Peter Haviernik

Safety Concerns Related to Hematopoietic Stem Cell Gene Transfer Using Retroviral Vectors

Endogenous retroviruses have developed efficient methods during their life cycle for stable integration into the host genome. Because of this ability, retroviral vectors were designed with the goal of gene transfer into hematopoietic stem cells (HSCs). The ability to genetically modify HSCs provides a vehicle for durable expression of potentially therapeutic transgenes in all lineages of mature blood cells for the lifetime of the patient. Combined with bone marrow transplant, retroviral gene transfer has many potential applications for a wide range of blood diseases. Advances in the development of oncoretroviral vectors based on murine leukemia viruses (MLV) and more recent development of human immunodeficiency virus (HIV)-based vectors have greatly increased the gene transfer efficiency. Optimization of methods for gene transfer using MLV-based vectors has substantially improved marking levels in mice, with lower levels in large animals and in human clinical trials. With advances in gene transfer technology has also come renewed concern about insertional mutagenesis and activation of oncogenes. Advanced techniques for integration site analysis combined with sequence comparison using mouse and human genome databases has now made it possible to begin to understand the spectrum of possible integration sites for both MLV- and HIV-based vectors. Furthermore, other studies have shown positive and negative dosage-dependent effects of transgene expression in mouse and human cells. Therefore, vector design and safety testing are at the forefront of the field of gene therapy and this review discusses recent developments.

An ENU-induced recessive mutation in Mpl leads to thrombocytopenia with overdominance

OBJECTIVE The aim of this study was to identify and characterize the causative mutation in the thrombocytopenic mouse strain HLB219 that was generated at the Jackson Laboratory as part of a large-scale N-ethyl-N-nitrosourea mutagenesis screen. MATERIALS AND METHODS The HLB219 mutation was identified by interval mapping of F2 mice generated from intercross breeding of HLB219 to both BALB/cByJ (BALB) and 129/SvImJ (129/Sv). Mpl was identified as a candidate gene and sequenced. The mutation was characterized in vivo in mouse hematopoietic stem/progenitor cell assays and in cell culture by expression in Ba/F3 cells. RESULTS A novel mutation in the thrombopoietin (TPO) receptor Mpl in HLB219 mice caused a Cys-->Arg substitution at codon 40 in the extracellular region of the receptor. Mice homozygous for the Mpl(hlb219) mutation had an 80% decrease in the number of platelets in comparison to the wild-type C57BL/6J strain, low numbers of bone marrow megakaryocytes, high TPO levels, and decreased competitive repopulating ability, consistent with a loss-of-function mutation in the receptor. Mice heterozygous for Mpl(hlb219) however, showed an overdominance effect with a significant increase in platelet number. Functional analysis in vitro demonstrated that Ba/F3 cells expressing the mutant MPL(hlb219) protein failed to activate extracellular signal-regulated kinase and signal transducers and activators of transcription 5, but proliferated in the absence of TPO and required constitutive phosphorylation of RAC-alpha serine/threonine protein kinase (AKT) for cytokine-independent growth. CONCLUSION Thrombocytopenia in HLB219 mice is caused by a recessive mutation in Mpl that abrogates mitogen-activated protein kinase-extracellular signal regulated kinase and janus kinase-signal transducers and activators of transcription signaling.

Regulation of IL-2 expression by transcription factor BACH2 in umbilical cord blood CD4+ T cells

On activation, umbilical cord blood (UCB) CD4+ T cells demonstrate reduced expression of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), whereas maintaining equivalent interleukin-2 (IL-2) levels, as compared with adult peripheral blood (PB) CD4+ T cells. Nuclear factor of activated T cells (NFAT1) protein, a transcription factor known to regulate the expression of IL-2, TNF-α and IFN-γ, is reduced in resting and activated UCB CD4+ T cells. In contrast, expression of Broad-complex-Tramtrack-Bric-a-Brac and Cap‘n’collar homology 1 bZip transcription factor 2 (BACH2) was shown by gene array analyses to be increased in UCB CD4+ T cells and was validated by qRT-PCR. Using chromatin immunoprecipitation, BACH2 was shown binding to the human IL-2 proximal promoter. Knockdown experiments of BACH2 by transient transfection of UCB CD4+ T cells with BACH2 siRNA resulted in significant reductions in stimulated IL-2 production. Decreased IL-2 gene transcription in UCB CD4+ T cells transfected with BACH2 siRNA was confirmed by a human IL-2 luciferase assay. In summary, BACH2 maintains IL-2 expression in UCB CD4+ T cells at levels equivalent to adult PB CD4+ T cells despite reduced NFAT1 protein expression. Thus, BACH2 expression is necessary to maintain IL-2 production when NFAT1 protein is reduced, potentially impacting UCB graft CD4+ T-cell allogeneic responses.

Retroviral transduction of murine hematopoietic stem cells.

Hematopoietic stem cells (HSC) are inherently rare cell types that cannot be obtained in sufficient amounts for classical biochemical characterization. To facilitate functional studies of murine HSC and hematopoietic development, the technique of retroviral-mediated gene transfer provides a useful tool. The generation of high titer retroviral vectors permits transduction of stem cells with a variety of genes and leads to long-term marking in the blood of recipient mice. Optimized promoter/enhancers facilitate high-level transgene expression in mice transplanted with transduced bone marrow (BM) cells. The co-expression of reporter genes along with a gene of interest greatly facilitates tracking donor engraftment of transduced hematopoietic progeny following stem cell transplantation. This methodology can be used to reconstitute defective function in a mutant background or to study protein function during hematopoiesis by overexpression. Despite limitations such as integration site variegation and copy number-dependent effects, this approach is rapid and efficient compared with transgenic mouse technology. In this chapter, we review this broadly applicable technique for achieving high-level murine BM stem cell transduction. We also describe methods for transplantation and subsequent analysis of transplanted mice as a bona fide assay for the stem cell transduction efficiency.

Hematopoiesis in mice is extremely resilient to wide variation in TIMP/MMP balance

Tissue inhibitors of matrix metalloproteinases (TIMPs) are natural inhibitors of matrix metalloproteinases (MMPs) and are associated with normal and pathologic extracellular matrix turnover. Because the microenvironment is critical for normal hematopoietic stem/progenitor cell function, we aimed to determine whether alterations in the TIMP/MMP balance impact upon normal hematopoiesis in mice. We have used both overexpression and knockout mouse models to determine whether early hematopoiesis is susceptible to potentially pathologic changes in TIMP/MMP level. These studies used TIMP-1(-/-) mice and retroviral vectors co-expressing human TIMP-1 or TIMP-2 linked with the green fluorescent protein (GFP) transduced into bone marrow (BM) cells and transplanted into lethally-irradiated recipient mice. Loss of TIMP-1 in knockout mice or retroviral overexpression of TIMP-1 or TIMP-2 did not alter hematopoietic stem/progenitor function during steady-state hematopoiesis. Surprisingly, even when applying hematopoietic stress through mobilization, chemotaxis, or myelosuppression, murine hematopoiesis was not adversely affected by TIMP-1 or TIMP-2 level. We conclude that TIMP/MMP balance alone does not exert significant influence on blood cell development and homeostasis. An important corollary of these studies is that specific modulation using MMP inhibitors for cancer or immunologic therapy is unlikely to have adverse hematopoietic side effects.