Lentiviral Hematopoietic Stem Cell Gene Therapy Corrects Murine Pompe Disease.

Pompe disease is an autosomal recessive lysosomal storage disorder characterized by progressive muscle weakness. The disease is caused by mutations in the acid α-glucosidase (GAA) gene. Despite the currently available enzyme replacement therapy (ERT), roughly half of the infants with Pompe disease die before the age of 3 years. Limitations of ERT are immune responses to the recombinant enzyme, incomplete correction of the disease phenotype, lifelong administration, and inability of the enzyme to cross the blood-brain barrier. We previously reported normalization of glycogen in heart tissue and partial correction of the skeletal muscle phenotype by ex vivo hematopoietic stem cell gene therapy. In the present study, using a codon-optimized GAA (GAAco), the enzyme levels resulted in close to normalization of glycogen in heart, muscles, and brain, and in complete normalization of motor function. A large proportion of microglia in the brain was shown to be GAA positive. All astrocytes contained the enzyme, which is in line with mannose-6-phosphate receptor expression and the key role in glycogen storage and glucose metabolism. The lentiviral vector insertion site analysis confirmed no preference for integration near proto-oncogenes. This correction of murine Pompe disease warrants further development toward a cure of the human condition.

SUL-109 Protects Hematopoietic Stem Cells from Apoptosis Induced by Short-Term Hypothermic Preservation and Maintains Their Engraftment Potential.

The newly developed 6-hydroxychromanol derivate SUL-109 was shown to provide protection during hypothermic storage of several cell lines, but has not been evaluated in hematopoietic stem cells (HSCs). Hypothermic preservation of HSCs would be preferred over short-term cryopreservation to prevent cell loss during freezing/thawing and would be particularly useful for short-term storage, such as during conditioning of patients or transport of HSC transplants. Here we cultured human CD34+ umbilical cord blood (UCB) cells and lineage-depleted (Lin-) Balb/c bone marrow (BM) cells for up to 7 days in serum-free HSC expansion medium with hematopoietic growth factors. SUL-109-containing cultures were stored at 4°C for 3 to 14 days. The UCB cells were tested for viability, cell cycle, and reactive oxygen species (ROS). DMSO-cryopreserved Lin- BM cells or Lin- BM cells maintained for 14 days at 4°C were transplanted into RAG2-/- Balb/c mice and engraftment was followed for 6 months. The addition of SUL-109 during the hypothermic storage of expanded CD34+ UCB cells provided a significant improvement in cell survival of the immature CD34+/CD38- fraction after 7 days of hypothermic storage through scavenging of hypothermia-induced ROS and was able to preserve the multilineage capacity of human CD34+ UCB cells for up to 14 days of cold storage. In addition, SUL-109 protected murine BM Lin- cells from 14 days of hypothermic preservation and maintained their engraftment potential after transplantation in immune-deficient RAG2-/- mice. Our data indicate that SUL-109 is a promising novel chemical for use as a protective agent during cold storage of human and murine HSCs to prevent hypothermia-induced apoptosis and promote cell viability.

Preclinical Efficacy and Safety Evaluation of Hematopoietic Stem Cell Gene Therapy in a Mouse Model of MNGIE.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by thymidine phosphorylase (TP) deficiency resulting in systemic accumulation of thymidine (d-Thd) and deoxyuridine (d-Urd) and characterized by early-onset neurological and gastrointestinal symptoms. Long-term effective and safe treatment is not available. Allogeneic bone marrow transplantation may improve clinical manifestations but carries disease and transplant-related risks. In this study, lentiviral vector-based hematopoietic stem cell gene therapy (HSCGT) was performed in Tymp-/-Upp1-/- mice with the human phosphoglycerate kinase (PGK) promoter driving TYMP. Supranormal blood TP activity reduced intestinal nucleoside levels significantly at low vector copy number (median, 1.3; range, 0.2-3.6). Furthermore, we covered two major issues not addressed before. First, we demonstrate aberrant morphology of brain astrocytes in areas of spongy degeneration, which was reversed by HSCGT. Second, long-term follow-up and vector integration site analysis were performed to assess safety of the therapeutic LV vectors in depth. This report confirms and supplements previous work on the efficacy of HSCGT in reducing the toxic metabolites in Tymp-/-Upp1-/- mice, using a clinically applicable gene transfer vector and a highly efficient gene transfer method, and importantly demonstrates phenotypic correction with a favorable risk profile, warranting further development toward clinical implementation.

