Hemoglobin gene therapy for ß-thalassemia.

Allogeneic stem cell transplantation currently is the only curative option for severe ß-thalassemia and sickle cell disease. Human globin gene therapy with autotransplantation of transduced human hematopoietic stem cells is an exciting alternative approach to a potential cure. One patient with thalassemia has recently been reported to have clinical benefit after lentiviral human ß-globin gene therapy. He has not required blood transfusions for almost 2 years. Most of the patient's gene correction and new human ß-globin gene expression is caused by the expansion of a single clone in which the corrective transgene is inserted into an Hmga2 gene.

Transfusion independence and HMGA2 activation after gene therapy of human ß-thalassaemia.

The ß-haemoglobinopathies are the most prevalent inherited disorders worldwide. Gene therapy of ß-thalassaemia is particularly challenging given the requirement for massive haemoglobin production in a lineage-specific manner and the lack of selective advantage for corrected haematopoietic stem cells. Compound ß(E)/ß(0)-thalassaemia is the most common form of severe thalassaemia in southeast Asian countries and their diasporas. The ß(E)-globin allele bears a point mutation that causes alternative splicing. The abnormally spliced form is non-coding, whereas the correctly spliced messenger RNA expresses a mutated ß(E)-globin with partial instability. When this is compounded with a non-functional ß(0) allele, a profound decrease in ß-globin synthesis results, and approximately half of ß(E)/ß(0)-thalassaemia patients are transfusion-dependent. The only available curative therapy is allogeneic haematopoietic stem cell transplantation, although most patients do not have a human-leukocyte-antigen-matched, geno-identical donor, and those who do still risk rejection or graft-versus-host disease. Here we show that, 33 months after lentiviral ß-globin gene transfer, an adult patient with severe ß(E)/ß(0)-thalassaemia dependent on monthly transfusions since early childhood has become transfusion independent for the past 21 months. Blood haemoglobin is maintained between 9 and 10 g dl(-1), of which one-third contains vector-encoded ß-globin. Most of the therapeutic benefit results from a dominant, myeloid-biased cell clone, in which the integrated vector causes transcriptional activation of HMGA2 in erythroid cells with further increased expression of a truncated HMGA2 mRNA insensitive to degradation by let-7 microRNAs. The clonal dominance that accompanies therapeutic efficacy may be coincidental and stochastic or result from a hitherto benign cell expansion caused by dysregulation of the HMGA2 gene in stem/progenitor cells.

RD114 envelope proteins provide an effective and versatile approach to pseudotype lentiviral vectors.

Lentiviral vectors derived from the HIV-1 genome offer great promise for gene therapy due to their ability to transduce non-dividing cells and sustain long-term expression of transgenes. The majority of current lentiviral vectors are pseudotyped with the vesicular stomatitis viral envelope (VSV-G). VSV-G equips lentiviral vectors with a broad host cell tropism and increased stability. Increased particle stability enables viral supernatants to be concentrated by high-speed centrifugation to enhance their infectivity. Despite its efficacy, VSV-G is cytotoxic - a feature that prohibits the development of stable cell lines that constitutively express this envelope. Therefore, non-toxic envelope proteins are being investigated. RD114 is an attractive alternative because it also provides increased particle stability and its receptor is widely expressed on hematopoietic stem cells (HSCs). In this study, the packaging efficiency of three envelope proteins, RD114, RDpro and VSV-G, were evaluated with two lentiviral vectors (TRIP GFP and HPV-402). RDpro is an RD114-HIV chimera designed to pseudotype lentiviral vectors more efficiently. In transient systems, VSV-G generated titers of 10(8) and 10(7) viral particles/mL for TRIP GFP and HPV-402. RDpro possessed titers of 10(7) and 10(6), while RD114 titers were one log lower for each vector. Despite having relatively lower titers, RD114 proteins are less toxic; this was demonstrated in the extension of transient transfection reactions from 48 to 96 h. VSV-G transfections are generally limited to 48 h. In regard to gene therapy applications, we show that RDpro supernatants efficiently transduce peripheral blood HSCs. The versatility of RD114 envelopes was again demonstrated by using a 'mixed' expression system; composed of stably expressed RD114 envelope proteins to pseudotype lentiviral vectors generated in trans (titer range 10(3)-10(5)). Our data show that RD114 envelope proteins are effective and versatile constructs that could prove to be essential components of therapeutic lentiviral gene transfer systems.

