WO2023130911A1 - 一种适用地中海贫血症和镰刀形贫血基因治疗的慢病毒载体 - Google Patents

一种适用地中海贫血症和镰刀形贫血基因治疗的慢病毒载体 Download PDF

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WO2023130911A1
WO2023130911A1 PCT/CN2022/138330 CN2022138330W WO2023130911A1 WO 2023130911 A1 WO2023130911 A1 WO 2023130911A1 CN 2022138330 W CN2022138330 W CN 2022138330W WO 2023130911 A1 WO2023130911 A1 WO 2023130911A1
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beta globin
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lentiviral vector
expression cassette
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汪啸渊
凌思凯
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上海本导基因技术有限公司
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  • the invention belongs to the technical field of genetic engineering and relates to a lentiviral vector, in particular to a lentiviral vector suitable for gene therapy of thalassemia and sickle cell anemia.
  • Thalassemia thalassemia
  • thalassemia is a monogenic hereditary hemoglobinopathy with the widest distribution and the largest cumulative patient population in the world. It is the most common human hereditary disease among rare diseases.
  • Lentiviral vectors are inserted into the genome in an uncontrolled manner (about 75% are integrated into transcriptionally active gene coding regions), accompanied by a higher risk of activating oncogenes and inactivating tumor suppressor genes.
  • lentiviral vectors have been used to activate the HMGA2 gene during gene therapy for thalassemia, leading to massive expansion of transduced hematopoietic stem cell clones.
  • the abnormal expansion of this clone is due to the insulator element used in the lentiviral gene therapy vector, that is, the core chicken hypersensitivity site 4 (cHS4) of chicken beta globin deoxyribonuclease I (Cavazzana- Calvo, M. et al. Transfusion independence and HMGA2 activation after gene therapy of humanbeta-thalassaemia. Nature 467, 318-22(2010).).
  • cHS4 chicken hypersensitivity site 4
  • cHS4 chicken beta globin deoxyribonuclease I
  • Insulator elements are genetic elements that prevent gene silencing or prevent gene activation in their region.
  • the cHS4 insulator contains a secret RNA splicing signal, which causes clonal expansion, affects the safety of gene therapy, and makes the titer of lentivirus drop significantly (Cavazzana-Calvo, M. et al. Transfusion independence and HMGA2 activation after gene therapy of human beta -thalassaemia.Nature 467,318-22(2010).Negre,O.et al.Preclinical evaluation of efficacy and safety of animproved lentiviral vector for the treatment of beta-thalassemia and sicklecell disease.Curr Gene Ther 15,64 -81 (2015) .).
  • the insulator element included in the present invention comes from Foamy virus, only has a 36bp insulator sequence, does not contain a secret RNA splicing signal, and has the function of maintaining gene expression and preventing cancer gene activation in the region.
  • beta-globin gene HBB
  • LCR locus control region
  • minilocus consisting of core elements of the HS4, HS3, HS2 deoxyribonuclease I hypersensitive loci (miniLCR) resulted in a significant reduction in viral titer (Weber, L. et al. An Optimized Lentiviral Vector Efficiently Corrects the Human Sickle CellDisease Phenotype. Mol Ther Methods Clin Dev 10, 268-280 (2016).
  • the screened mini-locus of the present invention does not lead to a significant reduction in virus titer.
  • the technology for treating thalassemia based on lentiviral vectors has not yet been clinically used and commercialized.
  • the titer of viral vectors for treatment produced by existing technologies is too low, resulting in excessive production scale and production costs. It is difficult to produce a sufficient amount of viral vectors for clinical use and commercialization;
  • the therapeutic viral vectors produced by the prior art are used for treatment, the expression of beta globin is very limited, and it is difficult to achieve an effective therapeutic effect.
  • the present invention optimizes the carrier structure, and finds that after optimizing the No. 2 intron of the Beta globin gene (HBB), the expression of the gene can be improved to a certain extent, and the packaging yield of the virus can be greatly improved, so that it can be used clinically and industrialization become possible.
