Lentiviral Gene Therapy Combined with Low-Dose Busulfan in Infants with SCID-X1.

BACKGROUND: Allogeneic hematopoietic stem-cell transplantation for X-linked severe combined immunodeficiency (SCID-X1) often fails to reconstitute immunity associated with T cells, B cells, and natural killer (NK) cells when matched sibling donors are unavailable unless high-dose chemotherapy is given. In previous studies, autologous gene therapy with γ-retroviral vectors failed to reconstitute B-cell and NK-cell immunity and was complicated by vector-related leukemia. METHODS: We performed a dual-center, phase 1-2 safety and efficacy study of a lentiviral vector to transfer IL2RG complementary DNA to bone marrow stem cells after low-exposure, targeted busulfan conditioning in eight infants with newly diagnosed SCID-X1. RESULTS: Eight infants with SCID-X1 were followed for a median of 16.4 months. Bone marrow harvest, busulfan conditioning, and cell infusion had no unexpected side effects. In seven infants, the numbers of CD3+, CD4+, and naive CD4+ T cells and NK cells normalized by 3 to 4 months after infusion and were accompanied by vector marking in T cells, B cells, NK cells, myeloid cells, and bone marrow progenitors. The eighth infant had an insufficient T-cell count initially, but T cells developed in this infant after a boost of gene-corrected cells without busulfan conditioning. Previous infections cleared in all infants, and all continued to grow normally. IgM levels normalized in seven of the eight infants, of whom four discontinued intravenous immune globulin supplementation; three of these four infants had a response to vaccines. Vector insertion-site analysis was performed in seven infants and showed polyclonal patterns without clonal dominance in all seven. CONCLUSIONS: Lentiviral vector gene therapy combined with low-exposure, targeted busulfan conditioning in infants with newly diagnosed SCID-X1 had low-grade acute toxic effects and resulted in multilineage engraftment of transduced cells, reconstitution of functional T cells and B cells, and normalization of NK-cell counts during a median follow-up of 16 months. (Funded by the American Lebanese Syrian Associated Charities and others; LVXSCID-ND ClinicalTrials.gov number, NCT01512888.).

Resveratrol trimer enhances gene delivery to hematopoietic stem cells by reducing antiviral restriction at endosomes.

Therapeutic gene delivery to hematopoietic stem cells (HSCs) holds great potential as a life-saving treatment of monogenic, oncologic, and infectious diseases. However, clinical gene therapy is severely limited by intrinsic HSC resistance to modification with lentiviral vectors (LVs), thus requiring high doses or repeat LV administration to achieve therapeutic gene correction. Here we show that temporary coapplication of the cyclic resveratrol trimer caraphenol A enhances LV gene delivery efficiency to human and nonhuman primate hematopoietic stem and progenitor cells with integrating and nonintegrating LVs. Although significant ex vivo, this effect was most dramatically observed in human lineages derived from HSCs transplanted into immunodeficient mice. We further show that caraphenol A relieves restriction of LV transduction by altering the levels of interferon-induced transmembrane (IFITM) proteins IFITM2 and IFITM3 and their association with late endosomes, thus augmenting LV core endosomal escape. Caraphenol A-mediated IFITM downregulation did not alter the LV integration pattern or bias lineage differentiation. Taken together, these findings compellingly demonstrate that the pharmacologic modification of intrinsic immune restriction factors is a promising and nontoxic approach for improving LV-mediated gene therapy.

Evaluating the Safety of Retroviral Vectors Based on Insertional Oncogene Activation and Blocked Differentiation in Cultured Thymocytes.