On the road to gene therapy for beta-thalassemia and sickle cell anemia.

Human globin gene therapy is a potential cure for sickle cell disease and beta-thalassemia (Cooley anemia). A clinical trial of this treatment is currently under way in Paris using lentiglobin vectors.

AHSP: a novel hemoglobin helper.

Recently, the small protein alpha hemoglobin-stabilizing protein (AHSP) was identified and found to specifically bind alpha-globin, stabilize its structure, and limit the toxic effects of excess alpha-globin, which are manifest in the inherited blood disorder beta thalassemia. In this issue of the JCI, Yu, Weiss, and colleagues show that AHSP is also critical to the formation and stabilization of normal amounts of hemoglobin, even when alpha-globin is deficient, indicating unique and previously unidentified roles for this molecule.

Activating Notch1 mutations are an early event in T-cell malignancy of Ikaros point mutant Plastic/+ mice.

Ikaros and Notch1 genes are critical to T-cell differentiation through transcriptional activation of target genes and interaction with chromatin remodeling complexes. An Ikaros (Plastic) point mutation inhibits activity of normal Ikaros and Ikaros family members, and leads to T-cell lymphoma in heterozygotes (Plstc/+). Analysis revealed Notch1 activating mutations in 12 of 17 Plstc/+ lymphomas (70%), analogous to those in human T-ALL. Mice acquired Notch1 mutations in lymph nodes as early as 7 weeks. Thus, combined Notch1 and Ikaros dysfunction can be a significant early event in T-cell proliferation and tumorigenesis.

Regulation of human fetal hemoglobin: new players, new complexities.

The human globin genes are among the most extensively characterized in the human genome, yet the details of the molecular events regulating normal human hemoglobin switching and the potential reactivation of fetal hemoglobin in adult hematopoietic cells remain elusive. Recent discoveries demonstrate physical interactions between the beta locus control region and the downstream structural gamma- and beta-globin genes, and with transcription factors and chromatin remodeling complexes. These interactions all play roles in globin gene expression and globin switching at the human beta-globin locus. If the molecular events in hemoglobin switching were better understood and fetal hemoglobin could be more fully reactivated in adult cells, the insights obtained might lead to new approaches to the therapy of sickle cell disease and beta thalassemia by identifying specific new targets for molecular therapies.

Ikaros increases normal apoptosis in adult erythroid cells.

Ikaros is a critical transcriptional regulator of hematopoietic cell differentiation. In addition to its effects on the lymphoid system and hematopoietic stem-cell compartment, we have previously shown that Ikaros is also required for normal erythroid development. In this report, we compare Ikaros-dependent gene expression in erythroid cells of mice lacking the Ikaros protein with that of normal mice in purified adult bone-marrow erythroid cells (BMRC). Gene expression, measured by Affymetrix microarray analysis, indicates that in the BMRC of Ikaros-null mice, there is significant up-regulation of SMADs 6 and 7, serine protease inhibitor 3, and immediate-early protein 3 (IER3), all proteins that play a modulating role in apoptosis. We investigate the role of Ikaros in oxidative stress-induced apoptosis using Annexin-V staining and FACS analysis. We find a decrease in apoptosis in the BMRC of Ikaros-null mice compared to normal mice. This effect is also seen in nonerythroid cells but is stronger in BMRC. We conclude that normal Ikaros function increases normal apoptosis in erythroid cells. The data also suggest that Ikaros plays a role in apoptosis-mediated events in other normal hematopoietic cell lineages.

