Enhancement of mouse hematopoietic stem/progenitor cell function via transient gene delivery using integration-deficient lentiviral vectors.

Integration-deficient lentiviruses (IdLVs) deliver genes effectively to tissues but are lost rapidly from dividing cells. This property can be harnessed to express transgenes transiently to manipulate cell biology. Here, we demonstrate the utility of short-term gene expression to improve functional potency of hematopoietic stem and progenitor cells (HSPCs) during transplantation by delivering HOXB4 and Angptl3 using IdLVs to enhance the engraftment of HSPCs. Constitutive overexpression of either of these genes is likely to be undesirable, but the transient nature of IdLVs reduces this risk and those associated with unsolicited gene expression in daughter cells. Transient expression led to increased multilineage hematopoietic engraftment in in vivo competitive repopulation assays without the side effects reported in constitutive overexpression models. Adult stem cell fate has not been programmed previously using IdLVs, but we demonstrate that these transient gene expression tools can produce clinically relevant alterations or be applied to investigate basic biology.

Long-term persistence of a polyclonal T cell repertoire after gene therapy for X-linked severe combined immunodeficiency.

X-linked severe combined immunodeficiency (SCID-X1) is caused by mutations in the common cytokine receptor γ chain. These mutations classically lead to complete absence of functional T and natural killer cell lineages as well as to intrinsically compromised B cell function. Although human leukocyte antigen (HLA)-matched hematopoietic stem cell transplantation (HSCT) is highly successful in SCID-X1 patients, HLA-mismatched procedures can be associated with prolonged immunodeficiency, graft-versus-host disease, and increased overall mortality. Here, 10 children were treated with autologous CD34(+) hematopoietic stem and progenitor cells transduced with a conventional gammaretroviral vector. The patients did not receive myelosuppressive conditioning and were monitored for immunological recovery after cell infusion. All patients were alive after a median follow-up of 80 months (range, 54 to 107 months), and a functional polyclonal T cell repertoire was restored in all patients. Humoral immunity only partially recovered but was sufficient in some patients to allow for withdrawal of immunoglobulin replacement; however, three patients developed antibiotic-responsive acute pulmonary infection after discontinuation of antibiotic prophylaxis and/or immunoglobulin replacement. One patient developed acute T cell acute lymphoblastic leukemia because of up-regulated expression of the proto-oncogene LMO-2 from insertional mutagenesis, but maintained a polyclonal T cell repertoire through chemotherapy and entered remission. Therefore, gene therapy for SCID-X1 without myelosuppressive conditioning effectively restored T cell immunity and was associated with high survival rates for up to 9 years. Further studies using vectors designed to limit mutagenesis and strategies to enhance B cell reconstitution are warranted to define the role of this treatment modality alongside conventional HSCT for SCID-X1.

Hematopoietic stem cell gene therapy for adenosine deaminase-deficient severe combined immunodeficiency leads to long-term immunological recovery and metabolic correction.

Genetic defects in the purine salvage enzyme adenosine deaminase (ADA) lead to severe combined immunodeficiency (SCID) with profound depletion of T, B, and natural killer cell lineages. Human leukocyte antigen-matched allogeneic hematopoietic stem cell transplantation (HSCT) offers a successful treatment option. However, individuals who lack a matched donor must receive mismatched transplants, which are associated with considerable morbidity and mortality. Enzyme replacement therapy (ERT) for ADA-SCID is available, but the associated suboptimal correction of immunological defects leaves patients susceptible to infection. Here, six children were treated with autologous CD34-positive hematopoietic bone marrow stem and progenitor cells transduced with a conventional gammaretroviral vector encoding the human ADA gene. All patients stopped ERT and received mild chemotherapy before infusion of gene-modified cells. All patients survived, with a median follow-up of 43 months (range, 24 to 84 months). Four of the six patients recovered immune function as a result of engraftment of gene-corrected cells. In two patients, treatment failed because of disease-specific and technical reasons: Both restarted ERT and remain well. Of the four reconstituted patients, three remained off enzyme replacement. Moreover, three of these four patients discontinued immunoglobulin replacement, and all showed effective metabolic detoxification. All patients remained free of infection, and two cleared problematic persistent cytomegalovirus infection. There were no adverse leukemic side effects. Thus, gene therapy for ADA-SCID is safe, with effective immunological and metabolic correction, and may offer a viable alternative to conventional unrelated donor HSCT.

