with different promoters, and evaluation is performed between each group.

Gene therapy of B6/MPS II mice and NOG/MPS II mice is described below. Hematopoietic stem cells of B6/MPS II mice are infected with lentiviral vectors expressing 3 types of IDS and transplanted into B6/MPS II mice that have been lethally irradiated or anti-c-kit antibody (ACK2) + low-dose irradiation. . 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 open a bone marrow niche so that the transfected hematopoietic stem cells can engraft. Hematopoietic stem cells exist in the niche of the bone marrow through c-kit, etc. By existing in this niche, they maintain their functions as stem cells, that is, pluripotency and self-renewal ability (called dormancy) without cell division. ). Since it does not undergo cell division, it is resistant to radiation and anticancer drugs. Therefore, large doses of anticancer drugs/irradiation are required to kill the patient's hematopoietic stem cells. However, once hematopoietic stem cells withdraw from the niche, they begin to divide and become more sensitive to radiation and anticancer drugs. In other words, even low-dose radiation kills hematopoietic stem cells. Therefore, when an anti-c-Kit antibody is administered to a recipient, the recipient's hematopoietic stem cells leave the niche, start dividing, and become radiosensitive. This allows even low doses of radiation to potentially suffice as a pretreatment, avoiding aggressive pretreatment.

Human CD34-positive cells are infected with lentiviral vectors expressing 3 types of IDS and transplanted into NOG/MPS II mice. This is a more clinically relevant treatment because it targets human hematopoietic stem cells. Since the pretreatment is immunodeficient mice, a low dose of irradiation is sufficient.

Biochemical evaluation measures IDS activity and GAG in each site such as heart, liver, spleen, central nerve, and bone. 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. Mol Genet Metab 2016). Specifically, 2-sulfated iduronic acid present at the non-reducing end of GAG extracted and purified from tissues is cleaved as iduronic acid using recombinant IDS and iduronidase, and quantitatively analyzed in Multiple Reaction Monitoring (MRM) mode. I do.

For pathological evaluation, accumulation of GAG in nerve cells, etc., is performed using a conventional optical microscope or electron microscope. Regarding the brain, we will examine the number and area of LAMP2-positive cells, GFAP-positive cells, BSI-B4-positive cells, and GM3-positive cells that appear secondary to inflammatory changes in the brain using immunohistochemical techniques. We will also examine SCMAS-positive cells that are said to be associated with autophagy.

(1) Preparation of Vector Plasmid A basic vector of the lentiviral vector plasmid was prepared by the following procedure. Based on the pLV-SIN-CMV Neo vector (manufactured by Takara Bio Inc.), a hybrid LTR was created by replacing the U3 promoter region of the 5' HIV-1 LTR with the CMV promoter, and the residual Gag sequence downstream of the packaging signal was reduced. Remove promoter PCMVIE to neomycin resistance gene, change to mut9 sequence (X protein frameshift by single base insertion ) to suppress expression of X protein contained in WPRE sequence, cHS4 core insulator in 3' SIN LTR region A lentiviral vector plasmid pLGT1 carrying the sequence (forward orientation) was constructed. A vector plasmid was generated by a cloning operation that placed the full- length IDS cDNA into the vector plasmid pLGT1.

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

Substrate DNA (≦1 μg), Hind III (1 μl), BamHI (1 μl), 10×K Buffer (2 μl), and sterilized distilled water (20 μl) were mixed to prepare a reaction solution. After reacting at 37°C for 1 hour, loading buffer was added to half of the reaction solution (about 10 µl) and agarose gel electrophoresis was performed to confirm cleavage. A gel containing the target DNA fragment band was excised. Using NucleoSpin Gel and PCR Clean-up, DNA was purified from the gel and used as an insert DNA solution. The insert DNA was amplified by PCR using a primer with a restriction enzyme cleavage site added to the 5' side for ligation with the vector.

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

Using DNA Ligation Kit, 100 fmol of insert DNA solution, 50 ng (25 fmol) of plasmid DNA solution, 7.5 μl of Ligation Mix, and up to 15 μl of sterilized distilled water were mixed and allowed to react at 16° C. for 30 minutes. As a result, the insert DNA and the linearized plasmid were ligated to prepare the vector plasmid according to the present invention.

(2) Virus packaging Lentivirus packaging used the packaging plasmid pCAG-HIVgp, the VSVG/REV plasmid pCMV-VSV-G-RSV-Rev, and the SIN vector plasmid pLGT1 plasmid prepared above.

