WO2022199552A1 - 一种α-珠蛋白过表达载体及其应用 - Google Patents

一种α-珠蛋白过表达载体及其应用 Download PDF

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WO2022199552A1
WO2022199552A1 PCT/CN2022/082108 CN2022082108W WO2022199552A1 WO 2022199552 A1 WO2022199552 A1 WO 2022199552A1 CN 2022082108 W CN2022082108 W CN 2022082108W WO 2022199552 A1 WO2022199552 A1 WO 2022199552A1
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globin
gene
sequence
seq
hba2
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吴宇轩
程艳
徐赛娟
赵飞燕
王飞
席在喜
刘明耀
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华东师范大学
上海邦耀生物科技有限公司
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Definitions

  • the invention belongs to the field of biomedicine, and in particular relates to an alpha-globin overexpression vector and an application thereof.
  • Normal hemoglobin is a tetramer composed of two alpha-globin chains and two beta-globin chains.
  • Alpha-thalassemia ⁇ -thalassemia
  • ⁇ -thalassemia is an autosomal recessive disorder caused by the loss or dysfunction of the ⁇ -globin gene, which reduces or fails to synthesize the ⁇ -globin chain, resulting in an unbalanced synthesis of the ⁇ -chain and non- ⁇ -chains.
  • Sexually inherited diseases Common deletions on the ⁇ -globin gene cluster include ⁇ /--- SEA and - ⁇ 4.2 /- ⁇ 4.2 , etc.
  • the mechanism is due to the homologous and non-homologous recombination of one or two chromosomes of the ⁇ -globin gene.
  • the deletion of this gene can down-regulate the expression level of ⁇ -globin gene and reduce the production of ⁇ -globin chain.
  • alpha thalassemia is one of the most common single-gene genetic diseases in the world, especially common in the Mediterranean, Southeast Asia, Africa, the Middle East and the Indian subcontinent, and the incidence is higher in the regions south of the Yangtze River in my country.
  • WHO World Health Organization
  • 20% of the global population may have one or more alpha-globin gene deletions.
  • the most common deletion types are --SEA (SEA deletion), - ⁇ 4.2 (left deletion) and - ⁇ 3.7 (right deletion).
  • SEA deletion SEA deletion
  • - ⁇ 4.2 left deletion
  • - ⁇ 3.7 right deletion
  • CRISPR/Cas technology has become one of the hot spots in the scientific community.
  • Viral vectors have the characteristics of high infection rate and low cytotoxicity, and are still the best solution for many scientific researches and clinical trials.
  • the lentiviral vector is derived from HIV-1 lentivirus and comprises multiple helper elements for increasing transduction efficiency, viral packaging and/or elements for increasing therapeutic gene expression, and modified 5'LTR and/or 3'LTR, After such a vector is integrated into a target cell, RNA cannot be transcribed even if all viral proteins are present, making the virus replication-deficient to increase the safety of the lentiviral system.
  • Lentiviral vectors have the characteristics of being able to infect non-dividing cells, accommodating exogenous target gene fragments, strong stability, and high infection efficiency.
  • the third-generation lentiviral system, the gag, pol genes and rev or env genes were constructed on different plasmids, and a deletion was introduced into the 3'LTR of the viral genome to generate a self-inactivating (SIN) lentiviral vector, destroying the LTR Promoter/enhancer activity, which further improves safety and reduces carcinogenic risk.
  • SI self-inactivating
  • HSCs hematopoietic stem cells
  • the technical problem to be solved by the present invention is to provide an ⁇ -globin overexpression vector with moderate expression rate and its application in order to overcome the lack of an overexpression vector for expressing ⁇ -globin gene in the prior art.
  • the regulatory sequences of ⁇ -globin genes contain binding sites for multiple cis- and trans-acting factors, and their synergy ensures the organizational and temporal specificity of ⁇ -globin gene expression.
  • the complete replication of the natural process requires a large number of synthesis of a variety of functional region sequences, the process is very complex and difficult to achieve in terms of cost;
  • the expression of the source HBB gene is all regulated based on the regulatory elements of the natural HBB gene itself, while the selection of the expression regulatory elements of the heterologous ⁇ -globin gene has not been studied in the prior art.
  • the expression of ⁇ -globin gene is still a technical problem.
  • an ⁇ -globin overexpression vector which in turn comprises: (1) a locus control region, an ⁇ -globin promoter, HBA2 gene and HBA2 gene 3'sequence fragment, the locus control region is HS40;
  • locus control regions HS4, HS3 and HS2, ⁇ -globin promoter, HBA2 gene and HBB gene 3'sequence fragments are or (2) locus control regions HS4, HS3 and HS2, ⁇ -globin promoter, HBA2 gene and HBB gene 3'sequence fragments.
  • the sequential inclusion refers to sequential inclusion from the 5' end to the 3' end.
