Lentiviral Transfer of γ-Globin with Fusion Gene NUP98-HOXA10HD Expands Hematopoietic Stem Cells and Ameliorates Murine ß-Thalassemia.

Recently, an engineered Homeobox-nucleoporin fusion gene, NUP98-HOXA10HD or NA10HD, was reported to expand and maintain murine hematopoietic stem cells (HSCs). We postulated that NA10HD would increase the number of human γ-globin-expressing cells to therapeutic levels. We developed a double gene lentiviral vector encoding both human γ-globin and NA10HD, which was used to transduce human peripheral blood CD34+ cells and increased engraftment 2- to 2.5-fold at 15 weeks post-transplantation in immunodeficient mice. In ß-thalassemic mice transplanted with ß-thalassemic HSCs transduced with the γ-globin/NA10HD vector, the number of fetal hemoglobin (HbF)-expressing cells was significantly increased after 3 months, leading to resolution of the anemia. Furthermore, the increases in HbF were maintained at 6 months and persisted after secondary transplantation. In addition, NA10HD enrichment of transduced HSCs led to HbF increases without affecting homeostasis of the white blood cell lineages. Our results suggest that NA10HD increases the number of γ-globin-transduced HSCs that engraft, leading to an elevated number of fetal hemoglobin-containing red cells. These effects of NA10HD provide an improved platform for testing of the therapeutic efficacy of novel globin vectors and provide further impetus to develop safe and effective methods for selective expansion of genetically modified cells.

Fas-antisense long noncoding RNA is differentially expressed during maturation of human erythrocytes and confers resistance to Fas-mediated cell death.

Long noncoding RNAs (lncRNAs) interact with other RNAs, DNA and/or proteins to regulate gene expression during development. Erythropoiesis is one developmental process that is tightly controlled throughout life to ensure accurate red blood cell production and oxygen transport to tissues. Thus, homeostasis is critical and maintained by competitive outcomes of pro- and anti-apoptotic pathways. LncRNAs are expressed during blood development; however, specific functions are largely undefined. Here, a culture model of human erythropoiesis revealed that lncRNA Fas-antisense 1 (Fas-AS1 or Saf) was induced during differentiation through the activity of essential erythroid transcription factors GATA-1 and KLF1. Saf was also negatively regulated by NF-κB, where decreasing NF-κB activity levels tracked with increasing transcription of Saf. Furthermore, Saf over-expression in erythroblasts derived from CD34(+) hematopoietic stem/progenitor cells of healthy donors reduced surface levels of Fas and conferred protection against Fas-mediated cell death signals. These studies reveal a novel lncRNA-regulated mechanism that modulates a critical cell death program during human erythropoiesis.

Nfix is a novel regulator of murine hematopoietic stem and progenitor cell survival.

Hematopoietic stem cells are both necessary and sufficient to sustain the complete blood system of vertebrates. Here we show that Nfix, a member of the nuclear factor I (Nfi) family of transcription factors, is highly expressed by hematopoietic stem and progenitor cells (HSPCs) of murine adult bone marrow. Although short hairpin RNA-mediated knockdown of Nfix expression in Lineage(-)Sca-1(+)c-Kit(+) HSPCs had no effect on in vitro cell growth or viability, Nfix-depleted HSPCs displayed a significant loss of colony-forming potential, as well as short- and long-term in vivo hematopoietic repopulating activity. Analysis of recipient mice at 4 to 20 days posttransplant revealed that Nfix-depleted HSPCs are established in the bone marrow, but fail to persist due to increased apoptotic cell death. Gene expression profiling of Nfix-depleted HSPCs reveals that loss of Nfix expression in HSPCs is concomitant with a decrease in the expression of multiple genes known to be important for HSPCs survival, such as Erg, Mecom, and Mpl. These data reveal that Nfix is a novel regulator of HSPCs survival posttransplantation and establish a role for Nfi genes in the regulation of this cellular compartment.

Hydroxyurea therapy of a murine model of sickle cell anemia inhibits the progression of pneumococcal disease by down-modulating E-selectin.

