Renier Myburgh - Academia.edu (original) (raw)

Papers by Renier Myburgh

Research paper thumbnail of Engineering an HIV-1 resistant immune system

Gene knock-down using miRNA-based vector constructs is likely to become a prominent gene therapy ... more Gene knock-down using miRNA-based vector constructs is likely to become a prominent gene therapy approach. It was the aim of this study to improve the efficiency of gene knock-down through optimizing the structure of miRNA mimics. Knock-down of two target genes was analyzed: CCR5 and GFP. We describe here a novel and optimized miRNA mimic design called mirGE comprising a lower stem length of 13 base pairs, positioning of the targeting strand on the 5' side of the miRNA, together with nucleotide mismatches in upper stem positions 1 and 12 placed on the passenger strand. Our mirGE proved superior to miR-30 in four aspects: yield of targeting strand incorporation into RISC; incorporation into RISC of correct targeting strand; precision of cleavage by Drosha and ratio of targeting strand over passenger strand. A triple mirGE hairpin cassette targeting CCR5 was constructed. It allowed CCR5 knock-down with an efficiency of over 90% upon single copy transduction. Importantly, single copy expression of this construct rendered transduced target cells, including primary human macrophages, resistant to infection with a CCR5-tropic strain of HIV. Our results provide new insights for a better knock-down efficiency of constructs containing miRNA. Our results also provide the proof-of-principle that cells can be rendered HIV-resistant through single copy vector transduction, rendering this approach more compatible with clinical applications. iv) Review article (In preparation) Clinical applications of genetically modified hematopoietic stem cells

Research paper thumbnail of Anti-CD117 immunotherapy to eliminate hematopoietic and leukemia stem cells

Experimental Hematology

Precise replacement of diseased or dysfunctional organs is the goal of regenerative medicine and ... more Precise replacement of diseased or dysfunctional organs is the goal of regenerative medicine and has appeared to be a distant goal for a long time. In the field of hematopoietic stem cell transplantation, this goal is now becoming tangible as gene-editing technologies and novel conditioning agents are entering the clinical arena. Targeted immunologic depletion of hematopoietic stem cells (HSCs), which are at the very root of the hematopoietic system, will enable more selective and potentially more effective hematopoietic stem cell transplantation in patients with hematological diseases. In contrast to current conditioning regimes based on ionizing radiation and chemotherapy, immunologic conditioning will spare mature hematopoietic cells and cause substantially less inflammation and unspecific collateral damage to other organs. Biological agents that target the stem cell antigen CD117 are the frontrunners for this purpose and have exhibited preclinical activity in depletion of healthy HSCs. The value of anti-CD117 antibodies as conditioning agents is currently being evaluated in early clinical trials. Whereas mild, antibody-based immunologic conditioning concepts might be appropriate for benign hematological disorders in which incomplete replacement of diseased cells is sufficient, higher efficacy will be required for treatment and elimination of hematologic stem cell malignancies such as acute myeloid leukemia and myelodysplastic syndrome. Antibody−drug conjugates, bispecific T-cell engaging and activating antibodies (TEAs), or chimeric antigen receptor (CAR) T cells might offer increased efficacy compared with naked antibodies and yet higher tolerability and safety compared with current genotoxic conditioning approaches. Here, we summarize the current state regarding immunologic conditioning concepts for the treatment of HSC disorders and outline potential future developments.

Research paper thumbnail of EBV renders B cells susceptible to HIV-1 in humanized mice

Life Science Alliance

HIV and EBV are human pathogens that cause a considerable burden to worldwide health. In combinat... more HIV and EBV are human pathogens that cause a considerable burden to worldwide health. In combination, these viruses are linked to AIDS-associated lymphomas. We found that EBV, which transforms B cells, renders them susceptible to HIV-1 infection in a CXCR4 and CD4-dependent manner in vitro and that CXCR4-tropic HIV-1 integrates into the genome of these B cells with the same molecular profile as in autologous CD4+ T cells. In addition, we established a humanized mouse model to investigate the in vivo interactions of EBV and HIV-1 upon coinfection. The respective mice that reconstitute human immune system components upon transplantation with CD34+ human hematopoietic progenitor cells could recapitulate aspects of EBV and HIV immunobiology observed in dual-infected patients. Upon coinfection of humanized mice, EBV/HIV dual-infected B cells could be detected, but were susceptible to CD8+ T-cell–mediated immune control.