  • the ⁇ -globin overexpression vector comprises a cis-acting element, the HBA2 gene and the 3'sequence fragment of the HBA2 gene in sequence, and the cis-acting element is composed of the locus control region HS40 and the ⁇ -globin promoter. constitute.
  • the ⁇ -globin promoter of the present invention is preferably the HBA2 promoter; more preferably, its sequence comprises the sequence shown in SEQ ID NO: 2, or comprises the sequence shown in SEQ ID NO: 2 with 80 A sequence of % above sequence identity, for example, its sequence is shown in SEQ ID NO:2.
  • the locus control region HS40 of the present invention can be conventional in the art, and its sequence preferably comprises the sequence shown in SEQ ID NO: 1, or comprises a sequence that has more than 80% sequence identity with the sequence shown in SEQ ID NO: 1
  • the sequence, for example, is shown in SEQ ID NO:1.
  • the 3'sequence fragment of the HBA2 gene of the present invention can be conventional in the art, and its sequence preferably comprises the sequence shown in SEQ ID NO:4, or contains more than 80% sequence identity with the sequence shown in SEQ ID NO:4
  • the sequence of, for example, its sequence is shown in SEQ ID NO:4.
  • the ⁇ -globin promoter described in the present invention is preferably the HBB promoter; more preferably, its sequence comprises the sequence shown in SEQ ID NO: 8, or comprises the same sequence as the sequence shown in SEQ ID NO: 8 A sequence of more than 80% sequence identity, for example, its sequence is shown in SEQ ID NO:8.
  • the locus control region HS4 of the present invention can be conventional in the art, and its sequence preferably comprises the sequence shown in SEQ ID NO:5, or comprises more than 80% sequence identity with the sequence shown in SEQ ID NO:5
  • the sequence of, for example, its sequence is shown in SEQ ID NO:5.
  • the locus control region HS3 of the present invention can be conventional in the art, and its sequence preferably comprises the sequence shown in SEQ ID NO: 6, or comprises a sequence that has more than 80% sequence identity with the sequence shown in SEQ ID NO: 6
  • the sequence, for example, is shown in SEQ ID NO:6.
  • the locus control region HS2 of the present invention can be conventional in the art, and its sequence preferably comprises the sequence shown in SEQ ID NO: 7, or comprises a sequence that has more than 80% sequence identity with the sequence shown in SEQ ID NO: 7
  • the sequence, for example, is shown in SEQ ID NO:7.
  • the HBB gene 3'sequence fragment of the present invention can be conventional in the art, and its sequence preferably comprises the sequence shown in SEQ ID NO: 9, or contains more than 80% sequence identity with the sequence shown in SEQ ID NO: 9
  • the sequence of, for example, its sequence is shown in SEQ ID NO:9.
  • the sequence of the HBA2 gene of the present invention can be conventional in the art; its sequence preferably comprises the sequence shown in SEQ ID NO:3, or a sequence with more than 80% sequence identity with the sequence shown in SEQ ID NO:3 , for example, its sequence is shown in SEQ ID NO:3.
  • the plasmid vector of the ⁇ -globin overexpression vector of the present invention is preferably a Lenti vector, and the sequence of the Lenti vector preferably comprises the sequence shown in SEQ ID NO: 10, or comprises the same sequence as SEQ ID NO: 10
  • the sequence shown has a sequence identity of more than 80%, for example, the sequence of which is shown in SEQ ID NO: 10.
  • sequence identity above 80% includes 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98% or more than 99% sequence identity.
  • the second technical solution provided by the present invention is: a transformant comprising the above-mentioned ⁇ -globin overexpression vector.
  • the host cells of the transformants are preferably erythroid cells.
  • the erythroid cells are preferably HUDEP-2 or CD34 + hematopoietic stem cells.
  • the third technical solution provided by the present invention is: a lentiviral vector system comprising the above-mentioned overexpression vector.
  • the lentiviral vector system preferably further comprises psPAX2 and pCAG-VSVG plasmids.
  • the fourth technical solution provided by the present invention is: an application of the above-mentioned overexpression vector in the preparation of a preparation for treating thalassemia.
  • the thalassemia is preferably alpha-thalassemia.
  • the fifth technical solution provided by the present invention is: a HS40, ⁇ -globin promoter, HBA2 gene 3'sequence fragment, HS4, HS3, HS2, ⁇ -globin promoter or HBB gene 3 as defined in the above technical solution Application of 'sequence fragment in constructing HBA2 overexpression vector.
  • the vector is preferably a lentiviral vector.
  • the reagents and raw materials used in the present invention are all commercially available.
  • the ⁇ -globin overexpression vector constructed in the present invention can efficiently transduce the target gene, and can make the target gene express stably for a long time, with high infection efficiency and expression stability; the gene therapy mediated by the lentiviral vector in the present invention can Significantly increased the expression of ⁇ -globin gene in patients with deletional ⁇ -thalassemia, so that the expression level of ⁇ -globin gene reached a moderate state, neither too high nor too low, providing hope for the successful treatment of all ⁇ -thalassemia patients , has important clinical significance.