Sickle cell anemia is characterized by chronic hemolysis coupled with extensive vascular inflammation. This inflammatory state also mechanistically promotes a high risk of lethal, invasive pneumococcal infection. Current treatments to reduce vaso-occlusive complications include chronic hydroxyurea therapy to induce fetal hemoglobin. Because hydroxyurea also reduces leukocytosis, an understanding of the impact of this treatment on pneumococcal pathogenesis is needed. Using a sickle cell mouse model of pneumococcal pneumonia and sepsis, administration of hydroxyurea was found to significantly improve survival. Hydroxyurea treatment decreased neutrophil extravasation into the infected lung coincident with significantly reduced levels of E-selectin in serum and on pulmonary epithelia. The protective effect of hydroxyurea was abrogated in mice deficient in E-selectin. The decrease in E-selectin levels was also evident in human sickle cell patients receiving hydroxyurea therapy. These data indicate that in addition to induction of fetal hemoglobin, hydroxyurea attenuates leukocyte-endothelial interactions in sickle cell anemia, resulting in protection against lethal pneumococcal sepsis.

Transcriptional regulation of fetal to adult hemoglobin switching: new therapeutic opportunities.

In humans, embryonic, fetal, and adult hemoglobins are sequentially expressed in developing erythroblasts during ontogeny. For the past 40 years, this process has been the subject of intensive study because of its value to enlighten the biology of developmental gene regulation and because fetal hemoglobin can significantly ameliorate the clinical manifestations of both sickle cell disease and ß-thalassemia. Understanding the normal process of loss of fetal globin expression and activation of adult globin expression could potentially lead to new therapeutic approaches for these hemoglobin disorders. Herein, we briefly review the history of the study of hemoglobin switching and then focus on recent discoveries in the field that now make new therapeutic approaches seem feasible in the future. Erythroid-specific knockdown of fetal gene repressors or enforced expression of fetal gene activators may provide clinically applicable approaches for genetic treatment of hemoglobin disorders that would benefit from increased fetal hemoglobin levels.

Therapeutic levels of fetal hemoglobin in erythroid progeny of ß-thalassemic CD34+ cells after lentiviral vector-mediated gene transfer.

ß-Thalassemia major results from severely reduced or absent expression of the ß-chain of adult hemoglobin (α2ß2;HbA). Increased levels of fetal hemoglobin (α2γ2;HbF), such as occurs with hereditary persistence of HbF, ameliorate the severity of ß-thalassemia, raising the potential for genetic therapy directed at enhancing HbF. We used an in vitro model of human erythropoiesis to assay for enhanced production of HbF after gene delivery into CD34(+) cells obtained from mobilized peripheral blood of normal adults or steady-state bone marrow from patients with ß-thalassemia major. Lentiviral vectors encoding (1) a human γ-globin gene with or without an insulator, (2) a synthetic zinc-finger transcription factor designed to interact with the γ-globin gene promoters, or (3) a short-hairpin RNA targeting the γ-globin gene repressor, BCL11A, were tested. Erythroid progeny of normal CD34(+) cells demonstrated levels of HbF up to 21% per vector copy. For ß-thalassemic CD34(+) cells, similar gene transfer efficiencies achieved HbF production ranging from 45% to 60%, resulting in up to a 3-fold increase in the total cellular Hb content. These observations suggest that both lentiviral-mediated γ-globin gene addition and genetic reactivation of endogenous γ-globin genes have potential to provide therapeutic HbF levels to patients with ß-globin deficiency.

Cell Membrane-associated heparan sulfate is a receptor for prototype foamy virus in human, monkey, and rodent cells.

