Ute Modlich

Gene Therapy for Wiskott-Aldrich Syndrome—Long-Term Efficacy and Genotoxicity

Wiskott-Aldrich syndrome gene therapy is feasible, but γ-retroviral vectors contribute a substantial risk of leukemogenesis. Taking the Sting Out of Gene Therapy Wiskott-Aldrich syndrome (WAS) is a rare X-linked recessive disorder characterized by low platelet count, immune deficiency, autoimmunity, and high risk of cancer. WAS is primarily a disorder of blood cells, and hematopoietic stem cell transplantation (HSCT) has been the only hope of cure. However, HSCT is restricted to patients who can find matching donors. One way to overcome this limitation is through gene therapy that restores the function of the mutated protein in HSCs from the patient. Now, Braun et al. report correction of WAS protein (WASP) in 9 of 10 patients that underwent HSC gene therapy. The authors used a γ-retroviral vector to correct WASP expression in autologous HSCs. After transfer to patients, these cells engrafted and WASP was expressed in lymphoid and myeloid cells and platelets in 9 of 10 patients. What’s more, this therapy caused either partial or complete resolution of symptoms. However, seven patients developed acute leukemia, and further analysis revealed genetic alterations such as chromosomal translocations. These studies suggest that with improved vector design, gene therapy may be feasible and effective for patient with WAS. Wiskott-Aldrich syndrome (WAS) is characterized by microthrombocytopenia, immunodeficiency, autoimmunity, and susceptibility to malignancies. In our hematopoietic stem cell gene therapy (GT) trial using a γ-retroviral vector, 9 of 10 patients showed sustained engraftment and correction of WAS protein (WASP) expression in lymphoid and myeloid cells and platelets. GT resulted in partial or complete resolution of immunodeficiency, autoimmunity, and bleeding diathesis. Analysis of retroviral insertion sites revealed >140,000 unambiguous integration sites and a polyclonal pattern of hematopoiesis in all patients early after GT. Seven patients developed acute leukemia [one acute myeloid leukemia (AML), four T cell acute lymphoblastic leukemia (T-ALL), and two primary T-ALL with secondary AML associated with a dominant clone with vector integration at the LMO2 (six T-ALL), MDS1 (two AML), or MN1 (one AML) locus]. Cytogenetic analysis revealed additional genetic alterations such as chromosomal translocations. This study shows that hematopoietic stem cell GT for WAS is feasible and effective, but the use of γ-retroviral vectors is associated with a substantial risk of leukemogenesis.

Physiological Promoters Reduce the Genotoxic Risk of Integrating Gene Vectors

The possible activation of cellular proto-oncogenes as a result of clonal transformation is a potential limitation in a therapeutic approach involving random integration of gene vectors. Given that enhancer promiscuity represents an important mechanism of insertional transformation, we assessed the enhancer activities of various cellular and retroviral promoters in transient transfection assays, and also in a novel experimental system designed to measure the activation of a minigene cassette contained in stably integrating retroviral vectors. Retroviral enhancer-promoters showed a significantly greater potential to activate neighboring promoters than did cellular promoters derived from human genes, elongation factor-1alpha (EF1alpha) and phosphoglycerate kinase (PGK). Self-inactivating (SIN) vector design reduced but did not abolish enhancer interactions. Using a recently established cell culture assay that detects insertional transformation by serial replating of primary hematopoietic cells, we found that SIN vectors containing the EF1alpha promoter greatly decrease the risk of insertional transformation. Despite integration of multiple copies per cell, activation of the crucial proto-oncogene Evi1 was not detectable when using SIN-EF1alpha vectors. On the basis of several quantitative indicators, the decrease in transforming activity was highly significant (more than tenfold, P < 0.01) when compared with similarly designed vectors containing a retroviral enhancer-promoter with or without a well-characterized genetic insulator core element. In this manner, the insertional biosafety of therapeutic gene vectors can be greatly enhanced and proactively evaluated in sensitive cell-based assays.

Gene therapy - X-SCID transgene leukaemogenicity

Arising from: Woods, N.-B., Bottero, V., Schmidt, M., von Kalle, C. & Verma, I. M. 440, 1123 (2006); see also communication from Pike-Overzet et al.; Woods et al. replyGene therapy has been remarkably effective for the immunological reconstitution of patients with severe combined immune deficiency, but the occurrence of leukaemia in a few patients has stimulated debate about the safety of the procedure and the mechanisms of leukaemogenesis. Woods et al. forced high expression of the corrective therapeutic gene IL2RG, which encodes the γ-chain of the interleukin-2 receptor, in a mouse model of the disease and found that tumours appeared in a proportion of cases. Here we show that transgenic IL2RG does not necessarily have potent intrinsic oncogenic properties, and argue that the interpretation of this observation with respect to human trials is overstated.