Biochemical correction of X-CGD by a novel chimeric promoter regulating high levels of transgene expression in myeloid cells.

X-linked chronic granulomatous disease (X-CGD) is a primary immunodeficiency caused by mutations in the CYBB gene encoding the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase catalytic subunit gp91(phox). A recent clinical trial for X-CGD using a spleen focus-forming virus (SFFV)-based γ-retroviral vector has demonstrated clear therapeutic benefits in several patients although complicated by enhancer-mediated mutagenesis and diminution of effectiveness over time due to silencing of the viral long terminal repeat (LTR). To improve safety and efficacy, we have designed a lentiviral vector that directs transgene expression primarily in myeloid cells. To this end, we created a synthetic chimeric promoter that contains binding sites for myeloid transcription factors CAAT box enhancer-binding family proteins (C/EBPs) and PU.1, which are highly expressed during granulocytic differentiation. As predicted, the chimeric promoter regulated higher reporter gene expression in myeloid than in nonmyeloid cells, and in human hematopoietic progenitors upon granulocytic differentiation. In a murine model of stem cell gene therapy for X-CGD, the chimeric vector resulted in high levels of gp91(phox) expression in committed myeloid cells and granulocytes, and restored normal NADPH-oxidase activity. These findings were recapitulated in human neutrophils derived from transduced X-CGD CD34(+) cells in vivo, and suggest that the chimeric promoter will have utility for gene therapy of myeloid lineage disorders such as CGD.

Codon optimization of human factor VIII cDNAs leads to high-level expression.

Gene therapy for hemophilia A would be facilitated by development of smaller expression cassettes encoding factor VIII (FVIII), which demonstrate improved biosynthesis and/or enhanced biologic properties. B domain deleted (BDD) FVIII retains full procoagulant function and is expressed at higher levels than wild-type FVIII. However, a partial BDD FVIII, leaving an N-terminal 226 amino acid stretch (N6), increases in vitro secretion of FVIII tenfold compared with BDD-FVIII. In this study, we tested various BDD constructs in the context of either wild-type or codon-optimized cDNA sequences expressed under control of the strong, ubiquitous Spleen Focus Forming Virus promoter within a self-inactivating HIV-based lentiviral vector. Transduced 293T cells in vitro demonstrated detectable FVIII activity. Hemophilic mice treated with lentiviral vectors showed expression of FVIII activity and phenotypic correction sustained over 250 days. Importantly, codon-optimized constructs achieved an unprecedented 29- to 44-fold increase in expression, yielding more than 200% normal human FVIII levels. Addition of B domain sequences to BDD-FVIII did not significantly increase in vivo expression. These significant findings demonstrate that shorter FVIII constructs that can be more easily accommodated in viral vectors can result in increased therapeutic efficacy and may deliver effective gene therapy for hemophilia A.

Phosphorylation of WASp is a key regulator of activity and stability in vivo.

The Wiskott-Aldrich syndrome protein (WASp) is a key cytoskeletal regulator in hematopoietic cells. Covalent modification of a conserved tyrosine by phosphorylation has emerged as an important potential determinant of activity, although the physiological significance remains uncertain. In a murine knockin model, mutation resulting in inability to phosphorylate Y293 (Y293F) mimicked many features of complete WASp-deficiency. Although a phosphomimicking mutant Y293E conferred enhanced actin-polymerization, the cellular phenotype was similar due to functional dysregulation. Furthermore, steady-state levels of Y293E-WASp were markedly reduced compared to wild-type WASp and Y293F-WASp, although partially recoverable by treatment of cells with proteasome inhibitors. Consequently, tyrosine phosphorylation plays a critical role in normal activation of WASp in vivo, and is indispensible for multiple tasks including proliferation, phagocytosis, chemotaxis, and assembly of adhesion structures. Furthermore, it may target WASp for proteasome-mediated degradation, thereby providing a default mechanism for self-limiting stimulation of the Arp2/3 complex.

Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID-X1 patients.

X-linked SCID (SCID-X1) is amenable to correction by gene therapy using conventional gammaretroviral vectors. Here, we describe the occurrence of clonal T cell acute lymphoblastic leukemia (T-ALL) promoted by insertional mutagenesis in a completed gene therapy trial of 10 SCID-X1 patients. Integration of the vector in an antisense orientation 35 kb upstream of the protooncogene LIM domain only 2 (LMO2) caused overexpression of LMO2 in the leukemic clone. However, leukemogenesis was likely precipitated by the acquisition of other genetic abnormalities unrelated to vector insertion, including a gain-of-function mutation in NOTCH1, deletion of the tumor suppressor gene locus cyclin-dependent kinase 2A (CDKN2A), and translocation of the TCR-beta region to the STIL-TAL1 locus. These findings highlight a general toxicity of endogenous gammaretroviral enhancer elements and also identify a combinatorial process during leukemic evolution that will be important for risk stratification and for future protocol design.

Improvement of migratory defects in a murine model of Wiskott-Aldrich syndrome gene therapy.

Wiskott-Aldrich syndrome (WAS) is an X-linked hematological disease characterized by immunodeficiency, eczema, and thrombocytopaenia, and shows promise for treatment with hematopoietic stem cell gene therapy. The immunopathology of WAS is attributable at least in part to defects of cell migration and localization as a result of chemotactic, adhesive, and chemokinetic defects. Whereas previous studies using either gammaretroviral or lentiviral vectors have demonstrated variable correction of T-cell proliferation and dendritic cell (DC) cytoarchitecture, we have used a lentiviral vector expressing an eGFP-WASp fusion protein to test the potential for restoration of cell migratory defects. Multilineage expression of the fusion transgene was present for up to 10 months after primary engraftment, and also in secondary recipients analyzed after a further 9 months. Transduced bone marrow-derived dendritic cells (BMDCs) demonstrated recovery of podosome numbers and turnover, while B cells, BMDCs, and Langerhans cells (LCs) exhibited enhanced chemotactic responses to specific stimuli. As an indication of functionality in vivo, splenic marginal zone B cells and a cutaneous contact hypersensitivity (CHS) response to dinitrofluorobenzene (DNFB) were both partially restored. These proof of principle experiments demonstrate that WAS protein (WASp) transgene expression can be successfully maintained long term in primary and secondary recipients, and that it is associated with a significant repair of migratory defects.

Gene therapy of inherited immunodeficiencies.

BACKGROUND: Primary immunodeficiencies (PID) are a group of inherited diseases that affect the development or activity of the immune system. In severe cases allogeneic haematopoietic stem cell transplantation has proved to be a successful curative modality but it is limited by toxicity and reduced efficacy in mismatched donor settings. OBJECTIVE: Gene therapy for PID has been developed as an alternative strategy and has entered the clinical arena. In this review we discuss the outcomes of recent gene therapy trials and some of the problems that remain to be tackled. METHODS: Results from clinical trials for X-linked severe combined immunodeficiency (SCID-X1), adenosine deaminase deficient SCID (ADA-SCID), and X-linked chronic granulomatous disease (X-CGD) are discussed. In addition, other conditions are highlighted such as the Wiskott Aldrich Syndrome (WAS) for which gene therapy has shown considerable promise in preclinical studies, and are currently being translated into novel clinical approaches. RESULTS/CONCLUSION: Whilst these encouraging results demonstrate that gene therapy can be used successfully to treat monogenic PID, the occurrence of vector-related side effects has highlighted the need for accurate assessment of the associated risks and a requirement for improvements in vector design.

Self-inactivating gammaretroviral vectors for gene therapy of X-linked severe combined immunodeficiency.

