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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...

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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...

after the second round of selection and reached levels above 80% after the third round. The percentage of γ-globin-expressing cells was approximately 7-to 10-fold higher in erythroid Ter119 + cells versus nonerythroid Ter119cells in peripheral blood and bone marrow (Figure 1D). We used HPLC to measure the level of γ-globin protein in comparison with the adult mouse α-and β-globin chains (Figure 1E and Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/ JCI122836DS1). At week 18, these levels reached 10%-15% of adult mouse α-globin and β-major globin and approximately 25% of mouse β-minor globin. This was confirmed on the mRNA level by quantitative reverse transcription PCR (RT-qPCR), where human γ-globin mRNA was approximately 13% of mouse β-major mRNA (Figure 1F). To further demonstrate that primitive, long-term repopulating HSCs were transduced, we transplanted lineagedepleted (Lin-) bone marrow cells from in vivo-transduced/ selected mice into irradiated C57BL/6 mice. Engraftment levels analyzed in peripheral blood, bone marrow, and spleen were greater than 95% and stable over an observation period of 20 weeks (Supplemental Figure 2, A and B). Human γ-globin levels (compared with mouse α-globin) were similar in ("primary") in vivo-transduced mice (analyzed at week 18 after transduction) and secondary recipients analyzed at weeks 14 and 20 after transplantation (Supplemental Figure 2C). The in vivo HSPC transduction/selection approach does not change the SB100X-mediated random transgene integration pattern and does not alter hematopoiesis. We previously showed that in vivo transduction with the hybrid transposon/SB100X HDAd5/35++ system resulted in random transgene integration in HSPCs (6). To evaluate the effect of O 6 BG/BCNU in in vivo selection, we analyzed transgene integration in bone marrow Lincells at the end of the study, i.e., at week 20 in secondary recipients. Linear amplification-mediated PCR (LAM-PCR) followed by deep sequencing showed a random distribution pattern of integration sites in the mouse genome (Figure 2A). Data pooled from 5 mice demonstrated 2.23% integration into exons, 31.58% into introns, 65.17% into intergenic regions, and 1.04% into untranslated regions (Figure 2B). The level of randomness of integration was 99% without preferential integration in any given window of the whole mouse genome (Figure 2C). This indicates that in vivo selection and further expansion of cells in secondary recipients did not result in the emergence of dominant integration sites (Figure 2D). We measured, by qPCR, on average two γ-globin cDNA copies per bone marrow cell in a population containing both transduced and nontransduced cells. We then quantified the integrated transgene copy number on a single-cell level. To do this, we plated bone marrow Lincells from week 18 mice in methylcellulose, isolated individual progenitor colonies, and performed qPCR on genomic DNA. In transgene-positive colonies (n = 113), 86.7% of colonies had 2 or 3 integrated co...

MSC lose their immunomodulatory properties when infused in the inflammatory micromilieu of autoimmune arthritis. Conditioning of the recipient with bortezomib alters the disease microenvironment enabling MSC to modulate arthritis. Should milieu limitations also operate in human disease, this approach could serve as a strategy to treat RA by MSC.

The safety and efficacy of hematopoietic stem cell (HSC) mobilization was investigated in adult splenectomized (SPL) and non-SPL patients with thalassemia major, in two clinical trials, using different mobilization modes: granulocyte-colony-stimulating factor (G-CSF)-alone, G-CSF following pretreatment with hydroxyurea (HU), plerixafor-alone. G-CSF-mobilization was both safe and effective in non-SPL patients. However, in SPL patients the procedure resulted in excessive response to G-CSF, expressed as early hyperleukocytosis necessitating significant dose reduction, and suboptimal CD34(+) cells yields. One-month HU-pretreatment prevented hyperleukocytosis and allowed successful CD34(+) cell collections when an optimal washout period was maintained, but it significantly prolonged the mobilization procedure. Plerixafor resulted in rapid and effective mobilization in both SPL and non-SPL patients and was well-tolerated. For gene therapy of thalassemia, G-CSF or Plerixafor could be used as mobilization agents in non-SPL patients whereas Plerixafor appears to be the mobilization agent of choice in SPL adult thalassemics in terms of safety and efficacy.

In the present report, we carried out clinical-scale editing in adult mobilized CD34+ hematopoietic stem and progenitor cells (HSPCs) using zinc-finger nuclease-mediated disruption of BCL11a to upregulate the expression of γ-globin (fetal hemoglobin). In these cells, disruption of the erythroid-specific enhancer of the BCL11A gene increased endogenous γ-globin expression to levels that reached or exceeded those observed following knockout of the BCL11A coding region without negatively affecting survival or in vivo long-term proliferation of edited HSPCs and other lineages. In addition, BCL11A enhancer modification in mobilized CD34+ cells from patients with β-thalassemia major resulted in a readily detectable γ-globin increase with a preferential increase in G-gamma, leading to an improved phenotype and, likely, a survival advantage for maturing erythroid cells after editing. Furthermore, we documented that both normal and β-thalassemia HSPCs not only can be efficiently expanded ex vivo after editing but can also be successfully edited post-expansion, resulting in enhanced early in vivo engraftment compared with unexpanded cells. Overall, this work highlights a novel and effective treatment strategy for correcting the β-thalassemia phenotype by genome editing.