  • HBB No. 2 intron of the Beta globin gene
  • the use of lentiviral vectors in the gene therapy of thalassemia and sickle cell anemia has potential safety hazards and is not very efficient; the purpose of the present invention is to solve the problem that the titer of the vectors in the prior art is too low to reach the level of clinical application; Insufficient expression, unable to achieve a therapeutic effect; and the lack of insulator sequences in the vector, which easily activates the expression of adjacent genes, has defects such as greater safety risks, and provides a lentiviral vector suitable for gene therapy of thalassemia and sickle cell anemia.
  • One or more embodiments of the present invention relate to a lentiviral vector comprising a beta globin expression cassette, the expression cassette comprising:
  • A the mini-locus control region, for a section from the locus control region of beta globin 16kb, the mini-regulatory element that does not contain the HS1 region;
  • the minilocus control region is composed of HS2, HS3, HS4 deoxyribonuclease I hypersensitive site sequences after screening; or is composed of HS3, HS2 deoxyribonuclease The I hypersensitive site sequence is screened and combined.
  • the mini-locus control region is a mini-locus regulatory element composed of deoxyribonucleotide sequences intercepted from the HS4, HS3, and HS2 regions of the locus control region LCR of beta globin.
  • sequence of the mini-locus regulatory element is shown in SEQ ID NO.1.
  • the promoter sequence is shown in SEQ ID NO.4.
  • flanking sequence is shown in SEQ ID NO.5.
  • the insulator sequence is shown in SEQ ID NO.6.
  • the invention relates to the application of a lentiviral vector containing beta globin expression cassette in the preparation of gene therapy drugs for thalassemia and sickle cell anemia.
  • Figure 1 is a schematic diagram of carrier design; wherein, a is a schematic diagram of LGO4 carrier design, and b is a schematic diagram of LGO2 carrier design;
  • Figure 2 is a schematic diagram of the relative expression of each carrier hemoglobin T87Q ;
  • Figure 3 is a schematic diagram of the titer comparison of each carrier
  • Fig. 4 is a schematic diagram of the number of BFU-E (burst erythroid colony-forming units) formed after stem cells are infected with different vectors.
  • This example relates to an expression cassette of human beta globin, and then a lentiviral vector LGO4 containing the expression cassette for gene therapy was obtained.
  • the design of the LGO4 vector is shown in Figure 1(a), and the expression cassette includes the following elements: (1) the mini-locus control region (miniLCR), which is a non-selective gene selected from the 16kb locus control region of beta globin
  • mini-regulatory element containing the HS1 region is specifically composed of deoxyribonucleotide sequences with a length of about 380bp, 1900bp, and 720bp respectively intercepted from the HS4, HS3, and HS2 regions of the 16kb locus control region LCR of beta globin to form a total length of 3kb (sequence such as Shown in SEQ ID NO.1) the mini-locus regulatory element; (2) the gene sequence of beta globin.
  • exon 2 contains the T87Q mutation, and the sequence of intron 2 has been optimized;
  • the gene sequence of the beta globin is shown in SEQ ID NO.2;
  • the design diagram of the LGO2 vector is shown in Figure 1(b), and the selected beta
  • the gene sequence of globin is shown in SEQ ID NO.3;
  • the promoter sequence (sequence shown in SEQ ID NO.4) of the gene sequence upstream of the beta globin of 265-bp;
  • beta beads The downstream flanking 300-bp sequence of the gene sequence of the protein (sequence shown in SEQ ID NO.5);
  • the insulator sequence has the function of maintaining long-term gene expression and preventing gene activation near the integration site without reducing the titer of the lentiviral vector.
  • the sequence of the lentiviral vector containing the 3kb minilocus control region is shown in SEQ ID NO.7.
