WO2019228505A1 - Vecteur lentiviral utilisé pour le traitement de la leucodystrophie métachromatique, lentivirus, et son procédé de préparation et application associée - Google Patents

Vecteur lentiviral utilisé pour le traitement de la leucodystrophie métachromatique, lentivirus, et son procédé de préparation et application associée Download PDF

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WO2019228505A1
WO2019228505A1 PCT/CN2019/089548 CN2019089548W WO2019228505A1 WO 2019228505 A1 WO2019228505 A1 WO 2019228505A1 CN 2019089548 W CN2019089548 W CN 2019089548W WO 2019228505 A1 WO2019228505 A1 WO 2019228505A1
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lentiviral vector
cell
lentivirus
gene
arsa
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Zhangying ZHU
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Shenzhen Geno-Immune Medical Institute
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Definitions

  • the present application belongs to the field of genetic engineering technology and relates to a lentiviral vector pTYF used for the treatment of MLD, a lentivirus, and a preparation method and application thereof, and particularly relates to use of a lentiviral vector improved for optimizing the expression of ARSA gene in the preparation of a medicament for the treatment of metachromatic leukodystrophy.
  • Metachromatic leukodystrophy also known as ARSA (arylsulfatase A) deficiency
  • ARSA arylsulfatase A
  • CNS and PNS progressive neurologic dysfunction is the most significant.
  • Early symptoms include weakness, hypotonia and slurred speech.
  • Late symptoms include difficulty in speaking, deterioration of mental function, increased muscle tension, general or partial onset, and decreased vision, hearing and peripheral neuropathy.
  • Tonic spasm, decerebrate posture and loss of perception for surrounding environment may occur in the final stage.
  • the incidence rate is about 1 in 40,000, and the onset time is mainly based on the degree of brain nerve damage caused by sulfatide accumulation in the brain. Therefore, MLD is divided into three categories according to the age of onset: infantile type, juvenile type and adult type. In general, each relative of the infected patient has a probability of 25%of developing this disease, a probability of 50%of being a asymptomatic carrier, and a probability of 25%of being a non-patient or a non-carrier.
  • MLD an autosomal recessive inherited disease
  • a gene therapy can theoretically achieve complete treatment of the disease.
  • Direct injection of a viral vector carrying the normal ARSA gene into the brain can directly transfect the gene-deficient cells in the brain, to repair cells, secret the desired arylsulfatase A, reduce sulfatide accumulation in the brain, and repair surrounding cells or even cells throughout the brain by cross correction.
  • HSCs Hematopoietic stem cells
  • MSCs mesenchymal stem cells
  • HSCs and MSCs can be obtained from blood or bone marrow, and have ability to differentiate into a series of somatic cells and to renew various tissue cells. Therefore, transplantation of stem cells that have been modified in vitro is also an important way of gene therapy.
  • the present application provides a lentiviral vector used for the treatment of MLD, a lentivirus, and a preparation method and application thereof.
  • the lentiviral vector used for the treatment of MLD has higher transduction efficiency, stability and safety.
  • the application provides a lentiviral vector that is obtained by modifying a pTYF lentiviral vector at the 5'-end splice donor site, to be used for the treatment of MLD, wherein the specific modifications are as follows:
  • the lentiviral vector further comprises an ARSA gene.
  • the application provides a lentiviral vector that can be obtained by modifying a pTYF lentiviral vector at the 5'-end splice donor site and the gag AUG codon, wherein the specific modifications are as follows:
  • the lentiviral vector further comprises an ARSA gene.
  • the ARSA gene is a codon optimized and humanized sequence.
  • the 5'-end splice donor site is deleted or modified and the gag AUG may be deleted or modified so that the splice donor site of the lentiviral vector is not a potential site for homologous recombination between a packaging vector and the reference lentivirus packaging plasmids, that is, the lentiviral vector is unlikely to become pathogenic due to homologous recombination.
  • This allow the HIV-derived virus genetic materials to lose its self-replication function, thereby greatly improving the safety of the lentiviral vector used in gene therapy. This is a safety improvement that none of the other lentiviral vectors have, and in addition, this is the first application using pTYF derived vector expressing ARSA.
  • the modified lentiviral vector has higher transduction efficiency, high stability and improved safety, and it can express the delivered genes at higher efficiency during the gene therapy.
  • the ARSA gene is specifically cloned into the modified lentiviral vector which is then transfected into cells to produce lentiviral vector, which can infect cells to achieve a successful and stable expression of the ARSA gene in the target cells including stem cells, achieving a gene therapy of MLD with the lentiviral vector.
  • nucleotide sequences used in the deletion or modification of the 5'-end splice donor site of the lentiviral vector are listed below, for example:
  • the wild type 5' splice donor site GT is mutated to CA, wherein specific sequences are as follows:
  • Wild type (SEQ ID NO. 4) : GGCAAGAGGCGAGGGGCGGCGACTGGTGAGT ACGCCAAAAATTTTGACTAGCGGAGGCTA;
  • Mutant (SEQ ID NO. 5) : GGCAAGAGGCGAGGGGCGGCGACTGCAGAGTAC GCCAAAAATTTTGACTAGCGGAGGCTA.