  • Erythroid cell lines and hematopoietic stem cells infected with the vector lentivirus can overexpress alpha globin after erythroid differentiation, so the vector combined with hematopoietic stem cell transplantation is expected to treat patients with various mutant types of alpha thalassemia.
  • Figure 1 is a schematic diagram of the Lenti-A1 and Lenti-A2 lentiviral vectors.
  • Figure 2 shows that the expression level of ⁇ -globin increased after HUDEP-2 cells were infected with lentiviral erythroid differentiation by RT-qPCR.
  • Figure 3 shows that CD34 + cells were infected with lentiviral erythroid differentiation by RT-qPCR showing increased levels of ⁇ -globin expression.
  • Figure 4 shows that Lenti-A1 and Lenti-A2 lentivirus infection of ⁇ -thalassemia CD34 + hematopoietic stem cells can significantly increase the expression level of ⁇ -globin in differentiated erythrocytes by RT-qPCR (*, compared with the control group). ratio, P ⁇ 0.05; the expression level of ⁇ -globin increased after Lenti-A1 and Lenti-A2 lentivirus infection of cells).
  • Figure 5 shows that the expression level of ⁇ -globin increased by RT-qPCR in patients with ⁇ -thalassemia CD34 + hematopoietic stem cells infected with lentivirus (*, compared with the control group, P ⁇ 0.05; Lenti-A1 and Lenti-A2 slow ⁇ -globin expression levels were higher after the virus infected cells separately).
  • Figure 6 shows that the hematopoietic stem cell transplantation immunodeficient mice infected with Lenti-A1 and Lenti-A2 have similar homing efficiency to uninfected cells by flow analysis, reaching a humanization ratio of more than 85% .
  • Figure 7 shows that by flow analysis, hematopoietic stem cells after lentivirus infection transplanted into immunodeficient mice can successfully differentiate into various blood cells including B cells and myeloid cells in the mouse bone marrow.
  • Figure 8 shows that the Lenti-A1 and Lenti-A2 lentiviral vectors can successfully increase the expression of HBA in hematopoietic stem cells four months after transplantation in mice by RT-qPCR analysis.
  • * Compared with the Mock group, P ⁇ 0.05; the expression level of ⁇ -globin was significantly increased after Lenti-A1 and Lenti-A2 lentivirus infected cells respectively).
  • Lenti-A1 (Fig. 1) is an overexpression vector carrying human ⁇ -globin gene. It mainly contains DNase I high-sensitivity site HS40, the promoter and gene sequence of human HBA2 gene, about 8kb in length. Its own promoter and regulatory elements can express ⁇ -globin specifically in erythroid cells.
  • Lenti-A2 (Fig. 1) is another overexpression vector carrying human ⁇ -globin gene constructed by the present invention, which mainly includes human HBA2 gene, human ⁇ -globin gene promoter and a HS2, HS3 and HS4. mini-LCR, about 10kb long.
  • an LCR consisting of the HS2-HS4 core sequence to the hemoglobin A2 (HBA2) globin gene with a ⁇ -globin promoter the expression level of ⁇ -globin in erythrocytes is correlated with that of endogenous ⁇ -globin equivalent level.
  • HBA2 gene itself cannot be expressed at a high level in cultured erythrocytes, but when it is linked to ⁇ -LCR, it shows a high level of erythrocyte-specific expression.
  • HEK 293T cells were routinely cultured in DMEM high glucose medium containing 10% FBS. One day before transfection, take the cells in log phase condition and inoculate about 15ml-20ml of cell suspension in a 15cm dish, with 2.1 ⁇ 10 7 cells per 15cm dish. Transfection experiments were performed when the cell density was 80% to 90%. According to the instructions of PEI transfection reagent, the three plasmids of psPAX2, pCAG-VSVG and HBA overexpression vector were co-transfected into 293T cells, including 20 ⁇ g of the target gene plasmid, 13.3 ⁇ g of psPAX2, and 6.7 ⁇ g of pCAG-VSVG.
  • the complete medium (DMEM containing 10% FBS) was changed. After 48 hours, the virus solution was collected and ultracentrifuged for 2 hours at 24000 r/min. After centrifugation, the supernatant was discarded for concentration, and the virus was resuspended in 1 mL of X-VIVO to obtain a virus concentrate, which was stored at -80°C for later use.
  • HUDEP-2 Human Umbilical Cord Blood Derived Erythoid Progenitor-2
  • CD34 + hematopoietic stem cells by constructing a lentiviral vector, which is driven by cis-regulatory elements and produces high RBC-specific expression, thereby producing sufficient hemoglobin, reducing or eliminating the need for transfusion therapy.