Research paper thumbnail of Stereo- and regiodefined DNA-encoded chemical libraries enable efficient tumour-targeting applications

Research paper thumbnail of Anti-human CD117 CAR T-cells efficiently eliminate healthy and malignant CD117-expressing hematopoietic cells

Research paper thumbnail of BRAFV 600E or mutant MAP2K1 human CD34+ cells establish Langerhans cell–like histiocytosis in immune-deficient mice

Blood Advances

Key Points BRAFV600E or mutant MAP2K1 expression in human CB CD34+ HSPCs lead to Langerhans cell–... more Key Points BRAFV600E or mutant MAP2K1 expression in human CB CD34+ HSPCs lead to Langerhans cell–like histiocytosis in immune-deficient mice. BRAFV600E-expressing human CB CD34+ HSPCs did not generate hairy cell leukemia in xenograft mouse models.

Research paper thumbnail of Abstract 4229: Anti-P329G-CAR-T cells as a novel universal CAR-T cell platform

Clinical Research (Excluding Clinical Trials)

Research paper thumbnail of Impact of Ligand Size and Conjugation Chemistry on the Performance of Universal Chimeric Antigen Receptor TCells for Tumor Killing

Research paper thumbnail of Anti-Human CD117 CAR T-Cells Efficiently Eliminate Hematopoietic Stem and CD117-Positive AML Cells

Blood

Introduction: Acute Myeloid Leukemia (AML) is a clonal disease of the hematopoietic system that o... more Introduction: Acute Myeloid Leukemia (AML) is a clonal disease of the hematopoietic system that originates from immature hematopoietic stem and progenitor cells (HSPC). Because some AML-initiating cells are comparatively resistant to conventional cytotoxic agents, disease relapses are common with current treatment approaches. As an alternative, immunological eradication of leukemic cells by adoptively transferred chimeric-antigen receptor T-cells (CAR T-cells) might be considerably more efficient. To date, however, the search for AML-specific surface antigens has remained largely elusive. To circumvent this problem, we propose to target the stem cell antigen c-Kit (CD117) that is expressed by physiological HSPC as wells as by leukemic blasts in >90% of AML patients. For translation into a clinical setting, CAR T cell treatment must then be followed by depletion of CAR T-cells as well subsequent healthy/allogeneic HSC transplantation. Methods: A lentiviral vector was generated whi...

Research paper thumbnail of Development of a novel fully-human anti-CD123 antibody to target acute myeloid leukemia

Research paper thumbnail of Antiviral Activity of HIV gp120-Targeting Bispecific T Cell Engager Antibody Constructs

Journal of virology, Jul 15, 2018

Today's gold standard in HIV therapy is combined antiretroviral therapy (cART). It requires s... more Today's gold standard in HIV therapy is combined antiretroviral therapy (cART). It requires strict adherence by patients and lifelong medication, which can lower the viral load below detection limits and prevent HIV-associated immunodeficiency but cannot cure patients. The bispecific T cell-engaging (BiTE) antibody technology has demonstrated long-term relapse-free outcomes in patients with relapsed and refractory acute lymphocytic leukemia. Here, we generated BiTE antibody constructs that target the HIV-1 envelope protein gp120 (HIV gp120) using either the scFv B12 or VRC01, the first two extracellular domains (1 + 2) of human CD4 alone or joined to the single chain variable fragment (scFv) of the antibody 17b fused to an anti-human CD3ε scFv. These engineered human BiTE antibody constructs showed engagement of T cells for redirected lysis of HIV gp120-transfected CHO cells. Furthermore, they substantially inhibited HIV-1 replication in peripheral blood mononuclear cells (PBMCs...

