Evaluation of optimal expression cassette in retrovirus vector for beta-thalassemia gene therapy.
Trials of retroviral vector-mediated human beta-globin gene transfer were hampered by low titers, unstable vector transmission, and low-level expression of transferred gene. With the goal of optimizing the retrovirally encoded human beta-globin gene expression cassette for gene therapy of beta-thalassemia, we generated 3 series of vector constructs (a total of 12 constructs) and investigated the effects of the proximal promoter, 3' - enhancer, and derivatives from the beta-locus control region or alpha-major regulatory element on virus titer, vector transmission stability, and gene expression. The virus titers for 9 of the 12 vector constructs ranged between 2.8 x 10(4) cfu/mL and 1.0 x 10(6) cfu/mL. We found that proviral DNA was intact in most G418- resistant murine erythroleukemia (MEL) cell clones for 5 vector constructs, while obvious genetic instability was observed for 4 other vector constructs. MEL cells harboring the intact provirus were induced to differentiate, and human beta-globin gene expression was analyzed with RNase protection assay. The percentage of human beta-globin transcript relative to endogenous murine alpha-globin transcript were 101.8 +/- 64.3% (n = 10), 40.1 +/- 28.7% (n = 4), 31.1 +/- 31.9% (n = 12), 52.4 +/- 11.2% (n = 12), and 53.6 +/- 8.6% (n = 12) for the 5 constructs, respectively, demonstrating the development of optimized retroviral vectors for beta-globin gene therapy with murine erythroid cell lines as a model. Unexpectedly, we also documented that the point mutation 8700(C-->T) in DNase I hypersensitive site 2 (HS2) core fragment might contribute to low-level expression of the human beta-globin gene, based on a comparison of results from transfected and transduced MEL cells and sequence analysis of proviral DNA.
National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100005, People's Republic of China.
Wen-Ji
WJ
Dong
Bin
B
Li
De-Pei
DP
Liu
Zhen-Xiang
ZX
Zu
Jia
J
Li
De-Long
DL
Hao
Guang
G
Liu
Zhi-Chen
ZC
Guo
Chih-Chuan
CC
Liang
D000818:Animals; D001483:Base Sequence; D045744:Cell Line, Tumor; D041905:Erythroid Cells / Q000378:metabolism*; D015967:Gene Expression Regulation, Viral; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D005914:Globins / Q000096:biosynthesis* / Q000235:genetics*; D006801:Humans; D007668:Kidney / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D041681:NIH 3T3 Cells; D011786:Quality Control; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics*; D014162:Transfection / Q000379:methods; D017086:beta-Thalassemia / Q000235:genetics* / Q000378:metabolism* / Q000628:therapy
D011994:Recombinant Proteins; D005914:Globins