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.

Interleukin-7 receptor mutants initiate early T cell precursor leukemia in murine thymocyte progenitors with multipotent potential.

Early T cell precursor acute lymphoblastic leukemia (ETP-ALL) exhibits lymphoid, myeloid, and stem cell features and is associated with a poor prognosis. Whole genome sequencing of human ETP-ALL cases has identified recurrent mutations in signaling, histone modification, and hematopoietic development genes but it remains to be determined which of these abnormalities are sufficient to initiate leukemia. We show that activating mutations in the interleukin-7 receptor identified in human pediatric ETP-ALL cases are sufficient to generate ETP-ALL in mice transplanted with primitive transduced thymocytes from p19(Arf-/-) mice. The cellular mechanism by which these mutant receptors induce ETP-ALL is the block of thymocyte differentiation at the double negative 2 stage at which myeloid lineage and T lymphocyte developmental potential coexist. Analyses of samples from pediatric ETP-ALL cases and our murine ETP-ALL model show uniformly high levels of LMO2 expression, very low to undetectable levels of BCL11B expression, and a relative lack of activating NOTCH1 mutations. We report that pharmacological blockade of Jak-Stat signaling with ruxolitinib has significant antileukemic activity in this ETP-ALL model. This new murine model recapitulates several important cellular and molecular features of ETP-ALL and should be useful to further define novel therapeutic approaches for this aggressive leukemia.

Mouse transplant models for evaluating the oncogenic risk of a self-inactivating XSCID lentiviral vector.

Hematopoietic stem cell gene therapy requires the use of integrating retroviral vectors in order to stably transmit a therapeutic gene to mature blood cells. Human clinical trials have shown that some vector integration events lead to disrupted regulation of proto-oncogenes resulting in disordered hematopoiesis including T-cell leukemia. Newer vectors have been designed to decrease the incidence of these adverse events but require appropriate pre-clinical assays to demonstrate safety. We have used two distinct mouse serial transplant assays to evaluate the safety of a self-inactivating lentiviral vector intended for use in X-linked severe combined immunodeficiency (XSCID) gene therapy trials. These experiments entailed 28 months of total follow-up and included 386 mice. There were no cases in which the XSCID lentiviral vector clearly caused hematopoietic malignancies, although a single case of B cell malignancy was observed that contained the lentiviral vector as a likely passenger event. In contrast, a SFFV-DsRed γ-retroviral vector resulted in clonal transformation events in multiple secondary recipients. Non-specific pathology not related to vector insertions was noted including T cell leukemias arising from irradiated recipient cells. Overall, this comprehensive study of mouse transplant safety assays demonstrate the relative safety of the XSCID lentiviral vector but also highlight the limitations of these assays.

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.

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.

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.

Unique risk factors for insertional mutagenesis in a mouse model of XSCID gene therapy.

Although gene therapy can cure patients with severe combined immunodeficiency (SCID) syndromes, the clinical occurrence of T cell malignancies due to insertional mutagenesis has raised concerns about the safety of gene therapy. Several key questions have remained unanswered: (i) are there unique risk factors for X-linked SCID (XSCID) gene therapy that increase the risk of insertional mutagenesis; (ii) what other genetic lesions may contribute to transformation; and (iii) what systems can be used to test different vectors for their relative safety? To address these questions, we have developed an XSCID mouse model in which both the Arf tumor-suppressor gene and the gammac gene were ablated. Gene therapy in this animal model recapitulates the high incidence of integration-dependent, T cell tumors that was seen in the clinical trial. Ligation-mediated PCR analysis showed integration sites near or within established protooncogenes (Chd9, Slamf6, Tde1, Camk2b, and Ly6e), demonstrating that T cell transformation was associated with targeting of oncogene loci; however, no integrations within the Lmo2 locus were identified. The X-SCID background in transplanted cells was required for high rate transformation and was associated with expansion of primitive hematopoietic cells that may serve tumor precursors. This model should be useful for testing safety-modified vectors and for further exploring the risk factors leading to insertional mutagenesis in gene therapy trials.

Transient in vivo selection of transduced peripheral blood cells using antifolate drug selection in rhesus macaques that received transplants with hematopoietic stem cells expressing dihydrofolate reductase vectors.

One of the main obstacles for effective human gene therapy for hematopoietic disorders remains the achievement of an adequate number of genetically corrected blood cells. One approach to this goal is to incorporate drug resistance genes into vectors to enable in vivo selection of hematopoietic stem cells (HSCs). Although a number of drug resistance vectors enable HSC selection in murine systems, little is known about these systems in large animal models. To address this issue, we transplanted cells transduced with dihydrofolate resistance vectors into 6 rhesus macaques and studied whether selection of vector-expressing cells occurred following drug treatment with trimetrexate and nitrobenzylmercaptopurineriboside-phosphate. In some of the 10 administered drug treatment courses, substantial increases in the levels of transduced peripheral blood cells were noted; however, numbers returned to baseline levels within 17 days. Attempts to induce stem cell cycling with stem cell factor and granulocyte-colony stimulating factor prior to drug treatment did not lead to sustained enrichment for transduced cells. These data highlight an important species-specific difference between murine and nonhuman primate models for assessing in vivo HSC selection strategies and emphasize the importance of using drugs capable of inducing selective pressure at the level of HSCs.

Hematopoietic cells from mice that are deficient in both Bcrp1/Abcg2 and Mdr1a/1b develop normally but are sensitized to mitoxantrone.

Hematopoietic stem cells (HSCs) express Mdr1a/1b and Bcrp1/Abcg2, which are members of the ATP binding cassette transporter family. Mice lacking both Mdr1-type genes (Mdr1a and Mdr1b) or Bcrp1 had normal hematopoietic development, but it has been unclear whether Mdr1a/1b and Bcrp1 play redundant roles in hematopoiesis. We generated a mouse model lacking both Mdr1a/1b and Bcrp1 expression (M-/-B-/-). The M-/-B-/- mice had normal numbers of peripheral blood cells, bone marrow colony-forming cells (CFCs) and colony-forming units-spleen (CFU-S), and demonstrated normal hematopoietic development. There was a near total elimination of side population (SP) cells in the bone marrow of M-/-B-/- mice compared to M+/+B-/- mice, primarily in the subpopulation lacking other HSC markers, which indicated that Mdr1a/1b was responsible for a small portion of SP cells that were mainly mature cells. Hematopoietic progenitor cells from the bone marrow of M-/-B-/- mice were more sensitive to mitoxantrone in vitro compared to either M-/-B+/+ or M+/+B-/- mice, suggesting that Mdr1a/1b and Bcrp1 may provide additive protection to HSCs against genotoxic agents. These studies demonstrate the lack of functional redundancy between these transporters for HSC development and further clarify their contributing role to the SP phenotype in HSCs and to intrinsic drug resistance within hematopoietic progenitor cells.