Research paper thumbnail of 448. Lentivector Knock-Down of CCR5 in Hematopoietic Stem Cells Confers Functional and Persistent HIV-1 Resistance in Humanized Mice

Research paper thumbnail of 542. A Novel In Vitro/Vivo Selection/Suicide Strategy for Lentiviral Vectors

Research paper thumbnail of Long-term leukocyte reconstitution in NSG mice transplanted with human cord blood hematopoietic stem and progenitor cells

BMC immunology, Jan 30, 2017

Humanized mice (hu mice) are based on the transplantation of hematopoietic stem and progenitor ce... more Humanized mice (hu mice) are based on the transplantation of hematopoietic stem and progenitor cells into immunodeficient mice and have become important pre-clinical models for biomedical research. However, data about their hematopoiesis over time are scarce. We therefore characterized leukocyte reconstitution in NSG mice, which were sublethally irradiated and transplanted with human cord blood-derived CD34+ cells at newborn age, longitudinally in peripheral blood and, for more detailed analyses, cross-sectionally in peripheral blood, spleen and bone marrow at different time points. Human cell chimerism and absolute human cell count decreased between week 16 and 24 in the peripheral blood of hu mice, but were stable thereafter as assessed up to 32 weeks. Human cell chimerism in spleen and bone marrow was maintained over time. Notably, human cell chimerism in peripheral blood and spleen as well as bone marrow positively correlated with each other. Percentage of B cells decreased betw...

Research paper thumbnail of Homozygous Calreticulin mutations in patients with myelofibrosis lead to acquired myeloperoxidase deficiency

Blood, Jan 24, 2016

The pathogenesis of acquired myeloperoxidase (MPO) deficiency, a rare phenomenon observed in pati... more The pathogenesis of acquired myeloperoxidase (MPO) deficiency, a rare phenomenon observed in patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPN), is unknown. MPO is a glycoprotein chaperoned by Calreticulin (CALR) in the endoplasmic reticulum. Mutations inCALRare frequently found in patients with myelofibrosis (MF) and essential thrombocythemia (ET) with nonmutatedJanuskinase 2(JAK2). We hypothesized that acquired MPO deficiency in MPN could be associated with the presence ofCALRmutations. A cohort of 317 MPN patients (142 polycythemia vera (PV), 94 ET and 81 MF) was screened for MPO deficiency. MPO deficiency was observed in 6/81 MF patients (7.4%), but not in PV or ET patients. Susceptibility to infections had been documented in 2/6 (33%) MPO deficient patients. Five out of six patients with MPO deficiency carried a homozygousCALRmutation and were also deficient in eosinophilic peroxidase (EPX). In contrast, one MF patient with aJAK2-V617F mutation an...

Research paper thumbnail of Allelic variants of the Melanocortin 4 receptor (MC4R) gene in a South African study group

Molecular Genetics & Genomic Medicine, 2015

Obesity is a global epidemic that results in significant morbidity and mortality. Mutations in th... more Obesity is a global epidemic that results in significant morbidity and mortality. Mutations in the melanocortin 4 receptor (MC4R) gene, which codes for a G-protein-coupled receptor responsible for postprandial satiety signaling, have been associated with monogenic obesity. The prevalence of obesity is on the increase in South Africa, and it is hypothesized that mutations in MC4R are a contributing factor. The aim of this study was to perform a retrospective assessment of the relationship between allelic variants of MC4R and BMI in a South African study cohort. DNA was isolated from a demographically representative cohort of 297 individuals and the entire MC4R gene sequenced by Sanger sequencing. Eight previously reported MC4R variants were identified in 42 of the 297 (14.1%) study participants. The most frequently observed MC4R alleles were V103I (4.0%), I170V (1.5%), and I198I (1.2%), while the remaining five variants together constituted 1.18%. Five compound heterozygotes were also detected. Although MC4R variants were rare, the majority of variation was observed in individuals of Black African ancestry. No statistically significant associations with BMI were reported. Given that lifestyle interventions have limited success in decreasing obesity, there is an urgent need to perform largescale population studies to further elucidate the molecular underpinnings of this disease.

