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.
Dong
Wen-Ji
WJ
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.
Li
Bin
B
Liu
De-Pei
DP
Zu
Zhen-Xiang
ZX
Li
Jia
J
Hao
De-Long
DL
Liu
Guang
G
Guo
Zhi-Chen
ZC
Liang
Chih-Chuan
CC
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