Lentiviral vectors, cells and cell preparations

Abstract

To provide a lentiviral vector that can be used in gene therapy of GM1-gangliosidosis.SOLUTION: There is provided a lentiviral vector comprising LV-SMPUR-MCU3-cGLB1 and including a base sequence described in SEQ ID NO: 1. GLB1 being a responsible gene is introduced into the lentiviral vector of the present invention. The lentiviral vector of the present invention is introduced into a hematopoietic stem cell.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a lentiviral vector, a cell into which a responsible gene has been introduced by the lentiviral vector, and a cell preparation containing the cell. [Background technology]

[0002] GM1 gangliosidosis is a genetic metabolic disorder caused by a genetic mutation in the GLB1 gene, which encodes the lysosomal enzyme β-galactosidase (Non-Patent Documents 1 and 2). It is said to occur in 1 in 100,000 to 200,000 live births, and is classified into infantile, juvenile, and adult forms depending on the time of onset. Due to a deficiency in the β-galactosidase enzyme, GM1-ganglioside that cannot be broken down accumulates in cells throughout the body, primarily neurons. This accumulation causes secondary cholesterol accumulation and abnormalities in intracellular lipid transport, resulting in various symptoms (Non-Patent Documents 3 to 7).

[0003] In the infantile form, the most severe form, developmental delays occur by the age of six months, and hypotonia and hypersensitivity to sound may be observed. Tendon reflexes become hyperactive, rigid spasticity becomes stronger, and symptoms such as cherry-red spots on the fundus, hepatosplenomegaly, and generalized bone abnormalities progress, leading to death by around the age of two. In the juvenile form, symptoms begin around one year of age, and although symptoms are milder than in the infantile form, the prognosis is poor. Hepatosplenomegaly and cherry-red spots are less noticeable. In the adult form, intellectual disability is rare, but dysarthria is prominent in the early stages. Extrapyramidal symptoms such as gait disturbance and dystonia are prominent.

[0004] GM1 gangliosides can be diagnosed by measuring the β-galactosidase enzyme activity of peripheral lymphocytes or cultured skin fibroblasts, and if it is reduced (10% or less), this disease can be diagnosed. However, galactosialidosis also has reduced β-galactosidase enzyme activity, so it must be differentiated. GLB1 gene testing is also useful for diagnosis (Non-patent Document 7).

[0005] Krabbe disease, also known as globoid-cell leukodystrophy (GLD), is an autosomal recessive genetic disorder caused by a deficiency of galactocerebrosidase, which leads to demyelination (destruction of myelin) due to damage to oligodendroglia, which form central nerve fibers, and Schwann cells, which form peripheral nerve fibers, resulting in central and peripheral neuropathy. It is also thought that although galactocerebroside, the main substrate of galactocerebrosidase, does not accumulate, the accumulation of trace amounts of psychosine, a substrate, may cause cell damage.

[0006] Both Krabbe disease and GM1 ganglioside syndrome are classified as sphingolipidoses. Sphingolipidoses are disorders of sphingolipid metabolism. To date, approximately 10 types of sphingolipidoses caused by mutations in approximately 40 genes have been identified.

[0007] Gene therapy for sphingolipidoses using lentiviral vectors is being investigated. However, in Krabbe disease, this gene therapy has not been very effective in treating neurological disorders (Non-Patent Document 8). Specifically, a gene therapy approach is being considered in which a lentiviral vector incorporating a gene expressing the defective enzyme of the disease is constructed and administered systemically via the vein to a model mouse to examine its effectiveness.