Gene therapy for X-linked severe combined immunodeficiency (SCID-X1) has proven highly effective for long-term restoration of immunity in human subjects. However, the development of lymphoproliferative complications due to dysregulated proto-oncogene expression has underlined the necessity for developing safer vector systems. To reduce the potential for insertional mutagenesis, we have evaluated the efficacy of self-inactivating (SIN) gammaretroviral vectors in cellular and in vivo models of SCID-X1. Vectors incorporating an internal human elongation factor-1alpha regulatory element were capable of fully restoring the lymphoid differentiation potential of gammac-deficient lineage negative cells. Multilineage lymphoid reconstitution of a murine model was achieved at a similar level to that achieved by a conventional long-terminal repeat (LTR)-regulated vector used in previous clinical trials. Functional proliferative responses to mitogenic stimuli were also restored, and serum immunoglobulin levels were normalized. The reduced mutagenic potential conferred by SIN vector configurations and alternative non-LTR-based regulatory elements, together with proven efficacy in correction of cellular defects provides an important platform for development of the next phase of clinical trials for SCID-X1.

Bone morphogenetic protein 4 modulates c-Kit expression and differentiation potential in murine embryonic aorta-gonad-mesonephros haematopoiesis in vitro.

The transforming growth factor-beta-related factor bone morphogenetic protein 4 (BMP4) is expressed in the human embryonic aorta-gonad-mesonephros (AGM) coincident with the emergence of haematopoietic cells and influences postnatal mammalian haematopoietic stem cells in vitro. To investigate the role of BMP4 in mammalian embryonic haematopoiesis, cells were isolated from murine AGM and two populations of CD34(+) cells with different levels of c-Kit expression and multipotency were identified. CD34(+)/c-Kit(high) cells express CD45 and are haematopoietic-restricted progenitors. In contrast, CD34(+)/c-Kit(low) cells are Flk1+/CD45(neg) and generate adherent colonies in ex vivo culture that resemble haemangioblast colonies identified in other systems. The addition of BMP4 to AGM cells resulted in expansion of the CD34(+)/c-Kit(low) cell pool within 48 h, via a combination of down modulation of the c-Kit receptor in CD34(+)/c-Kit(high) cells and proliferation. In long-term culture, BMP4 increased the growth/survival of CD34(+)/c-Kit(high) haematopoietic progenitors, effects that were blocked by BMP inhibitors. CD34(+)/c-Kit(high) progenitors cultured with BMP4 also generated adherent colonies typical of c-Kit(low) cells. These results suggest that BMP4 regulates c-Kit expression and differentiation potential in CD34(+) AGM cells and supports a role for BMP signalling in the maintenance of multipotency during embryonic haematopoiesis, providing an insight into stem cell homeostasis within the mammalian haematopoietic niche.

Unregulated actin polymerization by WASp causes defects of mitosis and cytokinesis in X-linked neutropenia.

Specific mutations in the human gene encoding the Wiskott-Aldrich syndrome protein (WASp) that compromise normal auto-inhibition of WASp result in unregulated activation of the actin-related protein 2/3 complex and increased actin polymerizing activity. These activating mutations are associated with an X-linked form of neutropenia with an intrinsic failure of myelopoiesis and an increase in the incidence of cytogenetic abnormalities. To study the underlying mechanisms, active mutant WASp(I294T) was expressed by gene transfer. This caused enhanced and delocalized actin polymerization throughout the cell, decreased proliferation, and increased apoptosis. Cells became binucleated, suggesting a failure of cytokinesis, and micronuclei were formed, indicative of genomic instability. Live cell imaging demonstrated a delay in mitosis from prometaphase to anaphase and confirmed that multinucleation was a result of aborted cytokinesis. During mitosis, filamentous actin was abnormally localized around the spindle and chromosomes throughout their alignment and separation, and it accumulated within the cleavage furrow around the spindle midzone. These findings reveal a novel mechanism for inhibition of myelopoiesis through defective mitosis and cytokinesis due to hyperactivation and mislocalization of actin polymerization.

Gammaretrovirus-mediated correction of SCID-X1 is associated with skewed vector integration site distribution in vivo.