  • the carrier plasmid (13 ⁇ g) of the present invention was combined with a plasmid expressing membrane protein (such as 3.75 ⁇ g pMD2G (sequence shown in SEQ ID NO.8)), a plasmid expressing GagPol long-chain protein (usually 13 ⁇ g pMD1g/pRRE (sequence such as SEQ ID NO.8) Shown in ID NO.9)) and plasmids expressing REV protein (usually 3 ⁇ g pRSV-REV (sequence shown in SEQ ID NO.10)) are co-transfected with virus 4X10 6 production cells (such as 293T) to produce expression beta Globin Lentivirus LGO4.
  • a plasmid expressing membrane protein such as 3.75 ⁇ g pMD2G (sequence shown in SEQ ID NO.8)
  • a plasmid expressing GagPol long-chain protein usually 13 ⁇ g pMD1g/pRRE (sequence such as
  • the LGO2 vector design is shown in Figure 1(a), and the other steps are basically the same as above (refer to CN201910824134), to obtain lentivirus LGO2 expressing beta globin.
  • a vector plasmid expressing green fluorescent protein (pLV/PGK-EGFP, cited DOI: 10.1093/nar/gkt1163) was used to replace the vector plasmid of the present invention, and the other steps were the same as above to obtain a control virus vector. Enrichment or purification of lentivirus by ultracentrifugation or column chromatography.
  • hematopoietic stem cells were infected with the same amount of viral vectors in vitro. After the infection, the stem cells were induced to differentiate into erythroid. On the 14th day, RNA was extracted from each group of cells, and the integrated hemoglobin T87Q mRNA was analyzed by RT-Q-PCR using fluorescent quantitative PCR. The expression level of T87Q reflects the relative expression level of hemoglobin T87Q ( Figure 2), and the expression level of LG04 vector is the highest.
  • the stem cells After infecting human hematopoietic stem cells with the same amount of each virus vector, the stem cells were inoculated in a solid medium containing characteristic cytokines, and after 14 days of culture, the explosive erythrocyte colony forming units were counted under the microscope, and the number of colonies represents the differentiation ability of stem cells, as shown in the figure 4. It can be seen that the infection of LG04 carrier has basically no effect on the differentiation ability of hematopoietic stem cells.