  • the wild type 5' splice donor site GT is mutated to GG, wherein specific sequences are as follows:
  • Wild type (SEQ ID NO. 6) : GGCAAGAGGCGAGGGGCGGCGACTGGTGAGT ACGCCAAAAATTTTGACTAGCGGAGGCTA;
  • Mutant (SEQ ID NO. 7) : GGCAAGAGGCGAGGGGCGGCGACTGGGGAGTAC GCCAAAAATTTTGACTAGCGGAGGCTA.
  • the ARSA gene has the nucleotide sequence as shown in SEQ ID NO. 1, or a nucleotide sequence that shares at least 80%homology, preferably at least 85%homology, further preferably at least 95%homology therewith.
  • the ARSA gene has a nucleotide sequence that shares at least 80%homology with the nucleotide sequence as shown in SEQ ID NO. 1.
  • the ARSA gene has a nucleotide sequence that shares at least 82%homology with the nucleotide sequence as shown in SEQ ID NO. 1.
  • the ARSA gene has a nucleotide sequence that shares at least 85%homology with the nucleotide sequence as shown in SEQ ID NO. 1.
  • the ARSA gene has a nucleotide sequence that shares at least 88%homology with the nucleotide sequence as shown in SEQ ID NO. 1.
  • the ARSA gene has a nucleotide sequence that shares at least 90%homology with the nucleotide sequence as shown in SEQ ID NO. 1.
  • the ARSA gene has a nucleotide sequence that shares at least 92%homology with the nucleotide sequence as shown in SEQ ID NO. 1.
  • the ARSA gene has a nucleotide sequence that shares at least 95%homology with the nucleotide sequence as shown in SEQ ID NO. 1.
  • the sequence that shares at least 80%homology with the nucleotide sequence as shown in SEQ ID NO. 1 is a modified ARSA gene which still functions as an ARSA gene. It may be a shortened form of the ARSA protein or it may use only the functional domain sequence of the ARSA. Loading any one of these modified nucleotide sequences into the lentiviral vector can achieve the function of the ARSA gene to repair the ARSA gene.
  • the nucleotide sequence shown in SEQ ID NO. 1 is as follows:
  • a promoter sequence is further comprised in front of the ARSA gene, wherein the promoter sequence is EF1 ⁇ and/or CMV, preferably EF1 ⁇ .
  • any promoter can be used as long as it is capable of initiating ARSA gene expression.
  • the inventor has found that use of the EF1 ⁇ promoter achieves more efficient gene delivery while ensuring safety.
  • the EF1 ⁇ has the nucleotide sequence as shown in SEQ ID NO. 2, or a nucleotide sequence that shares at least 90%homology, preferably at least 95%homology therewith.
  • the EF1 ⁇ has a nucleotide sequence that shares at least 90%homology with the nucleotide sequence as shown in SEQ ID NO. 2.
  • the EF1 ⁇ has a nucleotide sequence that shares at least 92%homology with the nucleotide sequence as shown in SEQ ID NO. 2.
  • the EF1 ⁇ has a nucleotide sequence that shares at least 95%homology with the nucleotide sequence as shown in SEQ ID NO. 2.
  • the sequence that shares at least 90%homology with the nucleotide sequence as shown in SEQ ID NO. 2 is a modified EF1 ⁇ which still functions as a promoter. It may be a shortened form of the EF1 ⁇ . Loading any one of these modified nucleotide sequences into the lentiviral vector can achieve the function of the promoter to initiate the expression of the ARSA gene.
  • the nucleotide sequence shown in SEQ ID NO. 2 is as follows:
  • the present application provides a recombinant lentivirus that is obtained by co-transfecting a mammalian cell with the lentiviral vector pTYF according to the first aspect and packaging helper plasmids pNHP and pHEF-VSV-G.
  • the mammalian cell is a HEK293T cell and/or a TE671 cell.
  • the present application provides a method for preparing the lentivirus according to the second aspect, comprising the steps of:
  • the insertion site in step (2) may be any restriction site that can be synthesized by genetic engineering, although restriction sites BamHI and SpeI are preferably used in the present application.
  • the packaging helper plasmid in step (3) is pNHP and pHEF-VSV-G.
  • the mammalian cell is a HEK293T cell and/or a TE671 cell.
  • the co-transfected mammalian cell is cultured for 24-72 h, for example, 24 h, 25 h, 26 h, 27 h, 28 h, 29 h, 30 h, 31 h, 32 h, 33 h, 34 h, 35 h, 36 h, 37 h, 38 h, 39 h, 40 h, 41 h, 42 h, 43 h, 44 h, 45 h, 46 h, 47 h, 48 h, 50 h, 52 h, 55 h, 58 h, 60 h, 62 h, 65 h, 68 h, 70 h or 72 h.
  • the present application provides a recombinant cell which comprises the lentiviral vector according to the first aspect and/or the recombinant lentivirus according to the second aspect.
  • the recombinant cell is a recombinant stem cell and/or a progenitor cell, preferably a blood stem cell and/or a mesenchymal stem cell.
  • the lentivirus-transfected stem cells are capable of stably expressing the ARSA gene in a large amount.
  • the recombinant lentivirus may be introduced into peripheral blood stem cells and mesenchymal stem cells to form a double stem cell treatment strategy, which can further improve the delivery efficiency and expression level of the ARSA gene in the brain, thereby achieving a faster resolutionresolution of MLD symptoms and a more comprehensive and long-term gene therapy.
  • the present application provides a pharmaceutical composition which comprises any one selected from the group consisting of the lentiviral vector according to the first aspect, the recombinant lentivirus according to the second aspect, and the recombinant cell according to the forth aspect, or a combination of at least two selected therefrom.
  • the composition further comprises a pharmaceutically acceptable adjuvant which is any one selected from the group consisting of a growth-stimulating factor, an excipient, a diluent, a carrier, a flavoring agent, a binder and a filler, or a combination of at least two selected therefrom.