  • Analysis by RT-PCR showed that the two lentiviral vectors can express in HUDEP-2 cells (Kurita R, Suda N, Sudo K, Miharada K, Hiroyama T, et al.
  • Example 3 Construction of a deletion-type ⁇ -thalassaemia CD34 + cell model and the application of ⁇ -globin lentiviral vector in the model
  • deletion mutant ⁇ -thalassemia is divided into the following types: deletion of 1 ⁇ -globin gene (- ⁇ / ⁇ ) is Static type; deletion of 2 ⁇ -globin genes (--/ ⁇ or - ⁇ /- ⁇ ) is the standard type; deletion of 3 ⁇ -globin genes (--/- ⁇ ) is called hemoglobin H disease (HbH); 4 Hb Bart's fetal hydrops syndrome is defined as the deletion of all ⁇ -globin genes (--/--).
  • HbH hemoglobin H disease
  • 4 Hb Bart's fetal hydrops syndrome is defined as the deletion of all ⁇ -globin genes (--/--).
  • the main reason for the deletion of the ⁇ -globin gene is the deletion of large fragments of DNA, ranging from 2.7kb to the entire gene cluster.
  • sgRNA was designed for the exon region of HBA2 gene, and the purpose of deleting DNA fragments was achieved by inducing double-strand breaks at both ends of this region at the same time.
  • the CD34 + hematopoietic stem cells of patients infected with HbH disease (transfusion-dependent ⁇ -thalassemia, genotype -- SEA / ⁇ CS ) thalassemia were validated.
  • the CD34 + hematopoietic stem cells of the HbH disease patient were obtained by the following method: 10 ml of peripheral blood of the HbH thalassemia patient was taken, and after Ficoll was used to separate the mononuclear cells, the CD34 + cells were obtained with CD34 magnetic beads and resuspended in the medium. The results showed ( FIG.
  • the hematopoietic stem cells infected with the lentiviral vector in the experimental group were transplanted into immunodeficient mice, and flow cytometry analysis after four months showed that the homing efficiency was similar to that of uninfected cells (as shown in Figure 6, the Y-axis shows The proportion of human cells in mouse bone marrow), can successfully home; and can successfully differentiate into various blood cells including B cells and myeloid cells in mouse bone marrow (Figure 7); RT-qPCR after four months