Research paper thumbnail of Pharmacogenetics and Pharmacogenomics in the Developing World

Genomics and Health in the Developing World, 2012

Research paper thumbnail of Optimizing Synthetic miRNA Minigene Architecture for Efficient miRNA Hairpin Concatenation and Multi-target Gene Knockdown

Molecular Therapy - Nucleic Acids

Synthetic microRNA (miRNA) minigenes (SMIGs) have a major potential for molecular therapy; howeve... more Synthetic microRNA (miRNA) minigenes (SMIGs) have a major potential for molecular therapy; however, their optimal architecture still needs to be determined. We have previously optimized the stem structure of miRNA hairpins for efficient gene knockdown. Here, we investigate the overall architecture of SMIGs driven by polymerase II-dependent promoters. When miRNA hairpins were placed directly behind the promoter, gene knockdown was inefficient as compared with constructs containing an intercalated sequence ("spacer"). Spacer sequence was relevant for knockdown efficiency and concatenation potential: GFP-based sequences (even when truncated or including stop codons) were particularly efficient. In contrast, a spacer of similar length based on a CD4 intronic sequence was entirely inefficient. Spacer sequences influenced miRNA steady-state levels without affecting transcript stability. We demonstrate that with an optimized spacer, up to five concatenated hairpins targeting two different genes are efficiently expressed and able to knock down their respective targets. Transplantation of hematopoietic stem cells containing a CCR5 knockdown SMIG demonstrated a sustained in vivo efficacy of our approach. In summary, we have defined features that optimize SMIG efficiency. Based on these results, optimized knockdown of genes of interest, such as the HIV co-receptor CCR5 and the NADPH oxidase subunit p22, was achieved.

Research paper thumbnail of Cardiovascular pharmacogenetics

Pharmacology & Therapeutics, 2012

Human genetic variation in the form of single nucleotide polymorphisms as well as more complex st... more Human genetic variation in the form of single nucleotide polymorphisms as well as more complex structural variations such as insertions, deletions and copy number variants, is partially responsible for the clinical variation seen in response to pharmacotherapeutic drugs. This affects the likelihood of experiencing adverse drug reactions and also of achieving therapeutic success. In this paper, we review key studies in cardiovascular pharmacogenetics that reveal genetic variations underlying the outcomes of drug treatment in cardiovascular disease. Examples of genetic associations with drug efficacy and toxicity are described, including the roles of genetic variability in pharmacokinetics (e.g. drug metabolizing enzymes) and pharmacodynamics (e.g. drug targets). These findings have functional implications that could lead to the development of genetic tests aimed at minimizing drug toxicity and optimizing drug efficacy in cardiovascular medicine.

Research paper thumbnail of Lentivector knock-down of CCR5 in hematopoietic stem cells confers functional and persistent HIV-1 resistance in humanized mice

Journal of Virology, 2015

Gene-engineered CD34(+) hematopoietic stem and progenitor cells (HSPCs) can be used to generate a... more Gene-engineered CD34(+) hematopoietic stem and progenitor cells (HSPCs) can be used to generate an HIV-1-resistant immune system. However, a certain threshold of transduced HSPCs might be required for transplantation into mice for creating an HIV-resistant immune system. Here, we combined CCR5 knock-down by a highly efficient miRNA lentivector with pre-transplantation selection of transduced HSPCs to obtain a rather pure population of gene engineered CD34+ cells. Low-level transduction of HSPCs and subsequent sorting by flow cytometry yielded >70% transduced cells. Mice transplanted with these cells showed functional and persistent resistance to a CCR5-tropic HIV strain: viral load was significantly decreased over months, and human CD4(+) T-cells were preserved. In one mouse, viral mutations, resulting presumably in a CXCR4-tropic strain, overcame HIV resistance. Our results suggest that HSPC-based CCR5 knock-down may lead to efficient control of HIV in vivo. We overcome a major limitation of previous HIV gene therapy in humanized mice studies in which only a proportion of the cells in chimeric mice in vivo are anti-HIV engineered. Our strategy underlines the promising future of gene engineering HIV-resistant CD34(+) cells that produce a constant supply of HIV resistant progeny. Major issues in experimental long-term in vivo HIV gene therapy have been (i) low efficacy of cell transduction at the time of implantation and (ii) transduction resulting in multiple copies of heterologous DNA in target cells. In this study, we demonstrated the efficacy of a transplantation approach with a selection step for transduced cells that allows transplantation of an enriched population of HSPCs expressing a single (low) copy of a CCR5 miRNA. Efficient maintenance of CD4(+) T-cells and a low viral titer resulted only when at least 70% of the HIV target cells were genetically modified. These findings imply that clinical protocols of HIV gene therapy require a selective enrichment of genetically targeted cells because positive selection of modified cells is likely to be insufficient below this threshold. This selection approach may not only be beneficial for HIV patients, but also other patients requiring transplantation of genetically modified cells.