Mutant IDH1 promotes leukemogenesis in vivo and can be specifically targeted in human AML

Mutations in the metabolic enzymes isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) are frequently found in glioma, acute myeloid leukemia (AML), melanoma, thyroid cancer, and chondrosarcoma patients. Mutant IDH produces 2-hydroxyglutarate (2HG), which induces histone- and DNA-hypermethylation through inhibition of epigenetic regulators. We investigated the role of mutant IDH1 using the mouse transplantation assay. Mutant IDH1 alone did not transform hematopoietic cells during 5 months of observation. However, mutant IDH1 greatly accelerated onset of myeloproliferative disease-like myeloid leukemia in mice in cooperation with HoxA9 with a mean latency of 83 days compared with cells expressing HoxA9 and wild-type IDH1 or a control vector (167 and 210 days, respectively, P = .001). Mutant IDH1 accelerated cell-cycle transition through repression of cyclin-dependent kinase inhibitors Cdkn2a and Cdkn2b, and activated mitogen-activated protein kinase signaling. By computational screening, we identified an inhibitor of mutant IDH1, which inhibited mutant IDH1 cells and lowered 2HG levels in vitro, and efficiently blocked colony formation of AML cells from IDH1-mutated patients but not of normal CD34(+) bone marrow cells. These data demonstrate that mutant IDH1 has oncogenic activity in vivo and suggest that it is a promising therapeutic target in human AML cells.

Leukemia induction after a single retroviral vector insertion in Evi1 or Prdm16

Insertional activation of cellular proto-oncogenes by replication-defective retroviral vectors can trigger clonal dominance and leukemogenesis in animal models and clinical trials. Here, we addressed the leukemogenic potential of vectors expressing interleukin-2 receptor common γ-chain (IL2RG), the coding sequence required for correction of X-linked severe combined immunodeficiency. Similar to conventional γ-retroviral vectors, self-inactivating (SIN) vectors with strong internal enhancers also triggered profound clonal imbalance, yet with a characteristic insertion preference for a window located downstream of the transcriptional start site. Controls including lentivirally transduced cells revealed that ectopic IL2RG expression was not sufficient to trigger leukemia. After serial bone marrow transplantation involving 106 C57Bl6/J mice monitored for up to 18 months, we observed leukemic progression of six distinct clones harboring γ-retroviral long terminal repeat (LTR) or SIN vector insertions in Evi1 or Prdm16, two functionally related genes. Three leukemic clones had single vector integrations, and identical clones manifested with a remarkably similar latency and phenotype in independent recipients. We conclude that upregulation of Evi1 or Prdm16 was sufficient to initiate a leukemogenic cascade with consistent intrinsic dynamics. Our study also shows that insertional mutagenesis is required for leukemia induction by IL2RG vectors, a risk to be addressed by improved vector design.

Self-inactivating retroviral vectors with improved RNA processing

Three RNA features have been identified that elevate retroviral transgene expression: an intron in the 5′ untranslated region (5′UTR), the absence of aberrant translational start codons and the presence of the post-transcriptional regulatory element (PRE) of the woodchuck hepatitis virus in the 3′UTR. To include such elements into self-inactivating (SIN) vectors with potentially improved safety, we excised the strong retroviral promoter from the U3 region of the 3′ long terminal repeat (LTR) and inserted it either downstream or upstream of the retroviral RNA packaging signal (Ψ). The latter concept is new and allows the use of an intron in the 5′UTR, taking advantage of retroviral splice sites surrounding Ψ. Three LTR and four SIN vectors were compared to address the impact of RNA elements on titer, splice regulation and transgene expression. Although titers of SIN vectors were about 20-fold lower than those of their LTR counterparts, inclusion of the PRE allowed production of more than 106 infectious units per ml without further vector optimizations. In comparison with state-of-the-art LTR vectors, the intron-containing SIN vectors showed greatly improved splicing. With regard to transgene expression, the intron-containing SIN vectors largely matched or even exceeded the LTR counterparts in all cell types investigated (embryonic carcinoma cells, fibroblasts, primary T cells and hematopoietic progenitor cells).

Genotoxic Potential of Lineage-specific Lentivirus Vectors Carrying the β-Globin Locus Control Region