Foamy viruses (FVs) (spumaretroviruses) are good alternative to retroviruses as gene therapy vector. Despite four decades since the discovery of FV, its receptor molecule is still unknown. FV vector transduction of human CD34(+) cells was inhibited by culture with fibronectin. Because fibronectin contains heparin-binding domain, the interactions of fibronectin with heparan sulfate (HS) on cells might be inhibitory to FV transduction. These observations led us to investigate whether HS is a receptor for FV. Two mutant CHO cell lines (but not parental wild type) lacking cell surface HS but not chondroitin sulfate (CS) were largely resistant to FV attachment and transduction. Inhibition of HS expression using enzymes or chemicals greatly reduced FV transduction in human, monkey, and rodent cells. Raji cells, which lack HS and were largely resistant to FV, were rendered more permissive through ectopic expression of syndecan-1, which contains HS. In contrast, mutant syndecan-1-expressing cells were largely resistant to FV. Our findings indicate that cellular HS is a receptor for FV. Identifying FV receptor will enable better understanding of its entry process and optimal use as gene therapy vector to treat inherited and pathogenic diseases.

High-efficiency transduction of rhesus hematopoietic repopulating cells by a modified HIV1-based lentiviral vector.

Human immunodeficiency virus type 1 (HIV1) vectors poorly transduce rhesus hematopoietic cells due to species-specific restriction factors, including the tripartite motif-containing 5 isoformα (TRIM5α) which targets the HIV1 capsid. We previously developed a chimeric HIV1 (χHIV) vector system wherein the vector genome is packaged with the simian immunodeficiency virus (SIV) capsid for efficient transduction of both rhesus and human CD34(+) cells. To evaluate whether χHIV vectors could efficiently transduce rhesus hematopoietic repopulating cells, we performed a competitive repopulation assay in rhesus macaques, in which half of the CD34(+) cells were transduced with standard SIV vectors and the other half with χHIV vectors. As compared with SIV vectors, χHIV vectors achieved higher vector integration, and the transgene expression rates were two- to threefold higher in granulocytes and red blood cells and equivalent in lymphocytes and platelets for 2 years. A recipient of χHIV vector-only transduced cells reached up to 40% of transgene expression rates in granulocytes and lymphocytes and 20% in red blood cells. Similar to HIV1 and SIV vectors, χHIV vector frequently integrated into gene regions, especially into introns. In summary, our χHIV vector demonstrated efficient transduction for rhesus long-term repopulating cells, comparable with SIV vectors. This χHIV vector should allow preclinical testing of HIV1-based therapeutic vectors in large animal models.

A zinc-finger transcriptional activator designed to interact with the gamma-globin gene promoters enhances fetal hemoglobin production in primary human adult erythroblasts.

Fetal hemoglobin (HbF) is a potent genetic modifier of the severity of beta-thalassemia and sickle cell anemia. We used an in vitro culture model of human erythropoiesis in which late-stage erythroblasts are derived directly from human CD34(+) hematopoietic cells to evaluate HbF production. This system recapitulates expression of globin genes according to the developmental stage of the originating cell source. When cytokine-mobilized peripheral blood CD34(+) cells from adults were cultured, background levels of HbF were 2% or less. Cultured cells were readily transduced with lentiviral vectors when exposed to vector particles between 48 and 72 hours. Among the genetic elements that may enhance fetal hemoglobin production is an artificial zinc-finger transcription factor, GG1-VP64, designed to interact with the proximal gamma-globin gene promoters. Our data show that lentiviral-mediated, enforced expression of GG1-VP64 under the control of relatively weak erythroid-specific promoters induced significant amounts of HbF (up to 20%) in erythroblasts derived from adult CD34(+) cells without altering their capacity for erythroid maturation and only modestly reducing the total numbers of cells that accumulate in culture after transduction. These observations demonstrate the potential for sequence-specific enhancement of HbF in patients with beta-thalassemia or sickle cell anemia.

Therapeutic in vivo selection of thymic-derived natural T regulatory cells following non-myeloablative hematopoietic stem cell transplant for IPEX.

FOXP3 is critical for the development and function of CD4(+)CD25(bright) natural regulatory T cells (nTreg). Individuals harboring mutations in FOXP3 develop immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX). We describe a child diagnosed with IPEX who underwent a reduced intensity, T and B cell depleted, matched unrelated donor bone marrow transplant followed by clinical resolution. Using lineage-specific donor chimerism studies, we demonstrate that non-myeloablative HSCT resolves disease in the context of low level donor hematopoietic stem cell (HSC) engraftment. Despite low-levels of donor HSC, thymically-derived nTreg and to a lesser extent CD4(+) and CD8(+) T cells, exhibit a selective in vivo growth advantage for populations containing a functional FOXP3 gene. Moreover, nTreg from this patient show regulatory function directly ex vivo. These results have implications for improving clinical therapy for patients with IPEX and provide mechanistic insight into the in vivo development of human nTreg and unexpectedly, non-regulatory T cells.

