Mutagenesis of retroviral vectors transducing human beta-globin gene and beta-globin locus control region derivatives results in stable transmission of an active transcriptional structure.

Abstract

Retrovirus‐mediated gene transfer of the human beta‐globin gene into hematopoietic stem cells is an attractive approach to the therapy of human beta‐globin gene disorders. However, expression of the transduced beta‐globin gene linked to its proximal cis‐acting sequences (‐0.8 to +0.3 kb from the cap site) is considerably below the level required for a significant therapeutic effect. The discovery of the beta‐locus control region (beta‐LCR), organized in four major DNase I hypersensitive sites far upstream of the human beta‐like globin gene cluster, provided a potential means to achieve a high level of expression of a linked human beta‐globin gene, but initial attempts to incorporate beta‐LCR derivatives in retroviral vectors resulted in the production of low‐titer viruses with multiple rearrangements of the transmitted proviral structures. We now describe how extensive mutagenesis of the transduced beta‐globin gene, eliminating a 372 bp intronic segment and multiple reverse polyadenylation and splicing signals, increases viral titer significantly and restores stability of proviral transmission upon infection of cell lines and bone marrow‐repopulating cells. These optimized vectors have enabled us to analyze the expression properties of various retrovirally transduced beta‐LCR derivatives in dimethylsulfoxide‐induced murine erythroleukemia cells and to achieve ratios of human beta‐globin/murine beta maj‐globin mRNA, on a per gene basis, as high as 80%.

Physiological and tissue-specific vectors for treatment of inherited diseases

“…First attempts to achieve erythroid-specific expression of the b-globin gene involved the incorporation of the b-globin LCR and 3¢ enhancers in murine oncoretroviral vectors. [98][99][100][101][102] However, the presence of large regulatory sequences in oncoretroviral vectors was limited due to its limited packaging capacity (8 kb) and to the presence of RNA sequences that favor RNA processing and therefore block the production of full-length vector-RNA molecules in producer cell lines. The first two encouraging examples of lineage-specific expression of the b-globin transgene and therapeutic efficacy were provided by May et al 103 for b-thalassemia and by Pawliuk et al 104 for sickle cell disease using HIV-1-based LVs.…”

LCR-regulated transgene expression levels depend on the Oct-1 site in the AT-rich region of β-globin intron-2

“…31,32 The fragment was cloned into the BamHI site of Litmus 38 plasmid vector, using BamHI-linkers to produce the ATR construct. The footprint probe was produced using a modified protocol previously described 27 using restriction enzymes in the flanking polylinkers to radiolabel single DNA strands with 32 P(␣)dCTP (Amersham Pharmacia, Baie d'Urfe, Quebec, Canada) using Klenow fragment (Roche, Laval, Quebec, Canada).…”

“…The 521 BamHIDraI fragment containing the 372-bp ATR 19,31,32 was cloned into the Litmus 38 vector. Radiolabeled sense and antisense ATR fragments were incubated with mouse erythroleukemia (MEL) cell nuclear extract, briefly digested by DNaseI, and separated adjacent to a sequence ladder.…”

“…Hence, deletion of the ATR in globin retrovirus vectors improves virus titer 31,32 but compromises mean expression levels. 19 Our analysis of RNA extracted from transient transgenic mice precludes single-cell analysis to study possible variegation effects.…”

“…Although it is evident that high expression at all ectopic sites requires the ATR, it has proved difficult to incorporate the ATR in retrovirus vectors because it reduces titer by introducing premature polyadenylation signals, cryptic splice sites, and deletions into viral genomic RNA. 31,32 The function of the ATR may be to bind transcription factors that cooperate with LCR elements. Jackson et al have reported several transcription factor binding sites dispersed through the ␤-globin intron-2.…”