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.

Efficacy of lentivirus-mediated gene therapy in an Omenn syndrome recombination-activating gene 2 mouse model is not hindered by inflammation and immune dysregulation.

BACKGROUND: Omenn syndrome (OS) is a rare severe combined immunodeficiency associated with autoimmunity and caused by defects in lymphoid-specific V(D)J recombination. Most patients carry hypomorphic mutations in recombination-activating gene (RAG) 1 or 2. Hematopoietic stem cell transplantation is the standard treatment; however, gene therapy (GT) might represent a valid alternative, especially for patients lacking a matched donor. OBJECTIVE: We sought to determine the efficacy of lentiviral vector (LV)-mediated GT in the murine model of OS (Rag2R229Q/R229Q) in correcting immunodeficiency and autoimmunity. METHODS: Lineage-negative cells from mice with OS were transduced with an LV encoding the human RAG2 gene and injected into irradiated recipients with OS. Control mice underwent transplantation with wild-type or OS-untransduced lineage-negative cells. Immunophenotyping, T-dependent and T-independent antigen challenge, immune spectratyping, autoantibody detection, and detailed tissue immunohistochemical analyses were performed. RESULTS: LV-mediated GT allowed immunologic reconstitution, although it was suboptimal compared with that seen in wild-type bone marrow (BM)-transplanted OS mice in peripheral blood and hematopoietic organs, such as the BM, thymus, and spleen. We observed in vivo variability in the efficacy of GT correlating with the levels of transduction achieved. Immunoglobulin levels and T-cell repertoire normalized, and gene-corrected mice responded properly to challenges in vivo. Autoimmune manifestations, such as skin infiltration and autoantibodies, dramatically improved in GT mice with a vector copy number/genome higher than 1 in the BM and 2 in the thymus. CONCLUSIONS: Our data show that LV-mediated GT for patients with OS significantly ameliorates the immunodeficiency, even in an inflammatory environment.

The potential for chemical mixtures from the environment to enable the cancer hallmark of sustained proliferative signalling.

The aim of this work is to review current knowledge relating the established cancer hallmark, sustained cell proliferation to the existence of chemicals present as low dose mixtures in the environment. Normal cell proliferation is under tight control, i.e. cells respond to a signal to proliferate, and although most cells continue to proliferate into adult life, the multiplication ceases once the stimulatory signal disappears or if the cells are exposed to growth inhibitory signals. Under such circumstances, normal cells remain quiescent until they are stimulated to resume further proliferation. In contrast, tumour cells are unable to halt proliferation, either when subjected to growth inhibitory signals or in the absence of growth stimulatory signals. Environmental chemicals with carcinogenic potential may cause sustained cell proliferation by interfering with some cell proliferation control mechanisms committing cells to an indefinite proliferative span.

Assessing the carcinogenic potential of low-dose exposures to chemical mixtures in the environment: the challenge ahead.

Lifestyle factors are responsible for a considerable portion of cancer incidence worldwide, but credible estimates from the World Health Organization and the International Agency for Research on Cancer (IARC) suggest that the fraction of cancers attributable to toxic environmental exposures is between 7% and 19%. To explore the hypothesis that low-dose exposures to mixtures of chemicals in the environment may be combining to contribute to environmental carcinogenesis, we reviewed 11 hallmark phenotypes of cancer, multiple priority target sites for disruption in each area and prototypical chemical disruptors for all targets, this included dose-response characterizations, evidence of low-dose effects and cross-hallmark effects for all targets and chemicals. In total, 85 examples of chemicals were reviewed for actions on key pathways/mechanisms related to carcinogenesis. Only 15% (13/85) were found to have evidence of a dose-response threshold, whereas 59% (50/85) exerted low-dose effects. No dose-response information was found for the remaining 26% (22/85). Our analysis suggests that the cumulative effects of individual (non-carcinogenic) chemicals acting on different pathways, and a variety of related systems, organs, tissues and cells could plausibly conspire to produce carcinogenic synergies. Additional basic research on carcinogenesis and research focused on low-dose effects of chemical mixtures needs to be rigorously pursued before the merits of this hypothesis can be further advanced. However, the structure of the World Health Organization International Programme on Chemical Safety 'Mode of Action' framework should be revisited as it has inherent weaknesses that are not fully aligned with our current understanding of cancer biology.

