Hemophilia B, or factor IX deficiency, is an X-linked recessive disorder occurring in about 1 in 25,000 males. Affected individuals are at risk for spontaneous bleeding into many organs; treatment mainly consists of the transfusion of clotting factor concentrates prepared from human blood or recombinant sources after bleeding has started. Small- and large-animal models have been developed and/or characterized that closely mimic the human disease state. As a preclinical model for gene therapy, recombinant adeno-associated viral vectors containing the human or canine factor IX cDNAs were infused into the livers of murine and canine models of hemophilia B, respectively. There was no associated toxicity with infusion in either animal model. Constitutive expression of factor IX was observed, which resulted in the correction of the bleeding disorder over a period of over 17 months in mice. Mice with a steady-state concentration of 25% of the normal human level of factor IX had normal coagulation. In hemophilic dogs, a dose of rAAV that was approximately 1/10 per body weight that given to mice resulted in 1% of normal canine factor IX levels, the absence of inhibitors, and a sustained partial correction of the coagulation defect for at least 8 months.

Recombinant murine retroviruses are widely used as delivery vectors for gene therapy. However, once integrated into a chromosome, these vectors often suffer from profound position effects, with vector silencing observed in vitro and in vivo. To overcome this problem, we investigated whether the HS4 chromatin insulator from the chicken ␤-globin locus control region could protect a retrovirus vector from position effects. When used to flank a reporter vector, this element significantly increased the fraction of transduced cells that expressed the provirus in cultures and in mice transplanted with transduced marrow. These results demonstrate that a chromatin insulator can improve the expression performance of a widely used class of gene therapy vectors by protecting these vectors from chromosomal position effects.M ost gene therapy strategies involving hematopoietic stem cells require both a high level of gene transfer and persistent transgene expression in specific target lineages. Recent advances in nonhuman primate models demonstrate that gene transfer rates of approximately 10% in reconstituting hematopoietic stem cells can be routinely achieved with virus vectors based on murine leukemia virus and related oncoretroviruses (1-4). However, achieving persistent, uniform gene expression from murine leukemia virus-based vectors has been problematic. Much research has focused on defining the elements of the virus long terminal repeat (LTR) that are responsible for provirus silencing in vivo (5-7), and identifying the most appropriate promoters and enhancers(8, 9). Expression of integrated provirus is also affected by chromatin structure. Because the bulk of the mammalian genome is packaged into transcriptionally silent heterochromatin (10), and murine leukemia virusbased vectors insert at random sites in the genome, a large portion of murine leukemia virus insertions result in gene silencing. This can lead to highly variable expression among clones, with complete silencing of provirus expression in a significant fraction of clones either immediately after insertion or following cell expansion. The progeny of a single clone containing a unique integration event can also be affected by the surrounding chromatin to varying degrees (10), a phenomenon known as position effect variegation. Position-dependent silencing and position effect variegation are particularly troublesome for retrovirus vectors containing the human ␤-or ␥-globin genes (8,9,11,12).The mammalian genome is organized into discrete chromosomal domains, in part through the use of sequences termed chromatin insulators (13). These elements, first described in Drosophila and more recently in several vertebrate species, help define the boundary between differentially regulated loci and serve to shield promoters from the inf luence of neighboring regulatory elements (14,15). Insulators function in a polar manner (e.g., they must be located between the cis effectors and promoter) and do not have stimulatory or inhibitory transcriptional effects on their own, di...

We have previously described the development of oncoretrovirus vectors for human ␥-globin using a truncated ␤-globin promoter, modified ␥-globin cassette, and ␣-globin enhancer. However, one of these vectors is genetically unstable, and both vectors exhibit variable expression patterns in cultured cells, common characteristics of oncoretrovirus vectors for globin genes. To address these problems, we identified and removed the vector sequences responsible for genetic instability and flanked the resultant vector with the chicken ␤-globin HS4 chromatin insulator to protect expression from chromosomal position effects. After determining that flanking with the cHS4 element allowed higher, more uniform levels of ␥-globin expression in MEL cell lines, we tested these vectors using a mouse bone marrow transduction and transplantation model. When present, the ␥-globin cassettes from the uninsulated vectors were expressed in only 2% to 5% of red blood cells (RBCs) long term, indicating they are highly sensitive to epigenetic silencing. In contrast, when present the ␥-globin cassette from the insulated vector was expressed in 49% ؎ 20% of RBCs long term. RNase protection analysis indicated that the insulated ␥-globin cassette was expressed at 23% ؎ 16% per copy of mouse ␣-globin in transduced RBCs. These results demonstrate that flanking a globin vector with the cHS4 insulator increases the likelihood of expression nearly 10-fold, which in turn allows for ␥-globin expression approaching the therapeutic range for sickle cell anemia and ␤ thalassemia. IntroductionThe ␤ chain hemoglobinopathies ␤ thalassemia and sickle cell anemia constitute the most common class of hereditary, monogenic disorders in the human population, affecting hundreds of thousands of persons worldwide. 1 In ␤ thalassemia, a lack of ␤-globin synthesis results in the precipitation of free ␣-globin chains and the subsequent destruction of erythroid precursors in the marrow. 1 In sickle cell anemia, a single amino acid substitution in the ␤-globin chain leads to globin chain polymerization, red cell sickling, and subsequent vascular occlusions and red cell destruction. 2 Recent therapeutic interventions include the use of cytotoxic drugs to induce the synthesis of fetal ␥-globin, which can bind up free ␣-globin chains in ␤-thalassemia 3,4 and can interfere with globin chain polymerization in sickle cell anemia. [5][6][7] However, these agents have proven ineffective for the treatment of severe transfusion-dependent ␤ thalassemia, and safety concerns remain about the lifelong administration of cytotoxic drugs in patients with sickle cell disease. Allogeneic bone marrow transplantation can cure patients with ␤ chain hemoglobinopathies. 1,8,9 However, this procedure is limited by the availability of HLA-identical donors and morbidity and mortality risks that increase as the clinical phenotype of these diseases worsens with age. For these reasons, we and others have pursued the development of gene therapy for the treatment of the ␤ chain hemoglobinopathies.The...