[0008] However, no significant improvement in the lifespan of model mice has been reported using lentiviral vector-based gene therapy for sphingolipidosis. Furthermore, the combination of promoter and responsible gene in lentiviral vectors varies significantly depending on the individual disease and defective enzyme. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Yamashita, M., Yamazaki, M., Kusaka, H. et al.: A case of siblings with GM1 gangliosidosis type 3 showing marked differences in age of onset and clinical type. Clinical Neurology 33:631-636, 1993 [Non-patent document 2] Yoshida K, Ohshima A, Sakuraba H et al:GM1 Gangliosidosis in Adults:Clinical and Molecular Analysis of 16 Japanese Patients.Ann Neurol 31:328-332, 1992 [Non-patent document 3] Nishimoto, J.; Nanba, E.; Inui, K.; Okada, S.; Suzuki, K.: GM1-gangliosidosis (genetic beta-galactosidase deficiency): identification of four mutations in different clinical phenotypes among Japanese patients. Am. J. Hum. Genet. 49: 566-574, 1991 [Non-patent document 4] Yoshida, K.; Oshima, A.; Shimmoto, M.; Fukuhara, Y.; Sakuraba, H.; Yanagisawa, N.; Suzuki, Y. : Human beta-galactosidase gene mutations in G(M1)-gangliosidosis: a common mutation among Japanese adult / chronic cases. Am. J. Hum. Genet. 49: 435-442, 1991 [Non-Patent Document 5] Wenger DA et al. Adult GM1 gangiosidosis. Clin Genet 17: 323-334, 1980 [Non-patent document 6] Caciotti, A.; Bardelli, T.; Cunningham, J.; D'Azzo, A.; Zammarchi, E.; Morrone, A. : Modulating action of the new polymorphism L436F detected in the GLB1 gene of a type-II GM1 gangliosidosis patient. Hum. Genet. 113: 44-50, 2003 [Non-Patent Document 7] Masanori Ozaki, Taro Shimono, Takashi Hamasaki, Hiroyuki Tachikawa, Yukio Miki, A case of GM1 gangliosidosis type 2 with cerebellar atrophy, Clinical Radiology, Vol. 63, No. 4 (April 2018) [Non-patent document 8] Galbiati, F. et al. Combined hematopoietic and lentiviral gene-transfer therapies in newborn Twitcher mice reveal contemporaneous neurodegeneration and demyelination in Krabbe disease. J Neurosci Res 87, 1748-1759 (2009). Summary of the Invention [Problem to be solved by the invention]

[0010] The present invention has been made in view of these problems, and has as its object to provide a lentiviral vector that can be used in gene therapy for GM1 ganglioside. [Means for solving the problem]

[0011] The lentiviral vector of the present invention is a lentiviral vector consisting of LV-SMPUR-MCU3-cGLB1, and is a lentiviral vector containing the nucleotide sequence set forth in SEQ ID NO:1. [Effects of the Invention]

[0012] According to the present invention, a lentiviral vector that can be used for gene therapy of GM1 ganglioside is provided. Although there is no effective treatment for GM1 ganglioside, the present invention makes it possible to provide an effective treatment for GM1 ganglioside. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 shows a vector map of the lentiviral vector of the present invention. [Figure 2] FIG. 10 shows that a significant increase in enzyme activity was observed in plasma in the group treated with the lentiviral vector of the present invention compared to the untreated group. [Figure 3] FIG. 10 shows that a significant increase in enzyme activity was observed in the cerebrum in the group treated with the lentiviral vector of the present invention compared to the untreated group. [Figure 4] FIG. 10 shows that a significant decrease in GM1-ganglioside was observed in the cerebrum in the group treated with the lentiviral vector of the present invention compared to the untreated group. [Figure 5] FIG. 1 shows that a significant decrease in cholera toxin B (CTX-B)-positive cells was observed in the central nervous system in the group treated with the lentiviral vector of the present invention compared to the untreated group. [Figure 6] FIG. 10 shows that the GFAP-positive area in the central nervous system was significantly reduced in the group treated with the lentiviral vector of the present invention compared to the untreated group. [Figure 7] FIG. 1 shows that the MBP-positive area in the central nervous system was significantly reduced in the group treated with the lentiviral vector of the present invention compared to the untreated group. [Figure 8] FIG. 1 is a photograph of the apparatus used in the rotarod test. [Figure 9] FIG. 10 shows that the group treated with the lentiviral vector of the present invention had a faster falling speed from the rotarod test apparatus than the untreated group. DETAILED DESCRIPTION OF THE INVENTION

[0014] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, these embodiments are intended to facilitate understanding of the principles of the present invention, and the scope of the present invention is not limited to the following embodiments. 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.

[0015] The viral vector of the present invention is LV-SMPUR-MCU3-cGLB1. The sequence of LV-SMPUR-MCU3-cGLB1 is shown in SEQ ID NO: 1. The vector map of LV-SMPUR-MCU3-cGLB1 is shown in Figure 1.

[0016] Currently, there is no effective treatment for GM1 ganglioside. In the process of exploring a novel treatment for GM1 ganglioside using lentiviral vectors, the present inventors constructed a lentiviral vector using the PGK promoter and attempted gene therapy targeting hematopoietic stem cells. Gene therapy targeting hematopoietic stem cells using a PGK promoter-equipped lentiviral vector was performed in a mouse model, and β-gal activity and vector copy number in various organs were examined 20 weeks after treatment. βGal activity in the liver was 1.8±0.6 nmol / h / mg in the untreated group and 19.6±11.8 nmol / h / mg in the treated group, p=0.004; in the spleen, 3.2±0.4 nmol / h / mg in the untreated group and 414±269 nmol / h / mg in the treated group, p=0.0038; and in the brain, 1.7±0.3 nmol / h / mg in the untreated group and 1.9±0.3 mol / h / mg in the treated group, p=0.2. βGal enzyme activity was significantly elevated in peripheral organs, but not in the brain.