Lentiviral hematopoietic stem cell gene therapy in inherited metabolic disorders.

After more than 20 years of development, lentiviral hematopoietic stem cell gene therapy has entered the stage of initial clinical implementation for immune deficiencies and storage disorders. This brief review summarizes the development and applications, focusing on the lysosomal enzyme deficiencies, especially Pompe disease.

Pretransplant mobilization with granulocyte colony-stimulating factor improves B-cell reconstitution by lentiviral vector gene therapy in SCID-X1 mice.

Hematopoietic stem cell (HSC) gene therapy is a demonstrated effective treatment for X-linked severe combined immunodeficiency (SCID-X1), but B-cell reconstitution and function has been deficient in many of the gene therapy treated patients. Cytoreductive preconditioning is known to improve HSC engraftment, but in general it is not considered for SCID-X1 since the poor health of most of these patients at diagnosis and the risk of toxicity preclude the conditioning used in standard bone marrow stem cell transplantation. We hypothesized that mobilization of HSC by granulocyte colony-stimulating factor (G-CSF) should create temporary space in bone marrow niches to improve engraftment and thereby B-cell reconstitution. In the present pilot study supplementing our earlier preclinical evaluation (Huston et al., 2011), Il2rg(-/-) mice pretreated with G-CSF were transplanted with wild-type lineage negative (Lin(-)) cells or Il2rg(-/-) Lin(-) cells transduced with therapeutic IL2RG lentiviral vectors. Mice were monitored for reconstitution of lymphocyte populations, level of donor cell chimerism, and antibody responses as compared to 2 Gy total body irradiation (TBI), previously found effective in promoting B-cell reconstitution. The results demonstrate that G-CSF promotes B-cell reconstitution similar to low-dose TBI and provides proof of principle for an alternative approach to improve efficacy of gene therapy in SCID patients without adverse effects associated with cytoreductive conditioning.

Lentiviral gene transduction of mouse and human hematopoietic stem cells.

Lentiviral vectors can be used to genetically modify a broad range of cells. Hematopoietic stem cells (HSCs) are particularly suitable for lentiviral gene augmentation, because these cells can be enriched with relative ease from mouse bone marrow and human hematopoietic sources, and in principle require relatively limited cell numbers to completely reconstitute the hematopoietic system in vivo. Furthermore, lentiviral vectors are very efficient if pseudotyped with broad tropism envelope proteins. This chapter focuses on gene modification by the use of self-inactivating third-generation human immunodeficiency virus-derived lentiviral vectors for ex vivo HSC modification for both mouse and human application.

Recombination-activating gene 1 (Rag1)-deficient mice with severe combined immunodeficiency treated with lentiviral gene therapy demonstrate autoimmune Omenn-like syndrome.