  • the present invention has the following beneficial effects:
  • the insulator sequence from the foamy virus used in the present invention can maintain gene expression while preventing gene activation in the region.

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Abstract

本发明公开了一种适用地中海贫血症和镰刀形贫血基因治疗的慢病毒载体；包含：a、微基因座控制区，为一段从beta珠蛋白16kb的基因座控制区中筛选出来的不含HS1区的微型调控元件；b、beta珠蛋白的基因序列，该基因序列如SEQ ID NO.3所示；c、beta珠蛋白的基因序列上游侧翼的启动子序列；d、beta珠蛋白的基因序列下游的侧翼序列；e、来自泡沫病毒(Foamy virus)的绝缘子序列。

Description

一种适用地中海贫血症和镰刀形贫血基因治疗的慢病毒载体 技术领域
本发明属于基因工程技术领域，涉及一种慢病毒载体，尤其涉及一种适用地中海贫血症和镰刀形贫血基因治疗的慢病毒载体。
背景技术
迄今为止，人类共发现有7000余种罕见病，但仅5％的罕见病有药可用。目前，几乎所有的罕见病药物都需要终身使用。基因治疗则为罕见病提供了一次性治愈的可能。地中海贫血(地贫)是一种全球分布最广、累积患病人群最多的单基因遗传性血红蛋白病，是罕见病中最常见的人类遗传性疾病。
基因治疗具备良好的安全性是其能够成为常规疗法，使更多患者受益的前提。慢病毒载体以不可控的方式插入到基因组(约75％整合至转录活跃的基因编码区域)，伴随有较高的激活癌基因和失活抑癌基因的风险。事实上，慢病毒载体曾在基因治疗地中海贫血的过程中激活HMGA2基因，导致转导的造血干细胞克隆大量扩增。该克隆的异常扩增是由于慢病毒基因治疗载体中使用的绝缘子元件，也即鸡beta珠蛋白脱氧核糖核酸酶I超敏感部位4核心部位(core chicken hypersensitivity site 4，cHS4)造成的(Cavazzana-Calvo,M.etal.Transfusion independence and HMGA2 activation after gene therapy of humanbeta-thalassaemia.Nature 467,318-22(2010).)。
绝缘子元件是具有阻止基因沉默或者防止所在区域基因激活功能的基因元件。cHS4绝缘子含有隐秘的RNA剪接信号，造成克隆扩增，影响基因治疗的安全性，并且使得慢病毒的滴度大幅下降(Cavazzana-Calvo,M.et al.Transfusion independence and HMGA2activation after gene therapy of human beta-thalassaemia.Nature 467,318-22(2010).Negre,O.et al.Preclinical evaluation of efficacy and safety of animproved lentiviral vector for the treatment of beta-thalassemia and sicklecell disease.Curr Gene Ther 15,64-81(2015).)。本发明所包含的绝缘子元件来自泡沫病毒(Foamy virus)，仅36bp的绝缘子序列，不含有隐秘的RNA剪接信号，并具有维持基因表达和防止所在区域的癌基因激活的功能。