We treated 10 children with X-linked SCID (SCID-X1) using gammaretrovirus-mediated gene transfer. Those with sufficient follow-up were found to have recovered substantial immunity in the absence of any serious adverse events up to 5 years after treatment. To determine the influence of vector integration on lymphoid reconstitution, we compared retroviral integration sites (RISs) from peripheral blood CD3(+) T lymphocytes of 5 patients taken between 9 and 30 months after transplantation with transduced CD34(+) progenitor cells derived from 1 further patient and 1 healthy donor. Integration occurred preferentially in gene regions on either side of transcription start sites, was clustered, and correlated with the expression level in CD34(+) progenitors during transduction. In contrast to those in CD34(+) cells, RISs recovered from engrafted CD3(+) T cells were significantly overrepresented within or near genes encoding proteins with kinase or transferase activity or involved in phosphorus metabolism. Although gross patterns of gene expression were unchanged in transduced cells, the divergence of RIS target frequency between transduced progenitor cells and post-thymic T lymphocytes indicates that vector integration influences cell survival, engraftment, or proliferation.

Generation of trispecific cytotoxic T cells recognizing cytomegalovirus, adenovirus, and Epstein-Barr virus: an approach for adoptive immunotherapy of multiple pathogens.

Cytomegalovirus (CMV), adenovirus (Ad), and Epstein-Barr virus (EBV) are a major cause of morbidity and mortality after allogeneic stem cell transplantation (SCT). Adoptive immunotherapy with donor-derived cytotoxic T cells (CTLs) directed against EBV or CMV prevents the clinical manifestations of these viruses. We have designed a protocol for the simultaneous generation of polyclonal CTL specific for CMV, Ad, and EBV, which could be used to restore immunity to multiple viruses after SCT. EBV-transformed lymphoblastoid cell lines (LCLs), transduced with an adenoviral vector carrying a transgene for the immunodominant CMV antigen pp65 (Ad5f35-pp65GFP), were used to stimulate peripheral blood mononuclear cells in 6 normal donors. We detected the simultaneous presence of CD8 CTL recognizing peptide epitopes from all 3 viruses by pentamer staining. Enzyme-linked immunospot assays demonstrated a median 29-fold (8 to 248), 47-fold (2 to 137), or 18-fold (5 to 29) increase in cells secreting interferon-gamma in response to CMV, adenoviral, or EBV antigens, respectively, compared with unmanipulated peripheral blood mononuclear cell, with concomitant loss of alloreactivity. The CTL lines showed cytotoxicity against autologous LCL alone and increased cytotoxicity to autologous LCLs pulsed with CMV pp65 peptides or infected with Ad. In summary, we have developed a protocol for the generation of CTL with trivirus specificity, enabling adoptive transfer of CTL recognizing multiple viruses to restore cellular immunity after SCT.

Lentiviral vectors containing an enhancer-less ubiquitously acting chromatin opening element (UCOE) provide highly reproducible and stable transgene expression in hematopoietic cells.

Ubiquitously acting chromatin opening elements (UCOEs) consist of methylation-free CpG islands encompassing dual divergently transcribed promoters of housekeeping genes that have been shown to confer resistance to transcriptional silencing and to produce consistent and stable transgene expression in tissue culture systems. To develop improved strategies for hematopoietic cell gene therapy, we have assessed the potential of the novel human HNRPA2B1-CBX3 UCOE (A2UCOE) within the context of a self-inactivating (SIN) lentiviral vector. Unlike viral promoters, the enhancer-less A2UCOE gave rise to populations of cells that expressed a reporter transgene at a highly reproducible level. The efficiency of expression per vector genome was also markedly increased in vivo compared with vectors incorporating either spleen focus-forming virus (SFFV) or cytomegalovirus (CMV) promoters, suggesting a relative resistance to silencing. Furthermore, an A2UCOE-IL2RG vector fully restored the IL-2 signaling pathway within IL2RG-deficient human cells in vitro and successfully rescued the X-linked severe combined immunodeficiency (SCID-X1) phenotype in a mouse model of this disease. These data indicate that the A2UCOE displays highly reliable transcriptional activity within a lentiviral vector, largely overcoming insertion-site position effects and giving rise to therapeutically relevant levels of gene expression. These properties are achieved in the absence of classic enhancer activity and therefore may confer a high safety profile.