  • the HBA expression was analyzed and detected. Compared with the uninfected Mock group, the HBA expression of hematopoietic stem cells infected with Lenti-A1 and Lenti-A2 was significantly increased (Figure 8).

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Abstract

本发明公开了一种α-珠蛋白过表达载体及其应用，所述α-珠蛋白过表达载体依次包含：(1)基因座控制区、α-珠蛋白启动子、HBA2基因和HBA2基因3'sequence片段，所述基因座控制区为HS40；或(2)基因座控制区HS4、HS3和HS2、β-珠蛋白启动子、HBA2基因和HBB基因3'sequence片段。本发明所提供的过表达载体能够高效转导目的基因，并且能够使目的基因长期稳定的表达，具有高感染效率及表达稳定性。

Description

一种α-珠蛋白过表达载体及其应用
本申请要求申请日为2021/3/22的中国专利申请2021103040176的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明属于生物医药领域，具体涉及一种α-珠蛋白过表达载体及其应用。
背景技术
正常血红蛋白是由两条α-珠蛋白链和两条β-珠蛋白链组成的四聚体。有两个功能性α-血红蛋白基因，分别命名为HBA1和HBA2。α地中海贫血病(α地贫)是由于α珠蛋白基因缺失或功能障碍使α-珠蛋白链的合成减少或不能合成，造成α链和非α链合成不均衡所引起的一种常染色体隐性遗传病。α-珠蛋白基因簇上常见的缺失有αα/-- SEA和-α 4.2/-α 4.2等，其发生机理是由于α珠蛋白基因一条或两条染色体发生同源性和非同源性重组而缺失，该基因缺失可使α珠蛋白基因表达水平下调而降低α珠蛋白链产生。
据报道，α地中海贫血是世界上最常见的单基因遗传病之一，在地中海、东南亚、非洲、中东和印度次大陆尤其常见，在我国长江以南地区发病率较高。根据世界卫生组织(WHO)估计，全球20％的人口可能有一个或多个α-珠蛋白基因缺失。中国南方省份及东南亚地区，最常见的缺失类型为--SEA(东南亚缺失型)、-α 4.2(左缺失)和-α 3.7(右缺失)三种。在过去几十年中，由于人口结构的变化，北欧和北美的α地中海贫血发病率有所上升。目前，国内外对地中海贫血有效的治疗手段包括异源性造血细胞移植、规范性长期输血和去铁治疗，但是有排斥风险，并发症和其它毒性效应，因此基因治疗方案正在被评估作为一种新的方案。
近年来，由于基因工程技术的突飞猛进，CRISPR/Cas技术俨然已经成为科学界的热点之一。研究表明，CRISPR/Cas系统相较于其他基因编辑工具，在干细胞中有更高的切割效率，可用于构建疾病模型、筛选药物，制备治疗性干细胞等，为疾病治疗带来了新希望，但可能存在的脱靶风险不容忽视。病毒载体具有高感染率、低细胞毒性的特点，目前仍然是很多科学研究和临床试验的较优方案。慢病毒载体衍生自HIV-1慢病毒，包含多个用于增加转导效率的辅助元件、病毒包装和/或增加治疗基因表达的元件，以及经过修饰的5'LTR和/或3'LTR，这样的载体整合入目的细胞后，即使存在所有病毒蛋白也不能转录出RNA，使病毒变成复制缺陷型来增加慢病毒系统的安全性。慢病毒载体具有能感 染非分裂期细胞、容纳外源性目的基因片段大、稳定性强、感染效率高等特点。
第三代慢病毒系统，将gag、pol基因与rev或env基因分别构建在不同的质粒上，并且在病毒基因组的3'LTR引入缺失以产生自失活(SIN)慢病毒载体，破坏LTR的启动子/增强子活性，进一步提高了安全性，并降低致癌风险。目前有2/3的基因治疗临床试验是运用病毒载体来引入基因到靶细胞目前使用较多的是来源于HIV的慢病毒载体。通过慢病毒载体进行基因治疗为血红蛋白病带来了新的选择，其临床研究已经开展，结果令人鼓舞，但远期疗效和安全性仍待证明。体外基因治疗，通过载体转移和染色体整合的基因添加一个正常的珠蛋白基因连同合适的顺式连接的调节元件到造血干细胞(HSCs)仍然是选择的方法。慢病毒载体基因治疗需要非常高效的造血干细胞感染效率和长期稳定的α-珠蛋白表达才具有临床意义。
但目前现有技术中对珠蛋白过表达载体的研究几乎针对β-珠蛋白基因，因此急需一种表达率适于临床应用的α-珠蛋白过表达载体。
发明内容
本发明要解决的技术问题是为克服现有技术中缺乏表达α-珠蛋白基因的过表达载体，提供一种表达率适中的α-珠蛋白过表达载体及其应用。
在人体中，α-类珠蛋白基因的调控序列包含多种顺式和反式作用因子的结合位点，它们间的协同作用保证了α类珠蛋白基因表达的组织和时序特异性。而对于异源的α-珠蛋白基因的表达调控，完全复制天然的过程需大量合成多种功能区序列，过程十分复杂且从成本上讲也难以实现；而对于目前已经取得一定研究进展的异源HBB基因的表达，均是基于天然HBB基因本身的调控元件进行表达调控，而对于异源α-珠蛋白基因的表达调控元件的选择，现有技术却没有这方面的研究，因此，异源α-珠蛋白基因的表达目前仍为一个技术难题。