Research paper thumbnail of Engineering an HIV-1 resistant immune system

Gene knock-down using miRNA-based vector constructs is likely to become a prominent gene therapy ... more Gene knock-down using miRNA-based vector constructs is likely to become a prominent gene therapy approach. It was the aim of this study to improve the efficiency of gene knock-down through optimizing the structure of miRNA mimics. Knock-down of two target genes was analyzed: CCR5 and GFP. We describe here a novel and optimized miRNA mimic design called mirGE comprising a lower stem length of 13 base pairs, positioning of the targeting strand on the 5' side of the miRNA, together with nucleotide mismatches in upper stem positions 1 and 12 placed on the passenger strand. Our mirGE proved superior to miR-30 in four aspects: yield of targeting strand incorporation into RISC; incorporation into RISC of correct targeting strand; precision of cleavage by Drosha and ratio of targeting strand over passenger strand. A triple mirGE hairpin cassette targeting CCR5 was constructed. It allowed CCR5 knock-down with an efficiency of over 90% upon single copy transduction. Importantly, single copy expression of this construct rendered transduced target cells, including primary human macrophages, resistant to infection with a CCR5-tropic strain of HIV. Our results provide new insights for a better knock-down efficiency of constructs containing miRNA. Our results also provide the proof-of-principle that cells can be rendered HIV-resistant through single copy vector transduction, rendering this approach more compatible with clinical applications. iv) Review article (In preparation) Clinical applications of genetically modified hematopoietic stem cells

Research paper thumbnail of Anti-CD117 immunotherapy to eliminate hematopoietic and leukemia stem cells

Experimental Hematology

Precise replacement of diseased or dysfunctional organs is the goal of regenerative medicine and ... more Precise replacement of diseased or dysfunctional organs is the goal of regenerative medicine and has appeared to be a distant goal for a long time. In the field of hematopoietic stem cell transplantation, this goal is now becoming tangible as gene-editing technologies and novel conditioning agents are entering the clinical arena. Targeted immunologic depletion of hematopoietic stem cells (HSCs), which are at the very root of the hematopoietic system, will enable more selective and potentially more effective hematopoietic stem cell transplantation in patients with hematological diseases. In contrast to current conditioning regimes based on ionizing radiation and chemotherapy, immunologic conditioning will spare mature hematopoietic cells and cause substantially less inflammation and unspecific collateral damage to other organs. Biological agents that target the stem cell antigen CD117 are the frontrunners for this purpose and have exhibited preclinical activity in depletion of healthy HSCs. The value of anti-CD117 antibodies as conditioning agents is currently being evaluated in early clinical trials. Whereas mild, antibody-based immunologic conditioning concepts might be appropriate for benign hematological disorders in which incomplete replacement of diseased cells is sufficient, higher efficacy will be required for treatment and elimination of hematologic stem cell malignancies such as acute myeloid leukemia and myelodysplastic syndrome. Antibody−drug conjugates, bispecific T-cell engaging and activating antibodies (TEAs), or chimeric antigen receptor (CAR) T cells might offer increased efficacy compared with naked antibodies and yet higher tolerability and safety compared with current genotoxic conditioning approaches. Here, we summarize the current state regarding immunologic conditioning concepts for the treatment of HSC disorders and outline potential future developments.

Research paper thumbnail of EBV renders B cells susceptible to HIV-1 in humanized mice

Life Science Alliance

HIV and EBV are human pathogens that cause a considerable burden to worldwide health. In combinat... more HIV and EBV are human pathogens that cause a considerable burden to worldwide health. In combination, these viruses are linked to AIDS-associated lymphomas. We found that EBV, which transforms B cells, renders them susceptible to HIV-1 infection in a CXCR4 and CD4-dependent manner in vitro and that CXCR4-tropic HIV-1 integrates into the genome of these B cells with the same molecular profile as in autologous CD4+ T cells. In addition, we established a humanized mouse model to investigate the in vivo interactions of EBV and HIV-1 upon coinfection. The respective mice that reconstitute human immune system components upon transplantation with CD34+ human hematopoietic progenitor cells could recapitulate aspects of EBV and HIV immunobiology observed in dual-infected patients. Upon coinfection of humanized mice, EBV/HIV dual-infected B cells could be detected, but were susceptible to CD8+ T-cell–mediated immune control.

