One problem limiting the development of long-term gene replacement therapy is gene silencing. A variety of experiments have implicated DNA methylation and histone deacetylation in gene silencing and shown that the agents 5-azacytidine (5-Aza) and trichostatin A (TSA) are able to reverse these effects. To begin to investigate clinically relevant strategies to reverse silencing with these drugs, we transduced the MEL and FDCP-1 hematopoietic cell lines with Moloney murine leukemia virus (MMLV) and Harvey murine sarcoma virus (HMSV)-based retroviral vectors carrying the ␤-galactosidase/neomycin resistance fusion gene (␤-geo). Fifty-one clones were isolated under G418 selection over 2 weeks and then allowed to grow without selection as

Hormone-independent human breast cancer is characterized by estrogen receptor (ER) loss and the acquisition of high epidermal growth factor receptor (EGFR) levels. Despite the tendency for an inverse correlation between EGFR and ER, EGFR is a strong prognostic indicator for poor survival rate independent of ER status suggesting that EGFR overexpression is an important step in the progression to estrogen independence. We have previously shown that several DNase I hypersensitive sites which correspond to potential regulatory regions reside within the EGFR gene first intron exclusively in hormone-independent breast cancer cells. CAT assays investigating the transcriptional activity of the first intron of EGFR indicate that a 140 bp region has an enhancer ability specifically in these hormone-independent breast cancer cells. The DNA-protein interaction that occurs in this enhancer was localized to a 35 bp region and displayed enhancer-like activity in the same hormone-independent breast cancer cells. Furthermore, the protein that binds to this 35 bp region seems to be ubiquitous in the cell lines tested but in higher abundance in high EGFR expressing cells. Identifying the specific regulatory elements involved in EGFR up-regulation could lead to the development of therapies for preventing and treating estrogen-independent breast cancer.

The active elements of the ␤-globin locus control region (LCR) are located within domains of unique chromatin structure. These nuclease hypersensitive sites (HSs) are characterized by high DNase I sensitivity, erythroid specificity, similar nucleosomal structure, and evolutionarily conserved clusters of cis-acting elements that are required for the formation and function of the core elements. To determine the requirements for HS core formation in the setting of nuclear chromatin, we constructed a series of artificial HS cores containing binding sites for GATA-1, NF-E2, and Sp1. In contrast to the results of previous in vitro experiments, we found that when constructs were stably integrated in mouse erythroleukemia cells the binding sites for NF-E2, GATA-1, or Sp1 alone or in any combination were unable to form core HS structures. We subsequently identified two new cis-acting elements from the LCR HS4 core that, when combined with the NF-E2, Sp1, and tandem inverted GATA elements, result in core structure formation. Both new cis-acting elements bind Sp1, and one binds erythroid Kruppel-like factor (EKLF). We conclude that in vivo ␤-globin LCR HS core formation is more complex than previously thought and that several factors are required for this process to occur.

The cloning of the human β-globin genes more than 20 years ago led to predictions that β-thalassemia and sickle cell disease would be among the first monogenic diseases to be successfully treated by gene replacement therapy. However, despite the worldwide enrollment of more than 3,000 patients in approved gene transfer protocols, none have involved therapy for these diseases. This has been due to several technical hurdles that need to be overcome before gene replacement therapy for β-thalassemia and sickle cell disease can become practical. These problems include inefficient transduction of hematopoietic stem cells and an inability to achieve consistent, long-term, high-level expression of transferred β-like globin genes with current gene transfer vectors. In this review we highlight the relationships between understanding the fundamental mechanisms of β-globin gene locus function and basic vector biology and the development of strategies for β-globin gene replacement therapy. Despite slow initial progress in this field, recent advances in a variety of critical areas provide hope that clinical trials may not be far away.

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