The Optimization of Self-deleting Lentiviral Vector Carrying Human β-globin Gene and Promoter

doi:10.13523/j.cb.20180707

China Biotechnology

2018, Vol. 38

Issue (7): 50-57 DOI: 10.13523/j.cb.20180707

The Optimization of Self-deleting Lentiviral Vector Carrying Human β-globin Gene and Promoter

Ya-li HAN¹,Guang-heng YANG^1,²,Yan-wen CHEN¹,Xiu-li GONG¹,Jing-zhi ZHANG^1,^2,^**(

)

1 Shanghai Institute of Medical Genetics, Children’s Hospital of Shanghai, Children’s Hospital Affiliated to Shanghai Jiao Tong University, Shanghai 20040, China;
2 Key Laboratory of Embryo Molecular Biology, Ministry of Health, Shanghai Key Laboratory of Embryo and Reproduction Engineering, Shanghai 20040, China

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Abstract

Objective: Optimization of previous developed self-deleting lentiviral vector carrying human β-globin gene and promoter in terms of preparation titles and transgene expressing efficacy. Methods: Based on the comparison of the predictions of three on line promoter prediction softwares; three self-deleting lentiviral vectors carrying three different lengths of β-globin promoters with gene were constructed. The optimization was carried out in terms of preparation titles and transgene expressing efficacy. The optimized LVV was used to transduce murine -thalassemia iPSC;and resulted cells were used to generate chimeric mice. RT-PCR and Wright Giemsa staining were then carried out on the mice blood. Results: The viral particles prepared by the optimized LVV principally have no difference comparing with their parental virus, FUGW. The functional expression of normal spliced human β-globin gene was detected in the chimeric mice. And the morphologically normal of erythrocytes was observed. Conclusion: An optimized self-deletion lentiviral vector, FCB-P265, was made.

Key words： β-thalassemia Self-deleted lentivirus vector Optimization Gene therapy

Received: 22 March 2018 Published: 13 August 2018

ZTFLH:

Q819

Corresponding Authors: Jing-zhi ZHANG E-mail: jzhang38@hotmail.com

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	Ya-li HAN
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	Jing-zhi ZHANG

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Ya-li HAN,Guang-heng YANG,Yan-wen CHEN,Xiu-li GONG,Jing-zhi ZHANG. The Optimization of Self-deleting Lentiviral Vector Carrying Human β-globin Gene and Promoter. China Biotechnology, 2018, 38(7): 50-57.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20180707 OR https://manu60.magtech.com.cn/biotech/Y2018/V38/I7/50

Fig.1 Schematic presentation the mechanism of the self-deleted lentiviral vector

Table 1 List of primers

Table 2 Different bioinformatic prediction of human β-globin promoter

Fig.2 Bioinformatic prediction of human β-globin promoter

Fig.3 Schematic presentations of three different lengths of -globin promoter driven -globin expressing lentiviral vector(a) FCB-P1 700 (b)FCB-P675 (c) FCB-P265 P: β-globin promoter ; E:Exon; IN:Intron ; ▽:With a 372bp deletion in IVS2

Fig.4 Schematic presentations of the three pro-LVV after self-deletion (a) pro-1 700 (b)pro-675 (c)pro-265 P: β-globin promoter;E:Exon;IN:Intron; ▽: With a 372bp deletion in IVS2

Fig.5 The comparison of the expression among the three pro-LVV vectors after transfectionn=3, P=0.000 01<0.05

Fig.6 The comparison of the viral particles produced by above pseudo virusesn=3, P>0.05

Fig.7 Murine β-thalassaemia iPSC was successfully transduced with the FCB-P265 N: Negative control; P: Positive control;Number 1 to 12 are the iPSC clones, with which number 10 is the positive clone transduced with the FCB-265

Fig.8 The expression of the exogenous gene in the blood of the chimeric model mouse and the morphology of its red blood cells (a) RT-PCR analysis the expression of the exogenous gene in the blood of the chimeric model mouse S: The blood sample of the Chimeric model mouse; P:Positive control,normal human peripheral blood; N: Negative control,the peripheral blood sample from the β-654 model mouse;WT:The peripheral blood sample from the wild-type mouse (b) Blood smear and Wright Giemsa staining for observing the morphology of the red blood cells of the transgenic model mouse C: The blood sample of the Chimeric model mouse; 654: The peripheral blood sample of the β-654 model mouse;WT:The peripheral blood sample of the wild-type mouse


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