Lentiviral vectors for T-cell suicide gene therapy: preservation of T-cell effector function after cytokine-mediated transduction.

Retroviral transfer of the Herpes Simplex thymidine kinase (HSVTK) suicide gene to donor T cells has been used as a safety strategy against graft-versus-host disease following allogeneic stem cell transplantation. The feasibility of this strategy in human studies has been demonstrated, but a number of limitations have become apparent. Preactivation of donor lymphocytes using mitogens or monoclonal antibodies is essential for retroviral transduction, but can compromise subsequent T-cell function in vivo. We report the application of lentiviral vectors for transduction of T cells in cytokine culture, without activation through the T-cell receptor. Using vectors encoding either enhanced green fluorescent protein or a truncated CD34/mutant HSVTK fusion selection/suicide construct, we investigated the properties of T cells after gene modification. We found that following cytokine stimulation, a fraction of T cells undergoes division, and transgene expression occurred predominantly in these cells. Antiviral and alloreactive responses were preserved in these populations, and in contrast to fully activated T cells, there was minimal perturbation of regulatory T-cell numbers. We conclude that the use of interleukin-7 for lentiviral transduction offers the greatest potential for gene transfer to T cells without loss of function, and is favored for the clinical production of suicide gene modified T cells.

Capture and generation of adenovirus specific T cells for adoptive immunotherapy.

Adenoviral infections represent a major cause of morbidity and mortality following haematopoietic stem cell transplantation. Current anti-viral agents are virostatic and it is evident that elimination of adenovirus (ADV) infection is only achieved by recovery of cellular immunity. Using an interferon-gamma (IFN-gamma) secretion and capture assay to isolate ADV-specific T cells, followed by a 2 week expansion and restimulation protocol, we generated ADV T cells that may be used for cellular immunotherapy. In contrast to virus-specific T cells for cytomegalovirus or Epstein-Barr virus, the ADV response was dominated by CD4(+) T cells and the majority of captured cells exhibited an effector/memory immunophenotype. Highly specific antigen responses were demonstrated by intracellular IFN-gamma expression and cytotoxicity assays when the expanded cells underwent restimulation with ADV-pulsed target cells. Although T cells were initially generated in response to ADV species C, the expanded populations also showed strong activity against ADV species B, suggesting cross-reactivity across ADV species; a finding that has important clinical consequences in the paediatric setting, where the majority of infections are caused by ADV type B and C. The protocols can be readily translated to generate ADV-specific T cells suitable for clinical use and offer an effective immunotherapeutic strategy to control ADV infection.

Successful reconstitution of immunity in ADA-SCID by stem cell gene therapy following cessation of PEG-ADA and use of mild preconditioning.

Gene therapy is a promising treatment option for monogenic diseases, but success has been seen in only a handful of studies thus far. We now document successful reconstitution of immune function in a child with the adenosine deaminase (ADA)-deficient form of severe combined immunodeficiency (SCID) following hematopoietic stem cell (HSC) gene therapy. An ADA-SCID child who showed a poor response to PEG-ADA enzyme replacement was enrolled into the clinical study. Following cessation of enzyme replacement therapy, autologous CD34(+) HSCs were transduced with an ADA-expressing gammaretroviral vector. Gene-modified cells were reinfused following one dose of preconditioning chemotherapy. Two years after the procedure, immunological and biochemical correction has been maintained with progressive increase in lymphocyte numbers, reinitiation of thymopoiesis, and systemic detoxification of ADA metabolites. Sustained vector marking with detection of polyclonal vector integration sites in multiple cell lineages and detection of ADA activity in red blood cells suggests transduction of early hematopoietic progenitors. No serious side effects were seen either as a result of the conditioning procedure or due to retroviral insertion. Gene therapy is an effective treatment option for the treatment of ADA-SCID.