为解决上述技术问题，本发明提供的技术方案之一为：一种α-珠蛋白过表达载体，其依次包含：(1)基因座控制区、α-珠蛋白启动子、HBA2基因和HBA2基因3’sequence片段，所述基因座控制区为HS40；
或(2)基因座控制区HS4、HS3和HS2、β-珠蛋白启动子、HBA2基因和HBB基因3’sequence片段。
所述依次包含指的是从5’端到3’端的依次包含。
较佳地，所述的α-珠蛋白过表达载体依次包含顺式作用元件、HBA2基因和HBA2基因3’sequence片段，所述顺式作用元件由基因座控制区HS40和α-珠蛋白启动子构成。
本发明所述α-珠蛋白启动子较佳地为HBA2启动子；更佳地，其序列包含如SEQ ID NO:2所示的序列，或包含与如SEQ ID NO:2所示序列具有80％以上序列同一性的序列，例如，其序列如SEQ ID NO:2所示。
本发明所述基因座控制区HS40可为本领域常规，其序列优选包含如SEQ ID NO:1所示的序列，或包含与如SEQ ID NO:1所示序列具有80％以上序列同一性的序列，例如，其序列如SEQ ID NO:1所示。
本发明所述HBA2基因3’sequence片段可为本领域常规，其序列优选包含如SEQ ID NO:4所示的序列，或包含与如SEQ ID NO:4所示序列具有80％以上序列同一性的序列，例如，其序列如SEQ ID NO:4所示。
本发明中所述β-珠蛋白启动子较佳地为HBB启动子；更佳地，其序列包含如SEQ ID NO:8所示的序列，或包含与如SEQ ID NO:8所示序列具有80％以上序列同一性的序列，例如，其序列如SEQ ID NO:8所示。
在本发明所述基因座控制区HS4可为本领域常规，其序列优选包含如SEQ ID NO:5所示的序列，或包含与如SEQ ID NO:5所示序列具有80％以上序列同一性的序列，例如，其序列如SEQ ID NO:5所示。
本发明所述基因座控制区HS3可为本领域常规，其序列优选包含如SEQ ID NO:6所示的序列，或包含与如SEQ ID NO:6所示序列具有80％以上序列同一性的序列，例如，其序列如SEQ ID NO:6所示。
本发明所述基因座控制区HS2可为本领域常规，其序列优选包含如SEQ ID NO:7所示的序列，或包含与如SEQ ID NO:7所示序列具有80％以上序列同一性的序列，例如，其序列如SEQ ID NO:7所示。
本发明所述HBB基因3’sequence片段可为本领域常规，其序列优选包含如SEQ ID NO:9所示的序列，或包含与如SEQ ID NO:9所示序列具有80％以上序列同一性的序列，例如，其序列如SEQ ID NO:9所示。
本发明所述HBA2基因的序列可为本领域常规；其序列优选包含如SEQ ID NO:3所示的序列，或包含与如SEQ ID NO:3所示序列具有80％以上序列同一性的序列，例如，其序列如SEQ ID NO:3所示。
本发明所述的α-珠蛋白过表达载体的质粒载体较佳地为Lenti载体，所述Lenti载体的序列优选包含如SEQ ID NO:10所示的序列，或包含与如SEQ ID NO:10所示序列具有80％以上序列同一性的序列，例如，其序列如SEQ ID NO:10所示。
以上所述80％以上序列同一性包含81％、82％、83％、84％、85％、86％、87％、88％、 89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％以上序列同一性。
本发明提供的技术方案之二为：一种包含上述α-珠蛋白过表达载体的转化体。
所述转化体的宿主细胞较佳地为红系细胞。
所述红系细胞优选为HUDEP-2或CD34 +造血干细胞。
本发明提供的技术方案之三为：一种包含上述过表达载体的慢病毒载体系统。
所述慢病毒载体系统较佳地还包含psPAX2和pCAG-VSVG质粒。
本发明提供的技术方案之四为：一种上述过表达载体在制备治疗地中海贫血的制剂中的应用。
所述地中海贫血较佳地为α-地中海贫血。
本发明提供的技术方案之五为：一种如上技术方案所定义的HS40、α-珠蛋白启动子、HBA2基因3’sequence片段、HS4、HS3、HS2、β-珠蛋白启动子或HBB基因3’sequence片段在构建HBA2过表达载体中的应用。
所述载体较佳地为慢病毒载体。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：
本发明所构建的α-珠蛋白过表达载体能够高效转导目的基因，并且能够使目的基因长期稳定的表达，具有高感染效率及表达稳定性；本发明用慢病毒载体介导的基因治疗能够显著提升缺失型α-地中海贫血患者中α-珠蛋白基因的表达，使α-珠蛋白基因的表达量达到适中状态，既不过高也不过低，为成功治疗所有的α地贫患者提供了希望，具有重要的临床意义。经该载体慢病毒感染过的红系细胞系和造血干细胞诱导红系分化后，都能过表达α珠蛋白，因此该载体结合造血干细胞移植有望治疗各种突变类型的α地中海贫血患者。
附图说明
图1为Lenti-A1和Lenti-A2慢病毒载体示意图。
图2显示了通过RT-qPCR显示HUDEP-2细胞感染慢病毒红系分化后α-珠蛋白表达水平升高。
图3显示了通过RT-qPCR显示CD34 +细胞感染慢病毒红系分化后α-珠蛋白表达水平升高。