Beta珠蛋白基因(HBB)在发育过程中的特异性表达由一段位于其上游的基因座控制区(LCR)调控(Kim,A.&Dean,A.Chromatin loop formation in the beta-globin locusand its role in globin gene transcription.Mol Cells 34,1-5(2012).)。该段序列长达16kb，远远超出了慢病毒载体的运载能力。为了构建用于基因治疗的beta珠蛋白基因表达盒，必须从LCR区域中挑选出具有调控beta珠蛋白基因表达功能的、大小合适的核心元件。LCR由HS2、HS3、HS4等脱氧核糖核酸酶I(DNase I)超敏感位点(hypersensitive sites)组成。这些超敏感位点长度依然太大，必须对其进一步缩减才能包装进慢病毒载体。由于这些超敏感位点功能不同，必须相互配合才能发挥精准的调控功能，因此，必须找到有利beta珠蛋白基因高效和特异表达的组合。例如，由HS4、HS3、HS2脱氧核糖核酸酶I超敏感位点的核心元件组成的微基因座(miniLCR)导致病毒效价显著降低(Weber,L.et al.AnOptimized Lentiviral Vector Efficiently Corrects the Human Sickle CellDisease Phenotype.Mol Ther Methods Clin Dev 10,268-280(2018)。本发明的筛选的微基因座不会导致病毒效价的显著降低。
目前，基于慢病毒载体治疗地中海贫血技术仍未真正实现临床使用及商业化的原因有二：一，现有技术生产出的治疗用病毒载体滴度过低，导致生产规模和生产成本过大，难以生产出足量的病毒载体供应临床使用和商业化；二，现有技术生产出的治疗用病毒载体进行治疗后，其beta珠蛋白的表达量十分有限，难以起到有效的治疗效果。本发明对载体结构进行了优化，发现对Beta珠蛋白基因(HBB)的2号内含子优化后，既可以在一定程度提高基因的表达，又可以大幅度提高病毒包装产量，使其临床使用和产业化成为可能。
发明内容
现有技术中将慢病毒载体用于地中海贫血与镰刀型贫血基因治疗存在安全隐患，也并不十分高效；本发明的目的在于针对现有技术载体滴度过低，无法达到临床应用水平；基因表达量不足，无法起到治疗效果；以及载体缺乏绝缘子序列，容易激活临近基因表达，存在较大安全性风险等缺陷，提供一种适用地中海贫血症和镰刀形贫血基因治疗的慢病毒载体。
本发明的目的是通过以下技术方案实现的：
本发明其中一个或多个实施例涉及一种包含beta珠蛋白表达盒的慢病毒载体，所述表达盒包含：
a、微基因座控制区，为一段从beta珠蛋白16kb的基因座控制区中筛选出来的不 含HS1区的微型调控元件；
b、beta珠蛋白的基因序列；beta珠蛋白基因的2号内含子经优化，所述基因beta珠蛋白的基因序列如SEQ ID NO.2所示；
c、beta珠蛋白的基因序列上游侧翼的启动子序列；
d、beta珠蛋白的基因序列下游的侧翼序列；
e、来自泡沫病毒(Foamy virus)的绝缘子序列。
作为本发明的一个实施方案，所述微型基因座控制区是由HS2、HS3、HS4脱氧核糖核酸酶I超敏感位点序列经过筛选后，组合而成；或是由HS3、HS2脱氧核糖核酸酶I超敏感位点序列经过筛选后，组合而成。
作为本发明的一个实施方案，所述微基因座控制区是从beta珠蛋白的基因座控制区LCR的HS4、HS3、HS2区域分别截取的脱氧核糖核苷酸序列组成的微型基因座调控元件。
作为本发明的一个实施方案，所述微型基因座调控元件的序列如SEQ ID NO.1所示。
作为本发明的一个实施方案，所述启动子序列如SEQ ID NO.4所示。
作为本发明的一个实施方案，所述侧翼序列如SEQ ID NO.5所示。
作为本发明的一个实施方案，所述绝缘子序列如SEQ ID NO.6所示。
本发明涉及一种包含beta珠蛋白表达盒的慢病毒载体在制备地中海贫血和镰刀型贫血的基因治疗药物中的应用。
附图说明
通过阅读参照以下附图对非限制性实施例所作的详细描述，本发明的其它特征、目的和优点将会变得更明显：
图1为载体设计示意图；其中，a为LGO4载体设计示意图，b为LGO2载体设计示意图；
图2为各载体血红蛋白 T87Q相对表达示意图；
图3为各载体滴度对比示意图；
图4为不同载体感染干细胞后，形成BFU-E(爆式红细胞集落形成单位)的数量示意图。
具体实施方式
下面结合实施例对本发明进行详细说明。以下实施例将有助于本领域的技术人员进 一步理解本发明，但不以任何形式限制本发明。应当指出的是，对本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干调整和改进。这些都属于本发明的保护范围。
实施例
本实施例涉及一种人源beta珠蛋白的表达盒，进而获得含该表达盒的用于基因治疗的慢病毒载体LGO4。LGO4载体设计如图1(a)所示，该表达盒包含以下几种元件：(1)微基因座控制区(miniLCR)，为一段从beta珠蛋白16kb的基因座控制区中筛选出来的不含HS1区的微型调控元件，具体为从beta珠蛋白16kb的基因座控制区LCR的HS4、HS3、HS2区域分别截取长度约380bp、1900bp、720bp的脱氧核糖核苷酸序列组成总长3kb(序列如SEQ ID NO.1所示)的微型基因座调控元件；(2)beta珠蛋白的基因序列。