Use of the hereditary persistence of fetal hemoglobin 2 enhancer to increase the expression of oncoretrovirus vectors for human gamma-globin

Abstract

The development of oncoretrovirus vectors for human g-globin has been hampered by problems of low expression and gene silencing. In order to address these problems, we investigated an enhancer element identified from individuals with deletional hereditary persistence of fetal hemoglobin 2 (HPFH2), a genetic condition characterized by elevated levels of g-globin in adults. Plasmid transfection studies in erythroid MEL (murine erythroleukemia) cells demonstrated the HPFH2 element could function synergistically with the b-globin locus control region to enhance the expression of an A g-globin gene with a truncated À382 bp promoter. A series of oncoretrovirus vectors were subsequently generated that contain an expression cassette for A g-globin linked to various combinations of the HPFH2 enhancer, the a-globin HS40 enhancer, and several versions of the promoter from A g-globin or b-globin. Expression analysis in transduced MEL cell clones revealed very high levels of promoterautonomous silencing that was at least partially abrogated by the HPFH2 enhancer. The vector containing a combination of a À201 bp A g-globin gene promoter with the Greek HPFH À117 point mutation and both the HPFH2 and HS40 enhancers exhibited no signs of vector silencing and was expressed at 248799% per copy of mouse a-globin (62% of total a-globin). This represents a significant improvement over previously reported oncoretrovirus vectors for A g-globin, and demonstrates the capacity of the HPFH2 enhancer to abrogate sequence-autonomous silencing of the A g-globin promoter in the context of a gene transfer vector.

The New Self-Inactivating Lentiviral Vector for Thalassemia Gene Therapy Combining Two HPFH Activating Elements Corrects Human Thalassemic Hematopoietic Stem Cells

“…that GGHI was able to express c-globin at 282.1 -491.2% per copy of mouse a-globin. These results corroborate our previous findings (Fragkos et al, 2005) using oncoretroviral vectors, where c-globin from vector A c117HS40-HPFH2 was expressed at 248 -99% per copy of mouse a-globin. Last, as shown in Table 1, there was a relatively good correlation between c-globin expression from GGHI and the viral copy number per cell, as clones with higher copy number tend to produce higher levels of cglobin.…”

“…We designed and tested this LCR-free lentiviral vector, termed GGHI, which expresses the human c-globin gene under the control of regulatory elements that promote the continuous expression of c-globin in the adult environment and therefore prevent silencing of the c-globin gene, namely the -117 HPFH mutation in the A c promoter (Collins et al, 1985) and the HPFH-2 enhancer (Anagnou et al, 1995;Katsantoni et al, 2003;Fragkos et al, 2005). In the present study, we show that this improved vector exhibits high-level transduction of bone marrow progenitor cells from 20 patients with transfusion-dependent thalassemia major and leads to phenotypic as well as functional correction of transduced cells in the model of human thalassemia erythropoiesis in vitro.…”

“…This vector also contains the full-length (1200-bp) cHS4 (chicken hypersensitive site-4) insulator in the deleted U3 region, the polypurine tract (PPT) and the woodchuck hepatitis posttranscriptional regulatory element (WPRE). To generate vector GGHI, the 1700-bp HS40-117Ac cassette containing the A c-globin gene under the control of the Ac promoter carrying the -117 activating HPFH mutation from vector Ac117HS40-HPFH2 (Fragkos et al, 2005) was isolated as a HindIII fragment and cloned in the HindIII site of plasmid pBluescript II SK( + ), creating plasmid pBlue/117/Ac. The cglobin gene in this setting has a 713-bp deletion in the second intron compared with the original genomic sequence.…”

“…Moreover, when the HPFH-2 enhancer was combined with the -117 activating mutation in the context of oncoretroviral vectors, it demonstrated high levels of c-globin mRNA/ mouse a-globin mRNA and virtually no silencing of the transgene in MEL-585 cells (Fragkos et al, 2005).…”

Towards the genetic treatment of -thalassemia: new disease models, new vectors, new cells

“…Investigators were forced to either seek novel transcriptional control elements or explore alternative vector systems. Limited success was achieved by swapping the ␣-locus HS-40 regulatory region for the β-LCR, [41][42][43] and alternative erythroid specific promoters, such as the ankyrin 44,45 and mutant HPFH ␥-globin promoters, 46,47 in place of the ␤-globin promoter.…”