Insertional oncogenesis due to retroviral (RV) vector integration has caused recurrent leukemia in multiple gene therapy trials, predominantly due to vector integration effects at the LMO2 locus. While currently available preclinical safety models have been used for evaluating vector safety, none have predicted or reproduced the recurrent LMO2 integrations seen in previous X-linked severe combined immunodeficiency (X-SCID) and Wiskott-Aldrich clinical gene therapy trials. We now describe a new assay for assessing vector safety that recapitulates naturally occurring insertions into Lmo2 and other T-cell proto-oncogenes leading to a preleukemic developmental arrest in primary murine thymocytes cultured in vitro. This assay was used to compare the relative oncogenic potential of a variety of gamma-RV and lentiviral vectors and to assess the risk conferred by various transcriptional elements contained in these genomes. Gamma-RV vectors that contained full viral long-terminal repeats were most prone to causing double negative 2 (DN2) arrest and led to repeated cases of Lmo2 pathway activation, while lentiviral vectors containing these same elements were significantly less prone to activate proto-oncogenes or cause DN2 arrest. This work provides a new preclinical assay that is especially relevant for assessing safety in SCID disorders and provides a new tool for designing safer RV vectors.

Downregulation of Prdm16 mRNA is a specific antileukemic mechanism during HOXB4-mediated HSC expansion in vivo.

Overexpression of HOXB4 in hematopoietic stem cells (HSCs) leads to increased self-renewal without causing hematopoietic malignancies in transplanted mice. The molecular basis of HOXB4-mediated benign HSC expansion in vivo is not well understood. To gain further insight into the molecular events underlying HOXB4-mediated HSC expansion, we analyzed gene expression changes at multiple time points in Lin(-)Sca1(+)c-kit(+) cells from mice transplanted with bone marrow cells transduced with a MSCV-HOXB4-ires-YFP vector. A distinct HOXB4 transcriptional program was reproducibly induced and stabilized by 12 weeks after transplant. Dynamic expression changes were observed in genes critical for HSC self-renewal as well as in genes involved in myeloid and B-cell differentiation. Prdm16, a transcription factor associated with human acute myeloid leukemia, was markedly repressed by HOXB4 but upregulated by HOXA9 and HOXA10, suggesting that Prdm16 downregulation was involved in preventing leukemia in HOXB4 transplanted mice. Functional evidence to support this mechanism was obtained by enforcing coexpression of sPrdm16 and HOXB4, which led to enhanced self-renewal, myeloid expansion, and leukemia. Altogether, these studies define the transcriptional pathways involved in HOXB4 HSC expansion in vivo and identify repression of Prdm16 transcription as a mechanism by which expanding HSCs avoid leukemic transformation.

Clinical strategies for expansion of haematopoietic stem cells.

Haematopoietic stem cells (HSCs) give rise to all blood and immune cells and are used in clinical transplantation protocols to treat a wide variety of diseases. The ability to increase the number of HSCs either in vivo or in vitro would provide new treatment options, but the amplification of HSCs has been difficult to achieve. Recent insights into the mechanisms of HSC self-renewal now make the amplification of HSCs a plausible clinical goal. This article reviews the molecular mechanisms that control HSC numbers and discusses how these can be modulated to increase the number of HSCs. Clinical applications of HSC expansion are then discussed for their potential to address the current limitations of HSC transplantation.

ATP-binding cassette transporter Abcg2 lineage contributes to the cardiac vasculature after oxidative stress.

Due to their specialized location, stem and progenitor cells are often exposed to oxidative stress. Although ATP-binding cassette transporter subfamily G member 2 (Abcg2)-expressing cells have been implicated in cardiac protective mechanisms involving oxidative stress, there remains a lack of understanding regarding the behavior of cardiac Abcg2-expressing cells when exposed to ROS. The aim of the present study was to characterize the response of the cardiac Abcg2 lineage to oxidative stress. In vitro analysis demonstrated that the antioxidant program regulated by Abcg2 is dependent on a functional transporter. Delivery of paraquat dichloride (PQ), a systemic oxidative stress-inducing agent, to mice confirmed that Abcg2 provides a survival benefit. When exposed to PQ, reporter mice showed an increase in the Abcg2 lineage. Transcriptional and immunohistochemical analysis of Abcg2 lineage-positive cells revealed an enhanced vascular commitment after stress. Finally, preconditioning with PQ demonstrated a reduction in scar size and an increase in angiogenesis after permanent left coronary artery ligation. In conclusion, the data suggest that Abcg2 plays a cytoprotective role in response to in vivo oxidative stress. The contribution of the Abcg2 lineage to the vasculature in the heart is increased after PQ delivery.

Functional interactions between Lmo2, the Arf tumor suppressor, and Notch1 in murine T-cell malignancies.