  • a pharmaceutically acceptable adjuvant which is any one selected from the group consisting of a growth-stimulating factor, an excipient, a diluent, a carrier, a flavoring agent, a binder and a filler, or a combination of at least two selected therefrom.
  • the present application provides use of the lentiviral vector according to the first aspect, the recombinant lentivirus according to the second aspect, the recombinant cell according to the forth aspect, or the pharmaceutical composition according to the fifth aspect in the preparation of a medicament and/or an agent for the treatment of MLD.
  • peripheral blood of a patient is collected and stem cells are isolated therefrom which are then transduced with the lentiviral vector, followed by i. v. retransfusion into the patient for the treatment of MLD disease.
  • the lentiviral vector can be injected directly into the lesion cell site for the treatment of MLD disease.
  • the lentiviral vector is specifically modified so that the HIV virus lose its self-replication function, thereby greatly improving the safety performance of the lentiviral vector itself used in gene therapy.
  • the modified lentiviral vector has higher transduction efficiency, stability and safety, and it can more efficiently complete the delivery of normal genes during the gene therapy;
  • a human codon optimized ARSA gene is specifically connected into the modified lentiviral vector of the present invention under the EF1 ⁇ promoter, thereby achieving a more efficient gene delivery while ensuring safety, significantly increasing the expression level of the ARSA gene in transgenic brain-related cells, and more efficiently accomplishing the transfer of normal genes during the gene therapy of MLD;
  • the lentiviral vector can directly correct the functionally defect ARSA gene in cells, and can effectively improve the delivery efficiency and expression level of the ARSA gene in the brain, which has great significance in ensuring the effectiveness of gene therapy and lays foundation for a faster resolution of MLD symptoms and a more comprehensive and long-term gene therapy.
  • Figure 1 is a schematic diagram showing the modification of the lentiviral vector pTYF
  • Figure 2 is a schematic diagram showing the structure of the lentiviral vector
  • Figure 3 is a schematic diagram showing the purification process of the lentiviral vector
  • Figure 4 is a schematic diagram showing the treatment process of MLD by directly injecting a lentiviral vector carrying a functional ARSA gene into the brain;
  • Figure 5 is a schematic diagram showing the protein expression in CTL cells.
  • This example provides a method for constructing a lentiviral vector, which specifically includes the following steps:
  • Wild type (SEQ ID NO. 4) : GGCAAGAGGCGAGGGGCGGCGACTGGTGAGT ACGCCAAAAATTTTGACTAGCGGAGGCTA;
  • Mutant (SEQ ID NO. 5) : GGCAAGAGGCGAGGGGCGGCGACTGCAGAGTAC GCCAAAAATTTTGACTAGCGGAGGCTA;
  • the sequences of the normal ARSA gene (as shown in SEQ ID NO. 1) and the human EF1 ⁇ promoter (as shown in SEQ ID NO. 2) were synthesized by whole gene synthesis, which were then connected into the lentiviral vector TYF via restriction sites.
  • the obtained product was identified by sequencing and digestion with double enzymes (the NEB original recommendation was referred to for the best reaction condition; BamHI clone site (ggatccacc) -AUG was used for 5’a nd SpeI clone site (actagt) was used for 3’ ) to obtain a correctly linked lentiviral vector which carried the normal ARSA gene (as shown in SEQ ID NO. 3) inserted under the hEF1 ⁇ promoter.
  • the specific link position and the structure of the lentiviral vector are shown in Figure 2.
  • nucleotide sequence shown in SEQ ID NO. 1 is as follows:
  • nucleotide sequence shown in SEQ ID NO. 2 is as follows:
  • nucleotide sequence shown in SEQ ID NO. 3 is as follows:
  • the lentiviral vector prepared in Example 1 was further packaged, purified and concentrated to obtain a lentivirus.
  • the specific process is shown in FIG. 3, and the specific steps are as follows:
  • the constructed lentiviral vector and packaging helper plasmids pNHP and pHEF-VSV-G were co-transfected into mammalian cell HEK293T, and cultured for 24-72h;
  • the collected lentivirus carrying normal ARSA gene were used to transduce neuronal cells and glial cells which were then identified for protein expression to confirm the expression of the ARSA gene in neuronal cells.
  • the lentivirus carrying normal ARSA prepared in Example 2 was directly injected into the brain to treat MLD disease.
  • the schematic diagram of the treatment process is shown in Figure 4.
  • the site and specific coordinate in the brain at which the lentiviral vector was injected was determined by MRI or CT of the brain, and the lentiviral vector carrying normal ARSA gene was delivered into the patient's brain via direct intracranial injection for disease treatment.
  • Human peripheral blood T cells were transduced with lentivirus carrying a functional ARSA of the present application as prepared in Example 2.
  • Cellular proteins were harvested for Western blot analysis from 5-fold (5x) , 1-fold (1x) or un-transduced CTL cells.
  • the left panel in Figure 5 shows an antibody positive control.
  • the right panel shows 5x infected cells which has the highest expression, and un-transduced cells which has a low expression (normal cells also express ARSA) .
  • the lentiviral vector can directly repair the defective ARSA gene in cells, and can effectively improve the delivery efficiency and expression level of the ARSA gene in the brain, which has great significance in ensuring the effectiveness of gene therapy and lays foundation for a faster resolution of MLD symptoms and a more comprehensive and long-term gene therapy.