Role of intergenic human gamma-delta-globin sequences in human hemoglobin switching and reactivation of fetal hemoglobin in adult erythroid cells.

The details of the molecular events regulating normal human hemoglobin switching and reactivation of fetal hemoglobin in adult hematopoietic cells are unclear. The potential role of sequences between the human gamma- and delta-globin genes (intergenic gamma-delta sequences) in this process has been in question until the recent finding that two patients homozygous for the Corfu deletion, involving the loss of 7.2 kb of the intergenic gamma-delta region upstream of the delta gene, have 88% and 90% fetal hemoglobin, only mild anemia, and no transfusion requirements. These results provide the first strong evidence in humans that the gamma-delta intergenic sequences alone have a role in the reactivation of fetal hemoglobin in adult-type cells, and perhaps are involved in normal hemoglobin switching as well. The polypyrimidine (PYR) complex is a hematopoietic cell-specific and stage-specific chromatin remodeling complex that binds upstream of the human delta-globin gene within the Corfu deletion. Deletion of the PYR binding site has been shown to delay human gamma-to-beta globin switching. The PYR complex is present in adult human hematopoietic cells and absent in fetal-embryonic cells: properties of a globin-switching complex. Taken together, the data from patients with the Corfu deletion and the PYR complex results suggest that intergenic gamma-delta sequences are involved in human gamma-to-beta globin switching and reactivation of fetal hemoglobin in adult cells.

A phase I/II clinical trial of beta-globin gene therapy for beta-thalassemia.

Recent success in the long-term correction of mouse models of human beta-thalassemia and sickle cell anemia by lentiviral vectors and evidence of high gene transfer and expression in transduced human hematopoietic cells have led to a first clinical trial of gene therapy for the disease. A LentiGlobin vector containing a beta-globin gene (beta(A-T87Q)) that produces a hemoglobin (Hbbeta(A-T87Q)) that can be distinguished from normal hemoglobin will be used. The LentiGlobin vector is self-inactivating and contains large elements of the beta-globin locus control region as well as chromatin insulators and other features that should prevent untoward events. The study will be done in Paris with Eliane Gluckman as the principal investigator and Philippe Leboulch as scientific director.

Understanding globin regulation in beta-thalassemia: it's as simple as alpha, beta, gamma, delta.

A vast excess of alpha-globin production and inadequate gamma-globin compensation lead to the development of severe anemia in human beta-thalassemia. Newly identified modifiers of alpha- and gamma-globin synthesis and insights into the mechanisms of globin regulation provide the tools for potential new approaches to treating this and other red blood cell disorders. In the study by Han and colleagues in this issue of the JCI, the activity of a heme-regulated protein, HRI, is shown to modulate the accumulation of excess alpha-globin chains in murine beta-thalassemia and to decrease the severity of the disease.

Hematopoietic stem cell gene therapy: selecting only the best.

Hematopoietic stem cell (HSC) gene therapy can potentially cure a variety of human hematopoietic diseases, such as sickle cell disease. Selection and expansion of gene-corrected HSCs has now been accomplished for the first time using HSC from large animals - dogs and humans - with a novel drug-resistance gene, MGMT, which is not expressed in normal HSCs (see the related articles beginning on pages 1561 and 1581). Highly efficient lentiviral transfer and expression of MGMT into relatively few HSCs led to repopulation of most of the hematopoietic compartment with gene-corrected cells following suitable drug treatment. This selection system may be useful in human clinical trials to permit gene therapy in autologous and allogeneic bone marrow transplantation settings.

A stable murine-based RD114 retroviral packaging line efficiently transduces human hematopoietic cells.