Research paper thumbnail of Stereo- and regiodefined DNA-encoded chemical libraries enable efficient tumour-targeting applications

Research paper thumbnail of Anti-human CD117 CAR T-cells efficiently eliminate healthy and malignant CD117-expressing hematopoietic cells

Research paper thumbnail of BRAFV 600E or mutant MAP2K1 human CD34+ cells establish Langerhans cell–like histiocytosis in immune-deficient mice

Blood Advances

Key Points BRAFV600E or mutant MAP2K1 expression in human CB CD34+ HSPCs lead to Langerhans cell–... more Key Points BRAFV600E or mutant MAP2K1 expression in human CB CD34+ HSPCs lead to Langerhans cell–like histiocytosis in immune-deficient mice. BRAFV600E-expressing human CB CD34+ HSPCs did not generate hairy cell leukemia in xenograft mouse models.

Research paper thumbnail of Abstract 4229: Anti-P329G-CAR-T cells as a novel universal CAR-T cell platform

Clinical Research (Excluding Clinical Trials)

Research paper thumbnail of Impact of Ligand Size and Conjugation Chemistry on the Performance of Universal Chimeric Antigen Receptor TCells for Tumor Killing

Research paper thumbnail of Anti-Human CD117 CAR T-Cells Efficiently Eliminate Hematopoietic Stem and CD117-Positive AML Cells

Blood

Introduction: Acute Myeloid Leukemia (AML) is a clonal disease of the hematopoietic system that o... more Introduction: Acute Myeloid Leukemia (AML) is a clonal disease of the hematopoietic system that originates from immature hematopoietic stem and progenitor cells (HSPC). Because some AML-initiating cells are comparatively resistant to conventional cytotoxic agents, disease relapses are common with current treatment approaches. As an alternative, immunological eradication of leukemic cells by adoptively transferred chimeric-antigen receptor T-cells (CAR T-cells) might be considerably more efficient. To date, however, the search for AML-specific surface antigens has remained largely elusive. To circumvent this problem, we propose to target the stem cell antigen c-Kit (CD117) that is expressed by physiological HSPC as wells as by leukemic blasts in >90% of AML patients. For translation into a clinical setting, CAR T cell treatment must then be followed by depletion of CAR T-cells as well subsequent healthy/allogeneic HSC transplantation. Methods: A lentiviral vector was generated whi...

Research paper thumbnail of Development of a novel fully-human anti-CD123 antibody to target acute myeloid leukemia

Research paper thumbnail of Antiviral Activity of HIV gp120-Targeting Bispecific T Cell Engager Antibody Constructs

Journal of virology, Jul 15, 2018

Today's gold standard in HIV therapy is combined antiretroviral therapy (cART). It requires s... more Today's gold standard in HIV therapy is combined antiretroviral therapy (cART). It requires strict adherence by patients and lifelong medication, which can lower the viral load below detection limits and prevent HIV-associated immunodeficiency but cannot cure patients. The bispecific T cell-engaging (BiTE) antibody technology has demonstrated long-term relapse-free outcomes in patients with relapsed and refractory acute lymphocytic leukemia. Here, we generated BiTE antibody constructs that target the HIV-1 envelope protein gp120 (HIV gp120) using either the scFv B12 or VRC01, the first two extracellular domains (1 + 2) of human CD4 alone or joined to the single chain variable fragment (scFv) of the antibody 17b fused to an anti-human CD3ε scFv. These engineered human BiTE antibody constructs showed engagement of T cells for redirected lysis of HIV gp120-transfected CHO cells. Furthermore, they substantially inhibited HIV-1 replication in peripheral blood mononuclear cells (PBMCs...