LMO2 is a target of chromosomal translocations in T-cell tumors and was activated by retroviral vector insertions in T-cell tumors from X-SCID patients in gene therapy trials. To better understand the cooperating genetic events in LMO2-associated T-cell acute lymphoblastic leukemia (T-ALL), we investigated the roles of Arf tumor suppressor loss and Notch activation in murine models of transplantation. Lmo2 overexpression enhanced the expansion of primitive DN2 thymocytes, eventually facilitating the stochastic induction of clonal CD4(+)/CD8(+) malignancies. Inactivation of the Arf tumor suppressor further increased the self-renewal capacity of the primitive, preleukemic thymocyte pool and accelerated the development of aggressive, Lmo2-induced T-cell lympholeukemias. Notch mutations were frequently detected in these Lmo2-induced tumors. The Arf promoter was not directly engaged by Lmo2 or mutant Notch, and use of a mouse model in which activation of a mutant Notch allele depends on previous engagement of the Arf promoter revealed that Notch activation could occur as a subsequent event in T-cell tumorigenesis. Therefore, Lmo2 cooperates with Arf loss to enhance self-renewal in primitive thymocytes. Notch mutation and Arf inactivation appear to independently cooperate in no requisite order with Lmo2 overexpression in inducing T-ALL, and all 3 events remained insufficient to guarantee immediate tumor development.

Drug transporters on arachnoid barrier cells contribute to the blood-cerebrospinal fluid barrier.

The subarachnoid space, where cerebrospinal fluid (CSF) flows over the brain and spinal cord, is lined on one side by arachnoid barrier (AB) cells that form part of the blood-CSF barrier. However, despite the fact that drugs are administered into the CSF and CSF drug concentrations are used as a surrogate for brain drug concentration following systemic drug administration, the tight-junctioned AB cells have never been examined for whether they express drug transporters that would influence CSF and central nervous system drug disposition. Hence, we characterized drug transporter expression and function in AB cells. Immunohistochemical analysis showed P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) in mouse AB cells but not other meningeal tissue. The Gene Expression Nervous System Atlas (GENSAT) database and the Allen Mouse Brain Atlas confirmed these observations. Microarray analysis of mouse and human arachnoidal tissue revealed expression of many drug transporters and some drug-metabolizing enzymes. Immortalized mouse AB cells express functional P-gp on the apical (dura-facing) membrane and BCRP on both apical and basal (CSF-facing) membranes. Thus, like blood-brain barrier cells and choroid plexus cells, AB cells highly express drug transport proteins and likely contribute to the blood-CSF drug permeation barrier.

Abcg2 expression marks tissue-specific stem cells in multiple organs in a mouse progeny tracking model.

The side population phenotype is associated with the Hoechst dye efflux activity of the Abcg2 transporter and identifies hematopoietic stem cells (HSCs) in the bone marrow. This association suggests the direct use of Abcg2 expression to identify adult stem cells in various other organs. We have generated a lineage tracing mouse model based on an allele that coexpresses both Abcg2 and a CreERT2 expression cassette. By crossing these mice with lox-STOP-lox reporter lines (LacZ or YFP), cells that express Abcg2 and their progeny were identified following treatment with tamoxifen (Tam). In the liver and kidney, in which mature cells express Abcg2, reporter gene expression verified the expected physiologic expression pattern of the recombinant allele. Long-term marking of HSCs was seen in multiple peripheral blood lineages from adult mice, demonstrating that Abcg2(+) bone marrow HSCs contribute to steady-state hematopoiesis. Stem cell tracing patterns were seen in the small intestine and in seminiferous tubules in the testis 20 months after Tam treatment, proving that stem cells from these organs express Abcg2. Interstitial cells from skeletal and cardiac muscle were labeled, and some cells were costained with endothelial markers, raising the possibility that these cells may function in the repair response to muscle injury. Altogether, these studies prove that Abcg2 is a stem cell marker for blood, small intestine, testicular germ cells, and possibly for injured skeletal and/or cardiac muscle and provide a new model for studying stem cell activity that does not require transplant-based assays.

