Assays to evaluate the genotoxicity of retroviral vectors.
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Biomedical subjects
Arthur W Nienhuis
Publications and source records attributed to Arthur W Nienhuis.
Genotoxicity of retroviral integration in hematopoietic cells.
The experience of the past 3 years, since the first case of leukemia was reported in a child cured of X-linked severe combined immunodeficiency (X-SCID) by gene therapy, indicates that the potential genotoxicity of retroviral integration in hematopoietic cells will remain a consideration in evaluating the relative risks versus benefits of gene therapy for specific blood disorders. Although many unique variables may have contributed to an increased risk in X-SCID patients, clonal dominance or frank neoplasia in animal models, clonal dominance in humans with chronic granulomatous disease, and the ability of retroviral integration to immortalize normal bone marrow cells or convert factor-dependent cells to factor independence suggest that transduction of cells with an integrating retrovirus has the potential for altering their subsequent biologic behavior. The selective pressure imposed during in vitro culture or after engraftment may uncover a growth or survival advantage for cells in which an integration event has affected gene expression. Such cells then carry the risk that subsequent mutations may lead to neoplastic evolution of individual clones. Balancing that risk is that the vast majority of integration events seem to be neutral and that optimizing vector design may diminish the probability of altering gene expression by an integrated vector genome. Several cell culture systems and animal models designed to empirically evaluate the safety of vector systems are being developed and should provide useful data for weighing the relative risks and benefits for specific diseases and patient populations. Gene therapy interventions continue to have enormous potential for the treatment of disorders of the hematopoietic system. The future of such efforts seems bright as we continue to evolve and improve various strategies to make such interventions both effective and as safe as possible.
Adaptor Proteins, Signal Transducing↗
Acute myeloid leukemia is associated with retroviral gene transfer to hematopoietic progenitor cells in a rhesus macaque.
We report, for the first time, a replication-defective retroviral vector-associated neoplasia in a nonhuman primate. Five years after transplantation with CD34+ cells transduced with a retroviral vector expressing enhanced green fluorescent protein (eGFP) and a drug-resistant variant of the dihydrofolate reductase gene (L22Y), a rhesus macaque developed a fatal myeloid sarcoma, a type of acute myeloid leukemia. Tumor cells contained 2 clonal vector insertions. One insertion was found in BCL2-A1, an antiapoptotic gene. This event suggests that currently available retroviral vectors may have long-term side effects, particularly in hematopoietic stem and progenitor cells.
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WASP- mice exhibit defective immune responses to influenza A virus, Streptococcus pneumoniae, and Mycobacterium bovis BCG.
OBJECTIVE: To quantify the immune response of WASP- mice to three different pathogens: influenza A virus, Streptococcus pneumoniae, and Mycobacterium bovis. METHODS: Primary and secondary T-cell responses to influenza A virus were quantified via tetramer assays. Viral clearance from lung was also measured. Lethality of intranasal inoculation with luminescent S. pneumoniae was assessed by dose escalation and direct luminescence imaging. After intravenous inoculation with M. bovis, residual mycobacteria in lung, liver, and spleen were measured by standard culture methods. RESULTS: The reduced secondary T-cell response to influenza A virus correlates with a relative but not absolute loss of splenic T and B cells similar to that seen in clinical Wiskott-Aldrich Syndrome (WAS), and slower clearance of virus from lung. The reduced magnitude of the secondary T-cell response correlates with a progressive loss of influenza-specific T cells after primary inoculation. WASP- mice show an increased susceptibility to lethal pneumonia after intranasal inoculation with S. pneumoniae, which is among the most frequent causes of clinical complications in WAS patients. WASP- mice clear M. bovis bacille Calmette-Guerin (BCG) more slowly from lung, liver, and spleen. Bone marrow-derived macrophages, however, show normal ex vivo cytokine secretion in response to M. bovis. CONCLUSIONS: These results demonstrate that WASP- mice are functionally immunodeficient in regard to three different pathogens, and provide relevant end points for the study of treatment modalities in this model. They also suggest a specific physiologic mechanism, failure to accumulate memory T cells, for at least one of the defective immune responses.
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Mobilization and mechanism of transcription of integrated self-inactivating lentiviral vectors.