Insertional mutagenesis by long terminal repeat (LTR) enhancers in γ-retrovirus-based vectors (GVs) in clinical trials has prompted deeper investigations into vector genotoxicity. Experimentally, self-inactivating (SIN) lentivirus vectors (LVs) and GV containing internal promoters/enhancers show reduced genotoxicity, although strong ubiquitously-active enhancers dysregulate genes independent of vector type/design. Herein, we explored the genotoxicity of β-globin (BG) locus control region (LCR), a strong long-range lineage-specific-enhancer, with/without insulator (Ins) elements in LV using primary hematopoietic progenitors to generate in vitro immortalization (IVIM) assay mutants. LCR-containing LV had ~200-fold lower transforming potential, compared to the conventional GV. The LCR perturbed expression of few genes in a 300 kilobase (kb) proviral vicinity but no upregulation of genes associated with cancer, including an erythroid-specific transcription factor occurred. A further twofold reduction in transforming activity was observed with insulated LCR-containing LV. Our data indicate that toxicology studies of LCR-containing LV in mice will likely not yield any insertional oncogenesis with the numbers of animals that can be practically studied.Insertional mutagenesis by long terminal repeat (LTR) enhancers in gamma-retrovirus-based vectors (GVs) in clinical trials has prompted deeper investigations into vector genotoxicity. Experimentally, self-inactivating (SIN) lentivirus vectors (LVs) and GV containing internal promoters/enhancers show reduced genotoxicity, although strong ubiquitously-active enhancers dysregulate genes independent of vector type/design. Herein, we explored the genotoxicity of beta-globin (BG) locus control region (LCR), a strong long-range lineage-specific-enhancer, with/without insulator (Ins) elements in LV using primary hematopoietic progenitors to generate in vitro immortalization (IVIM) assay mutants. LCR-containing LV had approximately 200-fold lower transforming potential, compared to the conventional GV. The LCR perturbed expression of few genes in a 300 kilobase (kb) proviral vicinity but no upregulation of genes associated with cancer, including an erythroid-specific transcription factor occurred. A further twofold reduction in transforming activity was observed with insulated LCR-containing LV. Our data indicate that toxicology studies of LCR-containing LV in mice will likely not yield any insertional oncogenesis with the numbers of animals that can be practically studied.

Development of novel efficient SIN vectors with improved safety features for Wiskott-Aldrich syndrome stem cell based gene therapy.

Gene therapy is a promising therapeutic approach to treat primary immunodeficiencies. Indeed, the clinical trial for the Wiskott-Aldrich Syndrome (WAS) that is currently ongoing at the Hannover Medical School (Germany) has recently reported the correction of all affected cell lineages of the hematopoietic system in the first treated patients. However, an extensive study of the clonal inventory of those patients reveals that LMO2, CCND2 and MDS1/EVI1 were preferentially prevalent. Moreover, a first leukemia case was observed in this study, thus reinforcing the need of developing safer vectors for gene transfer into HSC in general. Here we present a novel self-inactivating (SIN) vector for the gene therapy of WAS that combines improved safety features. We used the elongation factor 1 alpha (EFS) promoter, which has been extensively evaluated in terms of safety profile, to drive a codon-optimized human WASP cDNA. To test vector performance in a more clinically relevant setting, we transduced murine HSPC as well as human CD34+ cells and also analyzed vector efficacy in their differentiated myeloid progeny. Our results show that our novel vector generates comparable WAS protein levels and is as effective as the clinically used LTR-driven vector. Therefore, the described SIN vectors appear to be good candidates for potential use in a safer new gene therapy protocol for WAS, with decreased risk of insertional mutagenesis.

Preventing and exploiting the oncogenic potential of integrating gene vectors

Gene therapy requires efficient gene delivery to cure or prevent disease by modifying the genome of somatic cells. However, gene vectors, which insert themselves into the host genome in order to achieve persistent protein expression, can trigger oncogenesis by upregulating cellular protooncogenes. This adverse event, known as insertional mutagenesis, has become a major hurdle in the field. Vectors developed on the basis of lentiviruses are considered to be less genotoxic than the hitherto used gamma-retroviral vectors. For their report in this issue of the JCI, Montini et al. utilized a tumor-prone mouse model to identify the genetic determinants of insertional mutagenesis (see the related article beginning on page 964). They report that the lentiviral integration pattern and additional improvements in vector design reduce the genotoxic risk. These findings will inform future vector design with the goal of limiting genotoxicity for gene therapy or increasing genotoxicity for protooncogene discovery.

Lentiviral Vector Induced Insertional Haploinsufficiency of Ebf1 Causes Murine Leukemia

Integrating vectors developed on the basis of various retroviruses have demonstrated therapeutic potential following genetic modification of long-lived hematopoietic stem and progenitor cells. Lentiviral vectors (LV) are assumed to circumvent genotoxic events previously observed with γ-retroviral vectors, due to their integration bias to transcription units in comparison to the γ-retroviral preference for promoter regions and CpG islands. However, recently several studies have revealed the potential for gene activation by LV insertions. Here, we report a murine acute B-lymphoblastic leukemia (B-ALL) triggered by insertional gene inactivation. LV integration occurred into the 8th intron of Ebf1, a major regulator of B-lymphopoiesis. Various aberrant splice variants could be detected that involved splice donor and acceptor sites of the lentiviral construct, inducing downregulation of Ebf1 full-length message. The transcriptome signature was compatible with loss of this major determinant of B-cell differentiation, with partial acquisition of myeloid markers, including Csf1r (macrophage colony-stimulating factor (M-CSF) receptor). This was accompanied by receptor phosphorylation and STAT5 activation, both most likely contributing to leukemic progression. Our results highlight the risk of intragenic vector integration to initiate leukemia by inducing haploinsufficiency of a tumor suppressor gene. We propose to address this risk in future vector design.