Amelioration of murine beta-thalassemia through drug selection of hematopoietic stem cells transduced with a lentiviral vector encoding both gamma-globin and the MGMT drug-resistance gene.

Correction of murine models of beta-thalassemia has been achieved through high-level globin lentiviral vector gene transfer into mouse hematopoietic stem cells (HSCs). However, transduction of human HSCs is less robust and may be inadequate to achieve therapeutic levels of genetically modified erythroid cells. We therefore developed a double gene lentiviral vector encoding both human gamma-globin under the transcriptional control of erythroid regulatory elements and methylguanine methyltransferase (MGMT), driven by a constitutive cellular promoter. MGMT expression provides cellular resistance to alkylator drugs, which can be administered to kill residual untransduced, diseased HSCs, whereas transduced cells are protected. Mice transplanted with beta-thalassemic HSCs transduced with a gamma-globin/MGMT vector initially had subtherapeutic levels of red cells expressing gamma-globin. To enrich gamma-globin-expressing cells, transplanted mice were treated with the alkylator agent 1,3-bis-chloroethyl-1-nitrosourea. This resulted in significant increases in the number of gamma-globin-expressing red cells and the amount of fetal hemoglobin, leading to resolution of anemia. Selection of transduced HSCs was also obtained when cells were drug-treated before transplantation. Mice that received these cells demonstrated reconstitution with therapeutic levels of gamma-globin-expressing cells. These data suggest that MGMT-based drug selection holds promise as a modality to improve gene therapy for beta-thalassemia.

Sustained high-level polyclonal hematopoietic marking and transgene expression 4 years after autologous transplantation of rhesus macaques with SIV lentiviral vector-transduced CD34+ cells.

We previously reported that lentiviral vectors derived from the simian immunodeficiency virus (SIV) were efficient at transducing rhesus hematopoietic repopulating cells. To evaluate the persistence of vector-containing and -expressing cells long term, and the safety implications of SIV lentiviral vector-mediated gene transfer, we followed 3 rhesus macaques for more than 4 years after transplantation with transduced CD34+ cells. All 3 animals demonstrated significant vector marking and expression of the GFP transgene in T cells, B cells, and granulocytes, with mean GFP+ levels of 6.7% (range, 3.3%-13.0%), 7.4% (4.2%-13.4%), and 5.6% (3.1%-10.5%), respectively. There was no vector silencing in hematopoietic cells over time. Vector insertion site analysis of granulocytes demonstrated sustained highly polyclonal reconstitution, with no evidence for progression to oligoclonality. A significant number of clones were found to contribute at both 1-year and 3- or 4-year time points. No vector integrations were detected in the MDS1/EVI1 region, in contrast to our previous findings with a gamma-retroviral vector. These data show that lentiviral vectors can mediate stable and efficient long-term expression in the progeny of transduced hematopoietic stem cells, with an integration profile that may be safer than that of standard Moloney murine leukemia virus (MLV)-derived retroviral vectors.

Transduction of human primitive repopulating hematopoietic cells with lentiviral vectors pseudotyped with various envelope proteins.

Lentiviral vectors are useful for transducing primitive hematopoietic cells. We examined four envelope proteins for their ability to mediate lentiviral transduction of mobilized human CD34(+) peripheral blood cells. Lentiviral particles encoding green fluorescent protein (GFP) were pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G), the amphotropic (AMPHO) murine leukemia virus envelope protein, the endogenous feline leukemia viral envelope protein or the feline leukemia virus type C envelope protein. Because the relative amount of genome RNA per ml was similar for each pseudotype, we transduced CD34(+) cells with a fixed volume of each vector preparation. Following an overnight transduction, CD34(+) cells were transplanted into immunodeficient mice which were sacrificed 12 weeks later. The average percentages of engrafted human CD45(+) cells in total bone marrow were comparable to that of the control, mock-transduced group (37-45%). Lenti-particles pseudotyped with the VSV-G envelope protein transduced engrafting cells two- to tenfold better than particles pseudotyped with any of the gamma-retroviral envelope proteins. There was no correlation between receptor mRNA levels for the gamma-retroviral vectors and transduction efficiency of primitive hematopoietic cells. These results support the use of the VSV-G envelope protein for the development of lentiviral producer cell lines for manufacture of clinical-grade vector.