图4显示了通过RT-qPCR显示Lenti-A1和Lenti-A2两种慢病毒感染α地贫CD34 +造血干细胞后可显著提高其分化后红细胞中α-珠蛋白表达水平(*，与对照组相比，P<0.05；Lenti-A1与Lenti-A2慢病毒感染细胞后，α-珠蛋白表达水平升高)。
图5显示了通过RT-qPCR显示α地贫病人CD34 +造血干细胞感染慢病毒后α-珠蛋白表达水平升高(*,与对照组相比，P<0.05；Lenti-A1与Lenti-A2慢病毒分别感染细胞后，α-珠蛋白表达水平较高)。
图6显示了通过流式分析，表明经Lenti-A1和Lenti-A2感染过后的造血干细胞移植免疫缺陷小鼠，具有和未感染细胞相似的归巢效率，达到了85％以上的人源化比例。
图7显示了通过流式分析，表明将慢病毒感染后的造血干细胞移植免疫缺陷小鼠，可以在小鼠骨髓中成功分化为包括B细胞和髓细胞在内的各种血液细胞。
图8显示了通过RT-qPCR分析，表明小鼠移植四个月后，Lenti-A1和Lenti-A2慢病毒载体可以成功提升造血干细胞中HBA表达量。(*,与Mock组相比，P<0.05；Lenti-A1与Lenti-A2慢病毒分别感染细胞后，α-珠蛋白表达水平显著提升)。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择；未注明具体来源的材料试剂，均可按照市售产品常规购买。
实施例1 α-珠蛋白过表达载体的构建
Lenti-A1(图1)是一种携带有人α-珠蛋白基因的过表达载体，主要包含DNase I高敏位点HS40、人HBA2基因的启动子及基因序列，长约8kb左右，主要是选择其本身的启动子和调控元件，可在红系细胞中特异性地表达α-珠蛋白。
Lenti-A2(图1)是本发明构建的另一种携带有人α-珠蛋白基因的过表达载体，主要包含人HBA2基因、人β-珠蛋白基因启动子和由HS2、HS3和HS4组成的mini-LCR，长约10kb左右。通过将一个由HS2～HS4核心序列组成的LCR与带有β-珠蛋白启动子的血红蛋白A2(HBA2)珠蛋白基因相连，使α珠蛋白在红细胞中表达水平与内源性α珠蛋白的表达水平相当。HBA2基因本身在培养的红细胞中不能高水平表达，但当与β-LCR连接后则表现出红细胞特异的高水平表达。
其中各元件的序列分别如SEQ ID NO:1～10所示。
实施例2 慢病毒载体系统的构建及其在HUDEP-2和CD34 +造血干细胞中的应用
慢病毒载体系统包装方法：HEK 293T细胞常规培养于含10％FBS的DMEM高糖培 养基。转染前1天，取对数期状态良好的细胞，15cm皿接种15ml-20ml左右细胞悬液，每15cm培养皿2.1×10 7细胞。待细胞密度为80％～90％时进行转染实验。按照PEI转染试剂说明进行操作，将psPAX2、pCAG-VSVG及HBA过表达载体三质粒共转染293T细胞，其中目的基因质粒20μg，psPAX2 13.3μg，pCAG-VSVG 6.7μg。
6h后换完全培养液(含10％FBS的DMEM)。48h后收集病毒液，超速离心2h，转速24000r/min，离心后弃去上清浓缩，再用1mL X-VIVO重悬病毒，获得病毒浓缩液，-80℃储存备用。
我们实验目的是通过构建慢病毒载体将功能性α-珠蛋白基因，导入HUDEP-2(Human Umbilical Cord Blood Derived Erythoid Progenitor-2)和CD34 +造血干细胞中，由顺式调节元件驱动，产生高的红细胞特异性表达，从而产生足够的血红蛋白，减少或消除输血治疗的必要性。通过RT-PCR分析显示，两种慢病毒载体可以在HUDEP-2细胞(Kurita R,Suda N,Sudo K,Miharada K,Hiroyama T,et al.(2013)Establishment of Immortalized Human Erythroid Progenitor Cell Lines Able to Produce Enucleated Red Blood Cells.PLoS ONE 8(3):e59890.doi:10.1371/journal.pone.0059890)和CD34 +细胞(来源于正常人骨髓)中红系分化18天后，可以提升α-珠蛋白的表达水平(图2和图3)。
实施例3 构建缺失型α-地贫CD34 +细胞模型及α-珠蛋白慢病毒载体在该模型中的应用
正常人有4个α珠蛋白基因(αα/αα，2个α2和2个α1)；缺失突变型α地贫分为以下几种类型：缺失1个α珠蛋白基因(-α/αα)为静止型；缺失2个α珠蛋白基因(--/αα或-α/-α)为标准型；缺失3个α珠蛋白基因(--/-α)称为血红蛋白H病(HbH)；4个α珠蛋白基因均缺失(--/--)的即为Hb Bart's胎儿水肿综合征。引起α珠蛋白基因缺失的原因主要是DNA大片段缺失，缺失范围从2.7kb到整个基因簇不等。实验通过构建-α 4.2缺失α-地中海贫血疾病模型，针对HBA2基因外显子区域设计了sgRNA，通过同时诱发此区域的两端发生双链断裂，达到删除DNA片段的目的。我们将该sgRNA与Cas9蛋白形成的RNP通过电转导入人CD34 +造血干细胞中，构建纯合型和杂合型细胞株，进而检验α-珠蛋白与β-珠蛋白的表达水平及它们之间的比例。体外红系分化18天后RT-qPCR检测α-珠蛋白表达水平，结果表明，α-珠蛋白表达水平显著下降，证明疾病模型构建成功(图4)。疾病模型构建成功后，感染Lenti-A1和Lenti-A2慢病毒载体，体外红系分化18天后检测α-珠蛋白表达水平。结果表明，两种慢病毒载体均可以显著升高α-珠蛋白表达水平(图4)，这说明这两种载体都有望成为α地贫疾病的基因治疗载体。