Research paper thumbnail of 448. Lentivector Knock-Down of CCR5 in Hematopoietic Stem Cells Confers Functional and Persistent HIV-1 Resistance in Humanized Mice

Research paper thumbnail of 542. A Novel In Vitro/Vivo Selection/Suicide Strategy for Lentiviral Vectors

Research paper thumbnail of Long-term leukocyte reconstitution in NSG mice transplanted with human cord blood hematopoietic stem and progenitor cells

BMC immunology, Jan 30, 2017

Humanized mice (hu mice) are based on the transplantation of hematopoietic stem and progenitor ce... more Humanized mice (hu mice) are based on the transplantation of hematopoietic stem and progenitor cells into immunodeficient mice and have become important pre-clinical models for biomedical research. However, data about their hematopoiesis over time are scarce. We therefore characterized leukocyte reconstitution in NSG mice, which were sublethally irradiated and transplanted with human cord blood-derived CD34+ cells at newborn age, longitudinally in peripheral blood and, for more detailed analyses, cross-sectionally in peripheral blood, spleen and bone marrow at different time points. Human cell chimerism and absolute human cell count decreased between week 16 and 24 in the peripheral blood of hu mice, but were stable thereafter as assessed up to 32 weeks. Human cell chimerism in spleen and bone marrow was maintained over time. Notably, human cell chimerism in peripheral blood and spleen as well as bone marrow positively correlated with each other. Percentage of B cells decreased betw...

Research paper thumbnail of Homozygous Calreticulin mutations in patients with myelofibrosis lead to acquired myeloperoxidase deficiency

Blood, Jan 24, 2016

The pathogenesis of acquired myeloperoxidase (MPO) deficiency, a rare phenomenon observed in pati... more The pathogenesis of acquired myeloperoxidase (MPO) deficiency, a rare phenomenon observed in patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPN), is unknown. MPO is a glycoprotein chaperoned by Calreticulin (CALR) in the endoplasmic reticulum. Mutations inCALRare frequently found in patients with myelofibrosis (MF) and essential thrombocythemia (ET) with nonmutatedJanuskinase 2(JAK2). We hypothesized that acquired MPO deficiency in MPN could be associated with the presence ofCALRmutations. A cohort of 317 MPN patients (142 polycythemia vera (PV), 94 ET and 81 MF) was screened for MPO deficiency. MPO deficiency was observed in 6/81 MF patients (7.4%), but not in PV or ET patients. Susceptibility to infections had been documented in 2/6 (33%) MPO deficient patients. Five out of six patients with MPO deficiency carried a homozygousCALRmutation and were also deficient in eosinophilic peroxidase (EPX). In contrast, one MF patient with aJAK2-V617F mutation an...

Research paper thumbnail of Allelic variants of the Melanocortin 4 receptor (MC4R) gene in a South African study group

Molecular Genetics & Genomic Medicine, 2015

Obesity is a global epidemic that results in significant morbidity and mortality. Mutations in th... more Obesity is a global epidemic that results in significant morbidity and mortality. Mutations in the melanocortin 4 receptor (MC4R) gene, which codes for a G-protein-coupled receptor responsible for postprandial satiety signaling, have been associated with monogenic obesity. The prevalence of obesity is on the increase in South Africa, and it is hypothesized that mutations in MC4R are a contributing factor. The aim of this study was to perform a retrospective assessment of the relationship between allelic variants of MC4R and BMI in a South African study cohort. DNA was isolated from a demographically representative cohort of 297 individuals and the entire MC4R gene sequenced by Sanger sequencing. Eight previously reported MC4R variants were identified in 42 of the 297 (14.1%) study participants. The most frequently observed MC4R alleles were V103I (4.0%), I170V (1.5%), and I198I (1.2%), while the remaining five variants together constituted 1.18%. Five compound heterozygotes were also detected. Although MC4R variants were rare, the majority of variation was observed in individuals of Black African ancestry. No statistically significant associations with BMI were reported. Given that lifestyle interventions have limited success in decreasing obesity, there is an urgent need to perform largescale population studies to further elucidate the molecular underpinnings of this disease.

Research paper thumbnail of Pharmacogenetics and Pharmacogenomics in the Developing World

Genomics and Health in the Developing World, 2012

Research paper thumbnail of Optimizing Synthetic miRNA Minigene Architecture for Efficient miRNA Hairpin Concatenation and Multi-target Gene Knockdown