Genetically modified CD34+ hematopoietic stem cells contribute to turnover of brain perivascular macrophages in long-term repopulated primates.

Studies in rodents have shown that brain perivascular macrophages are derived from bone marrow precursors. Less is known about the origin and turnover of perivascular cells in the human central nervous system. We took advantage of non-human primates reconstituted with autologous CD34+ hematopoietic stem cells that had been transduced with a lentiviral vector expressing the enhanced green fluorescent protein (EGFP) to study the ontogeny of brain macrophages of rhesus macaques. Flow cytometry and immunohistochemistry/fluorescence microscopy showed long-term reconstitution of monocytes/macrophages in the blood, lymphoid, and brain tissues 4 years post-transplant. In the brain, EGFP+ cells were detected in the choroid plexus, cerebellum, and cerebrum, where the percent engraftment between animals reflected the percentage of EGFP+ monocytes in the blood. Morphology and location of brain EGFP+ cells exclusively in the vicinity of blood vessels were consistent with perivascular macrophages. Up to 85% of brain EGFP+ cells expressed CD163, a marker of perivascular macrophages, and greater than 70% were CD68+ macrophages. These findings clearly demonstrate that a subpopulation of CD163+/CD68+ brain perivascular macrophages in rhesus macaques are renewed by CD34+ hematopoietic stem cell-derived precursors and exhibit a continuous long-lasting turnover. Because perivascular macrophages are significant targets of productive HIV/simian immunodeficiency virus infection in the brain, these observations point to hematopoietic stem cells as targets of both HIV/simian immunodeficiency virus infection and potential gene therapy.

Hydroxyurea therapy requires HbF induction for clinical benefit in a sickle cell mouse model.

Hydroxyurea has proven clinical efficacy in patients with sickle cell disease. Potential mechanisms for the beneficial effects include fetal hemoglobin induction and the reduction of cell adhesive properties, inflammation and hypercoagulability. Using a murine model of sickle cell disease in which fetal hemoglobin induction does not occur, we evaluated whether hydroxyurea administration would still yield improvements in hematologic parameters and reduce end-organ damage. Animals given a maximally tolerated dose of hydroxyurea that resulted in significant reductions in the neutrophil and platelet counts showed no improvement in hemolytic anemia and end-organ damage compared to control mice. In contrast, animals having high levels of fetal hemoglobin due to gene transfer with a gamma-globin lentiviral vector showed correction of anemia and organ damage. These data suggest that induction of fetal hemoglobin by hydroxyurea is an essential mechanism for its clinical benefits.

Analysis of alpha hemoglobin stabilizing protein overexpression in murine ß-thalassemia.

Excess free alpha-globin is cytotoxic and contributes to the pathophysiology of b-thalassemia. Alpha hemoglobin stabilizing protein (AHSP) is a molecular chaperone that binds free alpha-globin to promote its folding and inhibit its ability to produce damaging reactive oxygen species. Reduced AHSP levels correlate with increased severity of b-thalassemia in some human cohorts, but causal mechanistic relationships are not established for these associations. We used transgenic and lentiviral gene transfer methods to investigate whether supraphysiologic AHSP levels could mitigate the severity of b-thalassemia intermedia by providing an increased sink for the excess pool of alpha-globin chains. We tested wild-type AHSP and two mutant versions with amino acid substitutions that confer 3- or 13-fold higher affinity for alpha-globin. Erythroid overexpression of these AHSP proteins up to 11-fold beyond endogenous levels had no major effects on hematologic parameters in b-thalassemic animals. Our results demonstrate that endogenous AHSP is not limiting for a-globin detoxification in a murine model of b-thalassemia.