SgRNA序列：GATGGAGAGCGTATGTTAAC。(SEQ ID NO:11)
实施例4  α-珠蛋白过表达慢病毒载体在HbH病地贫患者CD34 +中的应用
将两种α-珠蛋白过表达载体包装成慢病毒后分别感染了HbH病(输血依赖性α地贫，基因型为-- SEA/αα CS)地贫患者的CD34 +造血干细胞进行验证。所述HbH病患者CD34 +造血干细胞通过以下方法获得：取HbH病地贫患者的外周血10ml，经过Ficoll分离单个核细胞后，以CD34磁珠获取CD34 +细胞，重悬在培养基中。结果显示(图5)，感染病毒的细胞内表达的α-珠蛋白水平比未感染病毒的显著升高。从上述已经获得的研究结果表明，我们构建的α-珠蛋白过表达载体，包装成慢病毒后感染红系细胞系或CD34 +造血干细胞后，在其诱导分化来的红系细胞中可高效的表达α-珠蛋白。
实施例5 慢病毒载体感染效率及表达稳定性
将实验组中经慢病毒载体感染过后的造血干细胞移植免疫缺陷小鼠，四个月后流式分析检测，得出具有和未感染细胞相似的归巢效率(如图6所示，Y轴显示小鼠骨髓中人源细胞比例)，可以成功归巢；且可以在小鼠骨髓中成功分化为包括B细胞和髓细胞在内的各种血液细胞(图7)；四个月后RT-qPCR分析检测HBA表达量,与未感染病毒的Mock组相比，Lenti-A1和Lenti-A2感染过后的造血干细胞HBA表达量具有明显提升(图8)。以上结果表明，经Lenti-A1和Lenti-A2慢病毒载体感染过后，造血干细胞功能正常，其细胞分化未受到影响，且成功提升了造血干细胞中的HBA基因的表达水平，证明感染后的造血干细胞可以长期归巢和重建血液系统。

Claims (10)

  1. 一种α-珠蛋白过表达载体，其特征在于，其依次包含：(1)基因座控制区、α-珠蛋白启动子、HBA2基因和HBA2基因3’sequence片段，所述基因座控制区为HS40；
    或(2)基因座控制区HS4、HS3和HS2、β-珠蛋白启动子、HBA2基因和HBB基因3’sequence片段。
  2. 如权利要求1所述的α-珠蛋白过表达载体，其特征在于，其依次包含顺式作用元件、HBA2基因和HBA2基因3’sequence片段，所述顺式作用元件由基因座控制区HS40和α-珠蛋白启动子构成。
  3. 如权利要求1或2所述的α-珠蛋白过表达载体，其特征在于，所述α-珠蛋白启动子为HBA2启动子；较佳地，所述α-珠蛋白启动子包含如SEQ ID NO:2所示的序列；
    和/或，所述基因座控制区HS40包含如SEQ ID NO:1所示的序列；
    和/或，所述HBA2基因3’sequence片段包含如SEQ ID NO:4所示的序列。
  4. 如权利要求1所述的α-珠蛋白过表达载体，其特征在于，所述β-珠蛋白启动子为HBB启动子；较佳地，所述β-珠蛋白启动子包含如SEQ ID NO:8所示的序列；
    和/或，所述基因座控制区HS4包含如SEQ ID NO:5所示的序列，所述基因座控制区HS3包含如SEQ ID NO:6所示的序列，所述基因座控制区HS2包含如SEQ ID NO:7所示的序列；
    和/或，所述HBB基因3’sequence片段包含如SEQ ID NO:9所示的序列。
  5. 如权利要求1～4任一项所述的α-珠蛋白过表达载体，其特征在于，所述HBA2基因包含如SEQ ID NO:3所示的序列。
  6. 如权利要求1～5任一项所述的α-珠蛋白过表达载体，其特征在于，其质粒载体为Lenti载体；
    较佳地，所述Lenti载体包含如SEQ ID NO:10所示的序列。
  7. 一种包含如权利要求1～6任一项所述α-珠蛋白过表达载体的转化体；
    较佳地，所述转化体的宿主细胞为红系细胞；
    更佳地，所述转化体的宿主细胞为HUDEP-2或CD34 +造血干细胞。
  8. 一种包含如权利要求1～6任一项所述的α-珠蛋白过表达载体的慢病毒载体系统；
    较佳地，所述慢病毒载体系统还包含psPAX2和pCAG-VSVG质粒。
  9. 如权利要求1～6任一项所述的α-珠蛋白过表达载体在制备治疗地中海贫血的制剂中的应用；较佳地，所述地中海贫血为α-地中海贫血。
  10. 如权利要求1～3任一项所定义的HS40、α-珠蛋白启动子或HBA2基因3’sequence片段，如权利要求1或4所定义的HS4、HS3、HS2、β-珠蛋白启动子或HBB基因3’sequence片段在构建HBA2过表达载体中的应用；
    较佳地，所述载体为慢病毒载体。
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