BACKGROUND: Recombination-activating gene 1 (RAG1) deficiency results in severe combined immunodeficiency (SCID) caused by a complete lack of T and B lymphocytes. If untreated, patients succumb to recurrent infections. OBJECTIVES: We sought to develop lentiviral gene therapy for RAG1-induced SCID and to test its safety. METHODS: Constructs containing the viral spleen-focus-forming virus (SF), ubiquitous promoters, or cell type-restricted promoters driving sequence-optimized RAG1 were compared for efficacy and safety in sublethally preconditioned Rag1(-/-) mice undergoing transplantation with transduced bone marrow progenitors. RESULTS: Peripheral blood CD3(+) T-cell reconstitution was achieved with SF, ubiquitous promoters, and cell type-restricted promoters but 3- to 18-fold lower than that seen in wild-type mice, and with a compromised CD4(+)/CD8(+) ratio. Mitogen-mediated T-cell responses and T cell-dependent and T cell-independent B-cell responses were not restored, and T-cell receptor patterns were skewed. Reconstitution of mature peripheral blood B cells was approximately 20-fold less for the SF vector than in wild-type mice and often not detectable with the other promoters, and plasma immunoglobulin levels were abnormal. Two months after transplantation, gene therapy-treated mice had rashes with cellular tissue infiltrates, activated peripheral blood CD44(+)CD69(+) T cells, high plasma IgE levels, antibodies against double-stranded DNA, and increased B cell-activating factor levels. Only rather high SF vector copy numbers could boost T- and B-cell reconstitution, but mRNA expression levels during T- and B-cell progenitor stages consistently remained less than wild-type levels. CONCLUSIONS: These results underline that further development is required for improved expression to successfully treat patients with RAG1-induced SCID while maintaining low vector copy numbers and minimizing potential risks, including autoimmune reactions resembling Omenn syndrome.

In vivo expansion of co-transplanted T cells impacts on tumor re-initiating activity of human acute myeloid leukemia in NSG mice.

Human cells from acute myeloid leukemia (AML) patients are frequently transplanted into immune-compromised mouse strains to provide an in vivo environment for studies on the biology of the disease. Since frequencies of leukemia re-initiating cells are low and a unique cell surface phenotype that includes all tumor re-initiating activity remains unknown, the underlying mechanisms leading to limitations in the xenotransplantation assay need to be understood and overcome to obtain robust engraftment of AML-containing samples. We report here that in the NSG xenotransplantation assay, the large majority of mononucleated cells from patients with AML fail to establish a reproducible myeloid engraftment despite high donor chimerism. Instead, donor-derived cells mainly consist of polyclonal disease-unrelated expanded co-transplanted human T lymphocytes that induce xenogeneic graft versus host disease and mask the engraftment of human AML in mice. Engraftment of mainly myeloid cell types can be enforced by the prevention of T cell expansion through the depletion of lymphocytes from the graft prior transplantation.

Comprehensive investigation of parameter choice in viral integration site analysis and its effects on the gene annotations produced.

Introducing therapeutic genes into hematopoietic stem cells using retroviral vector-mediated gene transfer is an effective treatment for monogenic diseases. The risks of therapeutic gene integration include aberrant expression of a neighboring gene, resulting in oncogenesis at low frequencies (10(-7)-10(-6)/transduced cell). Mechanisms governing insertional mutagenesis are the subject of intensive ongoing studies that produce large amounts of sequencing data representing genomic regions flanking viral integration sites (IS). Validating and analyzing these data require automated bioinformatics applications. The exact methods used vary between applications, based on the requirements and preferences of the designer. The parameters used to analyze sequence data are capable of shaping the resulting integration site annotations, but a comprehensive examination of these effects is lacking. Here we present a web-based tool for integration site analysis, called Methods for Analyzing ViRal Integration Collections (MAVRIC), and use its highly customizable interface to look at how IS annotations can vary based on the analysis parameters. We used the integration data of the previously published adenosine deaminase severe combined immunodeficiency (ADA-SCID) gene therapy trials for evaluation of MAVRIC. The output illustrates how MAVRIC allows for direct multiparameter comparison of integration patterns. Careful analysis of the SCID data and reanalyses using different parameters for trimming, alignment, and repeat masking revealed the degree of variation that can be expected to arise due to changes in these parameters. We observed mainly small differences in annotation, with the largest effects caused by masking repeat sequences and by changing the size of the window around the IS.

Correction of murine Rag2 severe combined immunodeficiency by lentiviral gene therapy using a codon-optimized RAG2 therapeutic transgene.