Hemgn is a direct transcriptional target of HOXB4 and induces expansion of murine myeloid progenitor cells.

HOXB4, a member of the Homeobox transcription factor family, promotes expansion of hematopoietic stem cells and hematopoietic progenitor cells in vivo and ex vivo when overexpressed. However, the molecular mechanisms underlying this effect are not well understood. To identify direct target genes of HOXB4 in primary murine hematopoietic progenitor cells, we induced HOXB4 function in lineage-negative murine bone marrow cells, using a tamoxifen-inducible HOXB4-ER(T2) fusion protein. Using expression microarrays, 77 probe sets were identified with differentially changed expression in early response to HOXB4 induction. Among them, we show that Hemogen (Hemgn), encoding a hematopoietic-specific nuclear protein of unknown function, is a direct transcriptional target of HOXB4. We show that HOXB4 binds to the promoter region of Hemgn both ex vivo and in vivo. When we overexpressed Hemgn in bone marrow cells, we observed that Hemgn promoted cellular expansion in liquid cultures and increased self-renewal of myeloid colony-forming units in culture, partially recapitulating the effect of HOXB4 overexpression. Furthermore, down-regulation of Hemgn using an shRNA strategy proved that Hemgn contributes to HOXB4-mediated expansion in our myeloid progenitor assays. Our results identify a functionally relevant, direct transcriptional target of HOXB4 and identify other target genes that may also participate in the HOXB4 genetic network.

A self-inactivating lentiviral vector for SCID-X1 gene therapy that does not activate LMO2 expression in human T cells.

To develop safer and more effective vectors for gene therapy of X-linked severe combined immunodeficiency (SCID-X1), we have evaluated new self-inactivating lentiviral vectors based on the HIV virus. The CL20i4-hgamma(c)-Revgen vector contains the entire human common gamma chain (gamma(c)) genomic sequence driven by the gamma(c) promoter. The CL20i4-EF1alpha-hgamma(c)OPT vector uses a promoter fragment from the eukaryotic elongation factor alpha (EF1alpha) gene to express a codon-optimized human gamma(c) cDNA. Both vectors contain a 400-bp insulator fragment from the chicken beta-globin locus within the self-inactivating long-terminal repeat. Transduction of bone marrow cells using either of these vectors restored T, B, and natural killer lymphocyte development and function in a mouse SCID-X1 transplantation model. Transduction of human CD34(+) bone marrow cells from SCID-X1 patients with either vector restored T-cell development in an in vitro assay. In safety studies using a Jurkat LMO2 activation assay, only the CL20i4-EF1alpha-hgamma(c)OPT vector lacked the ability to transactivate LMO2 protein expression, whereas the CL20i4-hgamma(c)-Revgen vector significantly activated LMO2 protein expression. In addition, the CL20i4-EF1alpha-hgamma(c)OPT vector has not caused any tumors in transplanted mice. We conclude that the CL20i4-EF1alpha-hgamma(c)OPT vector may be suitable for testing in a clinical trial based on these preclinical demonstrations of efficacy and safety.

Cells previously identified as retinal stem cells are pigmented ciliary epithelial cells.

It was previously reported that the ciliary epithelium (CE) of the mammalian eye contains a rare population of cells that could produce clonogenic self-renewing pigmented spheres in culture. Based on their ability to up-regulate genes found in retinal neurons, it was concluded that these sphere-forming cells were retinal stem cells. This conclusion raised the possibility that CE-derived retinal stem cells could help to restore vision in the millions of people worldwide who suffer from blindness associated with retinal degeneration. We report here that human and mouse CE-derived spheres are made up of proliferating pigmented ciliary epithelial cells rather than retinal stem cells. All of the cells in the CE-derived spheres, including the proliferating cells, had molecular, cellular, and morphological features of differentiated pigmented CE cells. These differentiated cells ectopically expressed nestin when exposed to growth factors and low levels of pan-neuronal markers such as beta-III-tubulin. Although the cells aberrantly expressed neuronal markers, they retained their pigmented CE cell morphology and failed to differentiate into retinal neurons in vitro or in vivo. Our results provide an example of a differentiated cell type that can form clonogenic spheres in culture, self-renew, express progenitor cell markers, and initiate neuronal differentiation that is not a stem or progenitor cell. More importantly, our findings highlight the importance of shifting the focus away from studies on CE-derived spheres for cell-based therapies to restore vision in the degenerating retina and improving techniques for using ES cells or retinal precursor cells.