[0017] Therefore, the present inventors constructed a lentiviral vector using the MND promoter and attempted gene therapy targeting hematopoietic stem cells. MND stands for Moloney murine leukemia virus long terminal repeat (LTR) / myeloproliferative sarcoma virus enhancer. Gene therapy targeting hematopoietic stem cells was performed using a lentiviral vector carrying the MND promoter in a mouse model, and β-gal activity and vector copy number were examined in various organs 20 weeks after treatment. The use of the MND promoter is expected to increase the efficiency of gene transfer into hematopoietic stem cells. However, even with the MND promoter, the increase in activity in the brain was not sufficient.

[0018] Therefore, the present inventors attempted to use the lentiviral vector LV-SMPUR-MCU3-cGLB1 in the treatment of GM1 gangliosidosis using the MCU3 promoter. Surprising results were obtained in terms of increased enzyme activity, suppression of substrate accumulation, reduced inflammation, and decreased axonal loss. MCU3 is a modified version of MND, and can be expressed at high levels. The sequence of MCU3 is shown in SEQ ID NO: 2. cGLB1 is the gene deficient in GM1 gangliosidosis, i.e., the responsible gene. The sequence of cGLB1 is shown in SEQ ID NO: 3.

[0019] Lentiviral vectors are derived from the human immunodeficiency virus (HIV-1) and are a major tool for gene delivery in mammalian cells. An advantageous feature of lentiviral vectors is their ability to mediate potent transduction and stable expression in dividing and non-dividing cells both in vitro and in vivo. The HIV-1 genome contains nine open reading frames, which encode at least 15 different proteins involved in the infection cycle, including structural and regulatory proteins.

[0020] The lentiviral vector of the present invention is a second-generation lentiviral vector. Second-generation lentiviral vectors were developed by modifying accessory protein-encoding genes (Vif, Vpu, Vpr, or Nef). It is also possible to construct the lentiviral vector of the present invention using third-generation lentiviral vectors. The third-generation lentiviral vector system consists of four plasmids. The packaging vector is divided into two plasmids, one encoding Rev and the other Gag and Pol. Tat-independent plasmids were constructed by replacing the U3 promoter region of the 5'-LTR in the transfer plasmid with a strong viral promoter from CMV or RSV.

[0021] GM1 gangliosidosis is known to exist in several subtypes, including Type 1, which develops early and is characterized by growth retardation, hepatosplenomegaly, and bone changes, and Type 2, which develops after the age of one and has mild clinical symptoms.The lentiviral vector of the present invention can be used for gene therapy of either type of GM1 gangliosidosis.

[0022] Next, a method for producing the lentiviral vector of the present invention will be described.

[0023] The SMPUR-MND plasmid was used as a base vector, and the U3 region of the MND promoter was replaced with the TATA region of the human CMV promoter to create the vector plasmid SMPUR-MCU3.The GLB1 cDNA fragment was then cloned into the vector plasmid SMPUR-MCU3 to create the lentiviral vector plasmid LV-SMPUR-MCU3-cGLB1.

[0024] Next, usage modes of the lentiviral vector of the present invention will be described.

[0025] The lentiviral vector of the present invention is used to introduce the responsible gene GLB1 into cells. The cells of the present invention are cells into which the responsible gene GLB1 has been introduced by the lentiviral vector of the present invention. The cells into which the lentiviral vector system of the present invention is introduced are not particularly limited, and may be, for example, T cells, NK cells, or their precursor cells (hematopoietic stem cells, lymphoid stem cells, etc.), preferably hematopoietic stem cells.

[0026] Furthermore, the cell preparation of the present invention is a cell preparation containing a therapeutically effective amount of cells into which GLB1 has been introduced using the lentiviral vector of the present invention. For example, the cell preparation can contain 104 to 1010 cells for a single administration. Furthermore, the cell preparation may contain components such as dimethyl sulfoxide (DMSO) or serum albumin for cell protection, antibiotics for preventing bacterial contamination, and various components (vitamins, cytokines, growth factors, steroids, etc.) for cell activation, proliferation, or differentiation induction. [Example]

[0027] 1) Preparation of vector plasmid The SMPUR-MND plasmid was used as a base vector, and the U3 region of the MND promoter was replaced with the TATA region of the human CMV promoter to create the vector plasmid SMPUR-MCU3. The GLB1 cDNA fragment was cloned into the vector plasmid SMPUR-MCU3 to create the lentiviral vector plasmid LV-SMPUR-MCU3-cGLB1.