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Abstract

La présente invention concerne un vecteur lentiviral utilisé pour le traitement de la leucodystrophie métachromatique, un lentivirus, et son procédé de préparation et une application associée, le vecteur lentiviral pouvant être obtenu par l'application de pTYF ou la modification d'un vecteur lentiviral pTYF au niveau du site donneur d'épissage d'extrémité 5' et celui-ci comprenant en outre un gène ARSA. Le gène ARSA est relié spécifiquement au pTYF ou au vecteur lentiviral modifié selon la présente invention, ce qui permet d'obtenir une administration de gène plus efficace tout en garantissant une sécurité, une augmentation significative du niveau d'expression du gène ARSA dans des cellules transgéniques liées au cerveau, et un accomplissement plus efficace du transfert de gènes normaux pendant la thérapie génique de la leucodystrophie métachromatique.
PCT/CN2019/089548 2018-05-31 2019-05-31 Vecteur lentiviral utilisé pour le traitement de la leucodystrophie métachromatique, lentivirus, et son procédé de préparation et application associée WO2019228505A1 (fr)

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WO2020177618A1 (fr) * 2019-03-04 2020-09-10 Versitech Limited Vecteurs recombinés comprenant de l'arylsulfatase a et leurs utilisations dans la thérapie de cellules souches pour le traitement de la leucodystrophie métachromatique
CN109971787A (zh) * 2019-04-17 2019-07-05 北京美康基免生物科技有限公司 一种cybb慢病毒载体、慢病毒载体转染的干细胞及其制备方法和应用
CN111411110B (zh) * 2020-04-20 2022-04-29 东莞市东阳光生物药研发有限公司 慢病毒的滴度提高型转移质粒

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