Efficient construction of producer cell lines for a SIN lentiviral vector for SCID-X1 gene therapy by concatemeric array transfection.

Retroviral vectors containing internal promoters, chromatin insulators, and self-inactivating (SIN) long terminal repeats (LTRs) may have significantly reduced genotoxicity relative to the conventional retroviral vectors used in recent, otherwise successful clinical trials. Large-scale production of such vectors is problematic, however, as the introduction of SIN vectors into packaging cells cannot be accomplished with the traditional method of viral transduction. We have derived a set of packaging cell lines for HIV-based lentiviral vectors and developed a novel concatemeric array transfection technique for the introduction of SIN vector genomes devoid of enhancer and promoter sequences in the LTR. We used this method to derive a producer cell clone for a SIN lentiviral vector expressing green fluorescent protein, which when grown in a bioreactor generated more than 20 L of supernatant with titers above 10(7) transducing units (TU) per milliliter. Further refinement of our technique enabled the rapid generation of whole populations of stably transformed cells that produced similar titers. Finally, we describe the construction of an insulated, SIN lentiviral vector encoding the human interleukin 2 receptor common gamma chain (IL2RG) gene and the efficient derivation of cloned producer cells that generate supernatants with titers greater than 5 x 10(7) TU/mL and that are suitable for use in a clinical trial for X-linked severe combined immunodeficiency (SCID-X1).

3' UTR-truncated HMGA2 overexpression induces non-malignant in vivo expansion of hematopoietic stem cells in non-human primates.

Vector-mediated mutagenesis remains a major safety concern for many gene therapy clinical protocols. Indeed, lentiviral-based gene therapy treatments of hematologic disease can result in oligoclonal blood reconstitution in the transduced cell graft. Specifically, clonal expansion of hematopoietic stem cells (HSCs) highly expressing HMGA2, a chromatin architectural factor found in many human cancers, is reported in patients undergoing gene therapy for hematologic diseases, raising concerns about the safety of these integrations. Here, we show for the first time in vivo multilineage and multiclonal expansion of non-human primate HSCs expressing a 3' UTR-truncated version of HMGA2 without evidence of any hematologic malignancy >7 years post-transplantation, which is significantly longer than most non-human gene therapy pre-clinical studies. This expansion is accompanied by an increase in HSC survival, cell cycle activation of downstream progenitors, and changes in gene expression led by the upregulation of IGF2BP2, a mRNA binding regulator of survival and proliferation. Thus, we conclude that prolonged ectopic expression of HMGA2 in hematopoietic progenitors is not sufficient to drive hematologic malignancy and is not an acute safety concern in lentiviral-based gene therapy clinical protocols.

Marking of definitive HSC precursors in E7.5-E8.5 embryos using an Abcg2-CreER lineage-tracing mouse model.

Abcg2, a member of the ATP-binding cassette transporter family, is expressed in adult hematopoietic stem cells (HSCs) and is required for the side population phenotype of adult bone marrow HSCs and other adult tissue-specific stem cells. Lineage tracing in adult mice using the Abcg2-Cre mouse model showed that Abcg2 marks HSCs, intestinal stem cells, and spermatogonial stem cells. It is unclear whether definitive HSCs or their precursors in early embryonic development can be marked by Abcg2 expression. Here, we treated pregnant Abcg2 Cre/Cre RosaLSL-YFP mice with a single injection of 4-hydroxytamoxifen at embryonic day 7.5. Four months after birth, a small yellow fluorescent protein-positive (YFP+) cell population could be detected in all of the major white blood cell lineages and this was stable for 8 months. Transplant of bone marrow cells or Sca1+YFP+ cells from these mice showed continued multilineage marking in recipient mice at 4 months. These results demonstrate that Abcg2 expression marks precursors to adult long-term repopulating HSCs at E7.5 to E8.5 and contributes to a stable subpopulation of HSCs well into adulthood.