Several barriers exist to high-efficiency transfer of therapeutic genes into human hematopoietic stem cells (HSCs) using complex oncoretroviral vectors. Human clinical trials to date have used Moloney leukemia virus-based amphotropic and gibbon ape leukemia virus-based envelopes in stable retroviral packaging lines. However, retroviruses pseudotyped with these envelopes have low titers due to the inability to concentrate viral supernatants efficiently by centrifugation without damaging the virus and low transduction efficiencies because of low-level expression of viral target receptors on human HSC. The RD114 envelope from the feline endogenous virus has been shown to transduce human CD34+ cells using transient packaging systems and to be concentrated to high titers by centrifugation. Stable packaging systems have potential advantages over transient systems because greater and more reproducible viral productions can be attained. We have, therefore, constructed and tested a stable RD114-expressing packaging line capable of high-level transduction of human CD34+ cells. Viral particles from this cell line were concentrated up to 100-fold (up to 10(7) viral particles/ml) by ultracentrifugation. Human hematopoietic progenitors from cord blood and sickle cell CD34+ cells were efficiently transduced with a Neo(R)-containing vector after a single exposure to concentrated RD114-pseudotyped virus produced from this cell line. Up to 78% of progenitors from transduced cord blood CD34+ cells and 51% of progenitors from sickle cell CD34+ cells expressed the NeoR gene. We also show transfer of a human beta-globin gene into progenitor cells from CD34+ cells from sickle cell patients with this new RD114 stable packaging system. The results indicate that this packaging line may eventually be useful in human clinical trials of globin gene therapy.

Correction of phenotype in a thalassemia mouse model using a nonmyeloablative marrow transplantation regimen.

Gene therapy, the replacement of normal human beta- or gamma-globin genes into the hematopoietic stem cells of patients with homozygous beta-thalassemia, is a promising therapy for the future. High-level lineage-specific stable globin expression in transduced cells reinfused into patients in an autologous transplantation setting could be curative, if successful. Previous studies have shown high-level donor chimerism in nonmyeloablated non-thalassemic hosts. We have now studied the conditions for stable long-term engraftment of normal cells into a thalassemia mouse model that lead to high-level donor chimerism and correction of the abnormal phenotype. Thalassemic female mice treated with 0 to 300 cGy whole-body irradiation received transplantations of donor cells harvested from wild-type males. Engraftment of male cells was quantitated by Y-chromosome polymerase chain reaction analysis of blood and marrow progenitors, and changes in hemoglobin levels, red cell morphology, and spleen size were measured at various times posttransplantation. High-level stable donor cell engraftment was achieved in mice given 200 cGy and receiving transplants of 2 x 10(7) or more donor cells. The anemia, abnormal peripheral blood smears, and splenomegaly improved in the thalassemic mice that had successful engraftment. These studies demonstrate that stable and successful levels of engraftment of normal cells can correct the thalassemic phenotype without fully myeloablating the host. This animal model should allow us to test the amount of cytoreduction required and the level of engraftment and beta-globin expression needed in autologous transplantation of beta-globin gene-transduced cells to correct the abnormal phenotype in thalassemic mice, and it may be relevant to human clinical trials, as well.

Multiple hematopoietic defects and delayed globin switching in Ikaros null mice.

We have previously reported the structure of a chromatin remodeling complex (PYR complex) with Ikaros as its DNA binding subunit that is specifically present in adult murine and human hematopoietic cells. We now show that homozygous Ikaros "knockout" (null) mice lack the PYR complex, demonstrating the requirement for Ikaros in the formation of the complex on DNA. Heterozygous Ikaros null mice have about half as much PYR complex, indicating a dosage effect for both Ikaros and PYR complex. We also show that Ikaros null mice have multiple hematopoietic cell defects including anemia and megakaryocytic abnormalities, in addition to previously reported lymphoid and stem cell defects. The null mice also have a delay in murine embryonic to adult beta-globin switching and a delay in human gamma to beta switching, consistent with a previously suggested role for PYR complex in this process. Lastly, cDNA array analyses indicate that several hematopoietic cell-specific genes in all blood lineages are either up- or down-regulated in 14-day embryos from Ikaros null as compared with wild-type mice. These results indicate that Ikaros and PYR complex function together in vivo at many adult hematopoietic cell-specific genes and at intergenic sites, affecting their expression and leading to pleiotropic hematopoietic defects.