利用CD4迷你启动子/增强子的Mo-MLV逆转录病毒载体实现T细胞特异性表达

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"这篇学术论文探讨了T细胞特异性表达的研究，主要集中在含有CD4迷你启动子/增强子的Mo-MLV逆转录病毒载体上。由Jing Chao Zhao-Emonet、Gilles Marodon、Catherine Pioche-Durieu、François Loic Cosset和David Klatzmann等人共同完成，发表于与免疫病理学相关的实验室。文章介绍了如何通过改进的逆转录病毒载体来实现对T细胞的特异性基因治疗，这对于各种免疫系统疾病的基因疗法具有重要意义。文章首先阐述了背景，指出在治疗多种免疫学疾病时，具有T细胞特异性的逆转录病毒载体的重要性。传统的逆转录病毒载体通常会在多种细胞类型中表达，而研究人员的目标是设计出仅在T细胞中活跃的载体，以提高基因治疗的效率和安全性。接着，文章详细描述了他们构建的CD4迷你启动子/增强子元件。启动子是基因表达的起始点，而增强子则可以增加基因的转录活性。CD4蛋白是T细胞的标志物，因此将CD4相关序列整合到载体中，可以预期它将在T细胞中更有效地驱动基因表达。实验部分可能涉及了构建这些载体的过程，以及在不同细胞类型（包括T细胞）中的功能验证。可能包括了载体构建的技术细节，如克隆技术、分子生物学实验以及细胞培养方法。此外，作者可能通过实验展示了这些载体在非T细胞中的低表达或无表达，进一步证明了其T细胞特异性。在结果和讨论部分，研究团队可能报告了实验数据，分析了CD4迷你启动子/增强子如何影响载体在T细胞内的表达效率，以及可能存在的限制和潜在的应用前景。这可能涉及到对T细胞激活状态、病毒感染效率以及长期基因表达稳定性的影响。最后，论文总结了这项工作的创新点和对未来基因治疗策略的贡献，同时指出了未来研究的方向，可能包括优化载体设计以提高特异性，减少非靶向细胞的副作用，以及在临床前模型和潜在临床应用中的进一步验证。这篇研究论文为T细胞特异性基因疗法提供了一种新的工具，对于理解基因调控和开发更精确的遗传干预策略具有深远影响。"

RESEARCH ARTICLE

T Cell-speci®c expression from Mo-MLV retroviral

vectors containing a CD4 mini-promoter/enhancer

Jing Chao Zhao-Emonet

Gilles Marodon

Catherine Pioche-Durieu

FrancËois Loic Cosset

David Klatzmann

Laboratoire de Biologie et

The

rapeutique des Pathologies

Immunitaires UPMC-CNRS ESA

7087-CERVI-Ho

pital de la Pitie

83 Bd de l'Ho

pital,

75651 Paris Cedex 13, France

Laboratoire de Vectorologie

trovirale et The

rapie ge

nique,

Unite

de Virologie Humaine INSERM

U412, Ecole Normale Supe

rieure de

Lyon, 46 alle

e d'Italie, 69364 Lyon

Cedex 07, France

*Correspondence to: D. Klatzmann,

Laboratoire de Biologie et

The

rapeutique des Pathologies

Immunitaires UPMC-CNRS ESA

7087-CERVI-Ho

pital de la Pitie

83 Bd de l'Ho

pital,

75651 Paris Cedex 13, France.

E-mail: david.klatzmann@

psl.ap-hop-paris.fr

Received: 27 April 2000

Revised: 25 July 2000

Accepted: 8 August 2000

Published online: 15 September 2000

Abstract

Background Gene therapy of various immunological disorders will greatly

bene®t from improved retroviral vectors (RVs) with T cell speci®city. Such

vectors can be designed by placing a gene of therapeutic interest under the

control of tissue-speci®c transcriptional elements. However, low titers and

loss of speci®city are frequently encountered with tissue-speci®c vectors. The

aim of the present study was to develop a T cell-speci®c RV.

Methods We constructed a series of Moloney murine leukemia virus

(Mo-MLV)-based RVs expressing enhanced green ¯uorescent protein (EGFP)

under the control of a mini-promoter/enhancer cassette derived from the CD4

gene (CD4pmE) and tested them in cell lines and peripheral blood

lymphocytes. Expression of EGFP was monitored by ¯uorescence microscopy

and analyzed by ¯ow cytometry.

Results The CD4pmE cassette was inserted between the viral long terminal

repeats (LTRs) in self-inactivating vectors (SIN vectors) or was substituted to

the 3k U3 viral promoter/enhancer (hybrid vectors). High vector titers but

poor speci®c expression of EGFP were achieved when CD4pmE was inserted

in sense orientation in SIN vectors. Low titers but high speci®city were

observed when the CD4pmE cassette was in anti-sense orientation. In

contrast, high titers and good T cell speci®city were obtained with hybrid

vectors.

Conclusion An ef®cient T cell-speci®c retroviral vector was obtained.

Keywords retroviral vector (RV); tissue speci®city; CD4 promoter; EGFP;

gene therapy

Introduction

Retroviral vectors (RVs) derived from Moloney murine leukemia virus (Mo-

MLV) represent attractive tools for the treatment of genetic or acquired

immunode®ciency diseases affecting T cell functions, such as the adenosine

deaminase (ADA) de®ciency [1,2] or the X-linked severe combined

immunode®ciencies [3]. To reduce the potential risks or problems associated

with the presence of strong cis-regulatory elements in the retroviral long

terminal repeat (LTR) such as malignant transformation of transduced cells,

vector dissemination or loss of transgene expression in vivo [4±6], numerous

investigators have tried to obtain tissue-speci®c RVs. Several groups have

described RVs containing cellular promoter/enhancer elements that allow

tissue-speci®c expression of reporter genes in target cell lines [7±10].

However, low titers and full or partial loss of tissue speci®city from the

THE JOURNAL OF GENE MEDICINE

J Gene Med 2000; 2: 416±425.

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