Molecular Therapy - Nucleic Acids

Synthetic microRNA (miRNA) minigenes (SMIGs) have a major potential for molecular therapy; howeve... more Synthetic microRNA (miRNA) minigenes (SMIGs) have a major potential for molecular therapy; however, their optimal architecture still needs to be determined. We have previously optimized the stem structure of miRNA hairpins for efficient gene knockdown. Here, we investigate the overall architecture of SMIGs driven by polymerase II-dependent promoters. When miRNA hairpins were placed directly behind the promoter, gene knockdown was inefficient as compared with constructs containing an intercalated sequence ("spacer"). Spacer sequence was relevant for knockdown efficiency and concatenation potential: GFP-based sequences (even when truncated or including stop codons) were particularly efficient. In contrast, a spacer of similar length based on a CD4 intronic sequence was entirely inefficient. Spacer sequences influenced miRNA steady-state levels without affecting transcript stability. We demonstrate that with an optimized spacer, up to five concatenated hairpins targeting two different genes are efficiently expressed and able to knock down their respective targets. Transplantation of hematopoietic stem cells containing a CCR5 knockdown SMIG demonstrated a sustained in vivo efficacy of our approach. In summary, we have defined features that optimize SMIG efficiency. Based on these results, optimized knockdown of genes of interest, such as the HIV co-receptor CCR5 and the NADPH oxidase subunit p22, was achieved.

Research paper thumbnail of Cardiovascular pharmacogenetics

Pharmacology & Therapeutics, 2012

Human genetic variation in the form of single nucleotide polymorphisms as well as more complex st... more Human genetic variation in the form of single nucleotide polymorphisms as well as more complex structural variations such as insertions, deletions and copy number variants, is partially responsible for the clinical variation seen in response to pharmacotherapeutic drugs. This affects the likelihood of experiencing adverse drug reactions and also of achieving therapeutic success. In this paper, we review key studies in cardiovascular pharmacogenetics that reveal genetic variations underlying the outcomes of drug treatment in cardiovascular disease. Examples of genetic associations with drug efficacy and toxicity are described, including the roles of genetic variability in pharmacokinetics (e.g. drug metabolizing enzymes) and pharmacodynamics (e.g. drug targets). These findings have functional implications that could lead to the development of genetic tests aimed at minimizing drug toxicity and optimizing drug efficacy in cardiovascular medicine.

Research paper thumbnail of Lentivector knock-down of CCR5 in hematopoietic stem cells confers functional and persistent HIV-1 resistance in humanized mice

Journal of Virology, 2015

Gene-engineered CD34(+) hematopoietic stem and progenitor cells (HSPCs) can be used to generate a... more Gene-engineered CD34(+) hematopoietic stem and progenitor cells (HSPCs) can be used to generate an HIV-1-resistant immune system. However, a certain threshold of transduced HSPCs might be required for transplantation into mice for creating an HIV-resistant immune system. Here, we combined CCR5 knock-down by a highly efficient miRNA lentivector with pre-transplantation selection of transduced HSPCs to obtain a rather pure population of gene engineered CD34+ cells. Low-level transduction of HSPCs and subsequent sorting by flow cytometry yielded >70% transduced cells. Mice transplanted with these cells showed functional and persistent resistance to a CCR5-tropic HIV strain: viral load was significantly decreased over months, and human CD4(+) T-cells were preserved. In one mouse, viral mutations, resulting presumably in a CXCR4-tropic strain, overcame HIV resistance. Our results suggest that HSPC-based CCR5 knock-down may lead to efficient control of HIV in vivo. We overcome a major limitation of previous HIV gene therapy in humanized mice studies in which only a proportion of the cells in chimeric mice in vivo are anti-HIV engineered. Our strategy underlines the promising future of gene engineering HIV-resistant CD34(+) cells that produce a constant supply of HIV resistant progeny. Major issues in experimental long-term in vivo HIV gene therapy have been (i) low efficacy of cell transduction at the time of implantation and (ii) transduction resulting in multiple copies of heterologous DNA in target cells. In this study, we demonstrated the efficacy of a transplantation approach with a selection step for transduced cells that allows transplantation of an enriched population of HSPCs expressing a single (low) copy of a CCR5 miRNA. Efficient maintenance of CD4(+) T-cells and a low viral titer resulted only when at least 70% of the HIV target cells were genetically modified. These findings imply that clinical protocols of HIV gene therapy require a selective enrichment of genetically targeted cells because positive selection of modified cells is likely to be insufficient below this threshold. This selection approach may not only be beneficial for HIV patients, but also other patients requiring transplantation of genetically modified cells.