其中外显子2含T87Q突变，2号内含子序列经优化；该beta珠蛋白的基因序列如SEQ ID NO.2所示；LGO2载体的设计图如图1(b)，其选用的beta珠蛋白的基因序列如SEQ ID NO.3所示；(3)265-bp的beta珠蛋白的基因序列上游侧翼的启动子序列(序列如SEQ ID NO.4所示)；(4)beta珠蛋白的基因序列下游侧翼300-bp序列(序列如SEQ ID NO.5所示)；(5)将该表达盒置于慢病毒载体；在慢病毒载体的LTR U3区域添加一段来自泡沫病毒(Foamy virus)的仅36bp的绝缘子序列(序列如SEQ ID NO.6所示：aagggagacatctagtgatataagtgtgaactacac)。该绝缘子序列具有维持基因长期表达和阻止整合位点附近基因激活的功能，且不会降低慢病毒载体的滴度。含3kb微基因座控制区的慢病毒载体的序列如SEQ ID NO.7所示。将本发明载体质粒(13μg)与表达膜蛋白的质粒(如3.75μg pMD2G(序列如SEQ ID NO.8所示))、表达GagPol长链蛋白的质粒(通常为13μg pMDlg/pRRE(序列如SEQ ID NO.9所示))、表达REV蛋白(通常为3μg pRSV-REV(序列如SEQ ID NO.10所示))的质粒共转染病毒4X10 6生产细胞(如293T)，生产出表达beta珠蛋白的慢病毒LGO4。LGO2载体设计如图1(a)所示，其他步骤基本同上(参照CN201910824134)，获得表达beta珠蛋白的慢病毒LGO2。使用表达绿色荧光蛋白的载体质粒(pLV/PGK-EGFP，引用文献DOI:10.1093/nar/gkt1163)替换本发明载体质粒，其它步骤同上，获得对照病毒载体。慢病毒通过超速离心或者柱层析的方法的富集或者纯化。
在体外使用相同量的各个病毒载体感染人造血干细胞，感染完成后诱导干细胞向红系分化，在第14天提取各组细胞RNA，使用荧光定量PCR进行RT-Q-PCR分析整合的血红蛋白 T87QmRNA的表达水平，反应了血红蛋白 T87Q的相对表达量(图2)，LG04号载体表 达量最高。
使用293T细胞包装LG02和LG04载体，纯化后，感染293T，检测病毒感染滴度(图3)，结果显示，优化后的内含子可以提高病毒包装滴度。
使用相同量的各个病毒载体感染人造血干细胞后，将干细胞接种在含有特点细胞因子的固体培养基内，培养14天后，显微镜下统计爆式红细胞集落形成单位，集落数量代表干细胞分化能力，如图4，可以看出，LG04号载体感染对造血干细胞分化能力基本无影响。
与现有技术相比，本发明具有如下有益效果：
1)获得一种安全、高效的地中海贫血和镰刀型贫血的基因治疗方法；
2)提高了beta珠蛋白的基因的表达；
3)提高了病毒包装效率，增加了病毒滴度；
4)提高了病毒载体的安全性，降低了临床使用的安全风险；
5)本发明所使用的来自来自泡沫病毒的绝缘子序列，可以维持基因表达的同时防止所在区域基因激活。
以上对本发明的具体实施例进行了描述。需要理解的是，本发明并不局限于上述特定实施方式，本领域技术人员可以在权利要求的范围内做出各种变形或修改，这并不影响本发明的实质内容。

Claims (7)

  1. 一种包含beta珠蛋白表达盒的慢病毒载体，所述表达盒包含：
    a、微基因座控制区，为一段从beta珠蛋白16kb的基因座控制区中筛选出来的不含HS1区的微型调控元件；
    b、beta珠蛋白的基因序列，所述基因beta珠蛋白的基因序列如SEQ ID NO.2所示；
    c、beta珠蛋白的基因序列上游侧翼的启动子序列；
    d、beta珠蛋白的基因序列下游的侧翼序列；
    e、来自泡沫病毒Foamy virus的绝缘子序列。
  2. 根据权利要求1所述的包含beta珠蛋白表达盒的慢病毒载体，其特征在于，所述微型基因座控制区是由HS2、HS3、HS4脱氧核糖核酸酶I超敏感位点序列经过筛选后，组合而成。
  3. 根据权利要求2所述的包含beta珠蛋白表达盒的慢病毒载体，其特征在于，所述微型基因座调控元件的序列如SEQ ID NO.1所示。
  4. 根据权利要求1所述的包含beta珠蛋白表达盒的慢病毒载体，其特征在于，所述启动子序列如SEQ ID NO.4所示。
  5. 根据权利要求1所述的包含beta珠蛋白表达盒的慢病毒载体，其特征在于，所述侧翼序列如SEQ ID NO.5所示。
  6. 根据权利要求1所述的包含beta珠蛋白表达盒的慢病毒载体，其特征在于，所述绝缘子序列如SEQ ID NO.6所示。
  7. 一种如权利要求1所述的包含beta珠蛋白表达盒的慢病毒载体在制备地中海贫血和镰刀型贫血的基因治疗药物中的应用。
PCT/CN2022/138330 2022-01-06 2022-12-12 一种适用地中海贫血症和镰刀形贫血基因治疗的慢病毒载体 WO2023130911A1 (zh)

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