[0028] 2) Gene therapy in mice GM1 gangliosidosis model mice were genetically diagnosed, and bone marrow was extracted from the femur of 8-12-week-old donor model mice. After culturing, mouse hematopoietic stem cell lines isolated using the microbead method were infected with the purified viral vector LV-SMPUR-MCU3-cGLB1 at an MOI of 50 for gene transfer. The hematopoietic stem cells were CD34-positive. The bone marrow stem cells were then administered via tail vein injection to recipient model mice after lethal irradiation.

[0029] The mice were then followed up until 24 weeks of age, and the enzyme activity in the circulating blood, enzyme activity in various organs, particularly the central nervous system, liver, spleen, and bone marrow, and substrate accumulation in the central nervous system were analyzed. Immunohistochemical staining was also performed to histologically evaluate substrate accumulation and distribution in the central nervous system and inflammatory changes. Behavioral tests were also performed to evaluate motor function. These results were compared with those of untreated model mice.

[0030] 3) Measurement of enzyme (βgal) activity Frozen tissue and blood samples were used for β-gal activity analysis. Tissues were homogenized in six volumes of distilled water, centrifuged, and the supernatant was assayed for β-gal activity. β-gal activity was measured using 4-methylumbelliferyl-β-D-galactopyranoside as a synthetic chromogenic substrate.

[0031] As a result, as shown in FIG. 2, a significant increase in enzyme activity was observed in plasma compared to untreated model mice.

[0032] Furthermore, as shown in Figure 3, a significant increase in enzyme activity was observed in the cerebrum compared to untreated model mice, and enzyme activity was improved to approximately 10% of that in the normal group.

[0033] 4) Evaluation of substrate accumulation Liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used to quantify the substrate (GM1-ganglioside) in the cerebrum. As shown in Figure 4, GM1-ganglioside was reduced in the cerebrum of treated mice compared to untreated mice.

[0034] To evaluate the histological substrate, immunohistochemistry of the cerebrum was performed using cholera toxin B (CTX-B), which specifically stains GM1-ganglioside. The excised brains were soaked overnight in 4% paraformaldehyde and then fixed in 30% sucrose solution. After fixation, the brains were fixed at -80°C and sliced ​​into 40 μm sections, followed by the required staining. The stained specimens were imaged using an Olympus FV1200 confocal laser scanning inverted microscope. As shown in Figure 5, the number of CTX-B-positive cells in the central nervous system was reduced compared to untreated model mice.

[0035] 5) Evaluation of inflammatory changes and demyelination To evaluate neurons, glial cells, and axons in the brain, and to assess neuronal inflammatory changes and demyelination, immunohistochemistry was performed using combinations of antibodies (anti-NeuN, anti-GFAP, and anti-MBP). Samples were processed in the same manner as for the evaluation of matrix accumulation, and 40-μm-thick sections were prepared and imaged using a confocal laser scanning inverted microscope.

[0036] As a result, as shown in Figure 6, the area of ​​GFAP positivity was reduced compared to untreated model mice, suggesting that inflammatory changes were successfully suppressed.Furthermore, as shown in Figure 7, the area of ​​MBP positivity was also reduced compared to untreated model mice, suggesting that axons were protected from demyelination.

[0037] 6) Assessment of motor function To determine whether gene therapy can improve motor function, behavioral experiments were conducted using 32-week-old mice. The rotarod test was selected for the behavioral experiment to evaluate motor learning and coordination. As shown in Figure 8, the rotarod test involves placing a mouse on an accelerating device over a 5-minute period, with the speed increasing from 4 rpm to 40 rpm. The speed at which the mouse falls is recorded is recorded. The test consisted of a 1-minute practice session at 2 rpm (or two 30-second sessions at 4 rpm for more complex tasks). This was followed by a 15-20-minute rest period, followed by three trials on three consecutive days (a total of nine trials). The speed at which each mouse fell during each trial was recorded, and the fastest speed of any of the nine trials was calculated.

[0038] As a result, as shown in FIG. 9, the speed at which the mice fell from the rotarod was faster than that of the untreated model mice. [Industrial Applicability]

[0039] It can be used for gene therapy of GM1 ganglioside. [Sequence List Free Text]

[0040] SEQ ID NO: 1: Vector SEQ ID NO: 2: promoter SEQ ID NO: 3: Responsible gene

Claims

1. A lentiviral vector consisting of LV-SMPUR-MCU3-cGLB1, which contains the base sequence set forth in SEQ ID NO:

1.

2. The lentiviral vector of claim 1, wherein the promoter MCU3 has the sequence shown in SEQ ID NO:

2.

3. A lentiviral vector as described in claim 1, wherein cGLB1 is the sequence shown in sequence number 3.

4. A cell into which cGLB1 has been introduced by the lentiviral vector described in any one of claims 1 to 3.

5. The cell of claim 4 , wherein the cell is a hematopoietic stem cell.

6. A cell preparation comprising a therapeutically effective amount of the cells according to claim 4 or 5.

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