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

500 μl/dish of HBSS was prepared in a 1.5 ml eppendorf tube or a 5 ml polystyrene tube (Falcon 2058). The following plasmid DNAs were 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 allowed to stand at room temperature for 15-20 minutes. After mixing by pipetting several times with a Pipetman (P1000), a small amount was evenly seeded on a dish and cultured in a 37° C./5% or 10% CO ₂ incubator for 12 to 16 hours.

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

The virus-containing culture supernatant was recovered and passed through a 0.45 μm filter to remove floating cells and the like. When not concentrating, the filter filtrate was directly dispensed and stored at -80°C. When concentrating, the virus-containing culture supernatant passed through a 0.45 μm filter was transferred to Amicon Ultra-0.5 (100K), diluted with HBSS to the volume of the filter cup (~500 μl), and diluted to 14,000 x g (angle) at 4°C for 10 minutes. Centrifugation was performed for -30 minutes, and the filtrate in the lower tube was discarded.

The filter cup was set upside down in a new tube, and centrifugation was performed at 1,000 xg and 4°C for 2 minutes to collect the virus solution in a new tube below. ~100 µl (<4 µl/dish) of HBSS was added to the filter cup, and the membrane was washed several times by pipetting and collected in the lower tube.

In order to collect the virus fluid remaining in the filter cup, the filter cup was set upside down again and centrifuged at 1,000 xg at 4°C for 2 minutes to collect all the virus fluid in the lower tube. The recovered virus fluid was diluted to 10 ml with PBS(-) and ultracentrifuged at 50,000 xg (19,400 rpm for BECKMAN SW28) at 4°C for 2 hours. The supernatant was decanted or aspirated off and suspended in 1 ml of PBS using a Pipetman (P200). Transferred to a 50 ml centrifuge tube and vortexed for 3 hours.

Transfer the Vortex suspension to a 1.5 ml Eppendorf tube, centrifuge at 5000 rpm for 1 min to pellet debris, pipette the supernatant into screw-capped tubes and store at -80°C for 1 minute. I plan to use it within a year.

(3) Ex vivo gene therapy Ex vivo gene therapy was performed using the above three types of lentiviral vectors by the following method. First, MPSII model mice were genetically diagnosed, 8-week-old hemizygous male mice were pretreated, and the bone marrow was excised from the femur. After culturing, mouse hematopoietic stem cell lines separated and extracted by the microbead method or the like were infected with a purified viral vector at MOI=50 to introduce genes. The transduced bone marrow stem cells were administered to another hemizygous male mouse by tail vein injection after sublethal irradiation. After 20 weeks of age, the rats were followed up, and enzyme (IDS) activity in circulating blood, enzyme activity and substrate (GAG) accumulation in each organ, especially the central nervous system, bone, and heart, were analyzed. These tests were carried out using three types of viral vectors with different promoters, and evaluation was performed between each group.

As shown in FIG. 3, the IDS enzyme activity after 6 months of gene therapy showed a significant difference in serum, cerebellum, cerebrum, heart, liver, and spleen among the three groups treated with vectors carrying the MND promoter. showed high enzymatic activity. Six months after treatment, the serum, liver, spleen, and heart of this treatment group showed markedly higher enzyme activities, more than 10 times higher than those of the normal group.

As shown in FIG. 4, the amount of substrate (GAG) accumulated 6 months after gene therapy was normal in the cerebellum, cerebrum, heart, liver, and spleen in the group treated with the vector carrying the MND promoter among the three types. improved without significant difference. The cerebrum and cerebellum of the group treated with vectors containing the PGK promoter and CD11b promoter were significantly different from those of 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.

SEQ ID NOs: 1-3: vectors

Claims (6)

A vector plasmid consisting of CMV hybrid 5'LTR-Ψ-RRE-cPPT-PGK-IDS cDNA-WPREmut9-3'LTR and comprising the nucleotide sequence set forth in SEQ ID NO:1. A vector plasmid consisting of CMV hybrid 5'LTR-Ψ-RRE-cPPT-MND-IDS cDNA-WPREmut9-3'LTR and comprising the nucleotide sequence set forth in SEQ ID NO:3. A lentiviral vector system comprising the vector plasmid according to claim 1 or 2 . A cell transformed by introducing the lentiviral vector system according to claim 3 . A cell preparation comprising a therapeutically effective amount of the cells of claim 4 . A cell preparation for the prevention and/or treatment of Hunter's syndrome, comprising a therapeutically effective amount of the cells according to claim 4 .

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