Optimized lentiviral vector design improves titer and transgene expression of vectors containing the chicken beta-globin locus HS4 insulator element.

Hematopoietic cell gene therapy using retroviral vectors has achieved success in clinical trials. However, safety issues regarding vector insertional mutagenesis have emerged. In two different trials, vector insertion resulted in the transcriptional activation of proto-oncogenes. One strategy for potentially diminishing vector insertional mutagenesis is through the use of self-inactivating lentiviral vectors containing the 1.2-kb insulator element derived from the chicken beta-globin locus. However, use of this element can dramatically decrease both vector titer and transgene expression, thereby compromising its practical use. Here, we studied lentiviral vectors containing either the full-length 1.2-kb insulator or the smaller 0.25-kb core element in both orientations in the partially deleted long-terminal repeat. We show that use of the 0.25-kb core insulator rescued vector titer by alleviating a postentry block to reverse transcription associated with the 1.2-kb element. In addition, in an orientation-dependent manner, the 0.25-kb core element significantly increased transgene expression from an internal promoter due to improved transcriptional termination. This element also demonstrated barrier activity, reducing variability of expression due to position effects. As it is known that the 0.25-kb core insulator has enhancer-blocking activity, this particular insulated lentiviral vector design may be useful for clinical application.

Correction of murine sickle cell disease using gamma-globin lentiviral vectors to mediate high-level expression of fetal hemoglobin.

Increased levels of red cell fetal hemogloblin, whether due to hereditary persistence of expression or from induction with hydroxyurea therapy, effectively ameliorate sickle cell disease (SCD). Therefore, we developed erythroid-specific, gamma-globin lentiviral vectors for hematopoietic stem cell (HSC)-targeted gene therapy with the goal of permanently increasing fetal hemoglobin (HbF) production in sickle red cells. We evaluated two different gamma-globin lentiviral vectors for therapeutic efficacy in the BERK sickle cell mouse model. The first vector, V5, contained the gamma-globin gene driven by 3.1 kb of beta-globin regulatory sequences and a 130-bp beta-globin promoter. The second vector, V5m3, was identical except that the gamma-globin 3'-untranslated region (3'-UTR) was replaced with the beta-globin 3'-UTR. Adult erythroid cells have beta-globin mRNA 3'-UTR-binding proteins that enhance beta-globin mRNA stability and we postulated this design might enhance gamma-globin expression. Stem cell gene transfer was efficient and nearly all red cells in transplanted mice expressed human gamma-globin. Both vectors demonstrated efficacy in disease correction, with the V5m3 vector producing a higher level of gamma-globin mRNA which was associated with high-level correction of anemia and secondary organ pathology. These data support the rationale for a gene therapy approach to SCD by permanently enhancing HbF using a gamma-globin lentiviral vector.

Globin lentiviral vector insertions can perturb the expression of endogenous genes in beta-thalassemic hematopoietic cells.

Although hematopoietic cell gene therapy using retroviral vectors has recently achieved success in clinical trials, safety issues regarding vector insertional mutagenesis have emerged. Vector insertion, resulting in transcriptional activation of proto-oncogenes, played a role in the development of lymphoid leukemia in an X-linked severe combined immunodeficiency trial, and caused myeloid clonal dominance in a trial for chronic granulomatous disease. These events have raised the question of whether gene therapy for other disorders such as beta-thalassemia and sickle cell disease may hold a similar risk. In this study, we prospectively evaluated whether gamma-globin lentiviral vectors containing enhancer elements from the beta-globin locus control region could alter the expression of genes near the vector insertion. We studied this question in primary, clonal murine beta-thalassemic erythroid cells, where globin regulatory elements are highly active. We found an overall incidence of perturbed expression in 28% of the transduced clones, with 11% of all genes contained within a 600-kilobase region surrounding the vector-insertion site demonstrating altered expression. This rate was higher than that observed for a lentiviral vector containing a viral long-terminal repeat (LTR). This is the first direct evidence that lentiviral vectors can cause insertional dysregulation of cellular genes at a frequent rate.