Recombination activating gene 2 (RAG2) deficiency results in severe combined immunodeficiency (SCID) with complete lack of T and B lymphocytes. Initial gammaretroviral gene therapy trials for other types of SCID proved effective, but also revealed the necessity of safe vector design. We report the development of lentiviral vectors with the spleen focus forming virus (SF) promoter driving codon-optimized human RAG2 (RAG2co), which improved phenotype amelioration compared to native RAG2 in Rag2(-/-) mice. With the RAG2co therapeutic transgene, T-cell receptor (TCR) and immunoglobulin repertoire, T-cell mitogen responses, plasma immunoglobulin levels and T-cell dependent and independent specific antibody responses were restored. However, the thymus double positive T-cell population remained subnormal, possibly due to the SF virus derived element being sensitive to methylation/silencing in the thymus, which was prevented by replacing the SF promoter by the previously reported silencing resistant element (ubiquitous chromatin opening element (UCOE)), and also improved B-cell reconstitution to eventually near normal levels. Weak cellular promoters were effective in T-cell reconstitution, but deficient in B-cell reconstitution. We conclude that immune functions are corrected in Rag2(-/-) mice by genetic modification of stem cells using the UCOE driven codon-optimized RAG2, providing a valid optional vector for clinical implementation.

5-Androstene-3ß,17ß-diol promotes recovery of immature hematopoietic cells following myelosuppressive radiation and synergizes with thrombopoietin.

PURPOSE: 5-Androstene-3ß,17ß-diol (5-AED) stimulates recovery of hematopoiesis after exposure to radiation. To elucidate its cellular targets, the effects of 5-AED alone and in combination with (pegylated) granulocyte colony-stimulating factor and thrombopoietin (TPO) on immature hematopoietic progenitor cells were evaluated following total body irradiation. METHODS AND MATERIALS: BALB/c mice were exposed to radiation delivered as a single or as a fractionated dose, and recovery of bone marrow progenitors and peripheral blood parameters was assessed. RESULTS: BALB/c mice treated with 5-AED displayed accelerated multilineage blood cell recovery and elevated bone marrow (BM) cellularity and numbers of progenitor cells. The spleen colony-forming unit (CFU-S) assay, representing the life-saving short-term repopulating cells in BM of irradiated donor mice revealed that combined treatment with 5-AED plus TPO resulted in a 20.1-fold increase in CFU-S relative to that of placebo controls, and a 3.7 and 3.1-fold increase in comparison to 5-AED and TPO, whereas no effect was seen of Peg-G-CSF with or without 5-AED. Contrary to TPO, 5-AED also stimulated reconstitution of the more immature marrow repopulating (MRA) cells. CONCLUSIONS: 5-AED potently counteracts the hematopoietic effects of radiation-induced myelosuppression and promotes multilineage reconstitution by stimulating immature bone marrow cells in a pattern distinct from, but synergistic with TPO.

Keratinocyte growth factor and stem cell factor to improve thymopoiesis after autologous CD34+ cell transplantation in rhesus macaques.

Deficient thymopoiesis and retarded recovery of naive CD4(+) T cells are important determinants of insufficient immune-competence following hematopoietic stem cell transplantation (HSCT). Although keratinocyte growth factor (KGF) may protect the thymic epithelium, stem cell factor (SCF) is involved in early thymopoiesis. We evaluated whether KGF alone or combined with SCF would affect thymopoiesis and hematologic recovery following myeloablative autologous HSCT into rhesus macaques. Purpose-bred adult rhesus macaques received 10(6) autologous CD34(+)-selected mononuclear bone marrow cells (BMC)/kg after 9 Gy myeloablative conditioning. Animals were treated with phosphate-buffered saline (PBS) (n = 2), KGF alone (n = 2), or KGF combined with SCF (n = 2). KGF-treated animals showed accelerated hematologic recovery, improved thymopoiesis, and enhanced naive T-cell recovery following transplantation. Improved T cell recovery was not associated with protection against cytomegalovirus reactivation nor with improved antibody response to tetanus toxoid vaccination. Animals treated with KGF and SCF experienced severe adverse events that precluded evaluation of thymopoiesis and T cell recovery. Collectively, our data confirm that KGF may enhance thymopoiesis.