Permanent genetic modification of replicating primitive hematopoietic cells by an integrated vector has many potential therapeutic applications. Both oncoretroviral and lentiviral vectors have a predilection for integration into transcriptionally active genes, creating the potential for promoter activation or gene disruption. The use of self-inactivating (SIN) vectors in which a deletion of the enhancer and promoter sequences from the 3' long terminal repeat (LTR) is copied over into the 5' LTR during vector integration is designed to improve safety by reducing the risk of mobilization of the vector genome and the influence of the LTR on nearby cellular promoters. Our results indicate that SIN vectors are mobilized in cells expressing lentiviral proteins, with the frequency of mobilization influenced by features of the vector design. The mechanism of transcription of integrated vector genomes was evaluated using a promoter trap design with a vector encoding tat but lacking an upstream promoter in a cell line in which drug resistance depended on tat expression. In six clones studied, all transcripts originated from cryptic promoters either upstream or within the vector genome. We estimate that approximately 1 in 3,000 integrated vector genomes is transcribed, leading to the inference that activation of cryptic promoters must depend on local features of chromatin structure and the constellation of nearby regulatory elements as well as the nature of the regulatory elements within the vector.
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Distinct genomic integration of MLV and SIV vectors in primate hematopoietic stem and progenitor cells.
Murine leukemia virus (MLV)-derived vectors are widely used for hematopoietic stem cell (HSC) gene transfer, but lentiviral vectors such as the simian immunodeficiency virus (SIV) may allow higher efficiency transfer and better expression. Recent studies in cell lines have challenged the notion that retroviruses and retroviral vectors integrate randomly into their host genome. Medical applications using these vectors are aimed at HSCs, and thus large-scale comprehensive analysis of MLV and SIV integration in long-term repopulating HSCs is crucial to help develop improved integrating vectors. We studied integration sites in HSCs of rhesus monkeys that had been transplanted 6 mo to 6 y prior with MLV- or SIV-transduced CD34(+)cells. Unique MLV (491) and SIV (501) insertions were compared to a set of in silico-generated random integration sites. While MLV integrants were located predominantly around transcription start sites, SIV integrants strongly favored transcription units and gene-dense regions of the genome. These integration patterns suggest different mechanisms for integration as well as distinct safety implications for MLV versus SIV vectors.
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Extended beta-globin locus control region elements promote consistent therapeutic expression of a gamma-globin lentiviral vector in murine beta-thalassemia.
Since increased fetal hemoglobin diminishes the severity of beta-thalassemia and sickle cell anemia, a strategy using autologous, stem cell-targeted gene transfer of a gamma-globin gene may be therapeutically useful. We previously found that a gamma-globin lentiviral vector utilizing the beta-globin promoter and elements from the beta-globin locus control region (LCR) totaling 1.7 kb could correct murine beta-thalassemia. However, therapeutic consistency was compromised by chromosomal position effects on vector expression. In contrast, we show here that the majority of animals that received transplants of beta-thalassemic stem cells transduced with a new vector containing 3.2 kb of LCR sequences expressed high levels of fetal hemoglobin (17%-33%), with an average vector copy number of 1.3. This led to a mean 26 g/L (2.6 g/dL) increase in hemoglobin concentration and enhanced amelioration of other hematologic parameters. Analysis of clonal erythroid cells of secondary spleen colonies from mice that underwent transplantation demonstrated an increased resistance of the larger LCR vector to stable and variegating position effects. This trend was also observed for vector insertion sites located inside genes, where vector expression was often compromised, in contrast to intergenic sites, where higher levels of expression were observed. These data emphasize the importance of overcoming detrimental position effects for consistent therapeutic globin vector expression.
Anemia, Sickle Cell↗
Efficient gene transfer into rhesus repopulating hematopoietic stem cells using a simian immunodeficiency virus-based lentiviral vector system.
High-titer, HIV-1-based lentiviral vector particles were found to transduce cytokine-mobilized rhesus macaque CD34(+) cells and clonogenic progenitors very poorly (< 1%), reflecting the postentry restriction in rhesus cells to HIV infection. To overcome this barrier, we developed a simian immunodeficiency virus (SIV)-based vector system. A single exposure to a low concentration of amphotropic pseudotyped SIV vector particles encoding the green fluorescent protein (GFP) resulted in gene transfer into 68% +/- 1% of rhesus bulk CD34(+) cells and 75% +/- 1% of clonogenic progenitors. Polymerase chain reaction (PCR) analysis of DNA from individual hematopoietic colonies confirmed these relative transduction efficiencies. To evaluate SIV vector-mediated stem cell gene transfer in vivo, 3 rhesus macaques underwent transplantation with transduced, autologous cytokine-mobilized peripheral blood CD34(+) cells following myeloablative conditioning. Hematopoietic reconstitution was rapid, and an average of 18% +/- 8% and 15% +/- 7% GFP-positive granulocytes and monocytes, respectively, were observed 4 to 6 months after transplantation, consistent with the average vector copy number of 0.19 +/- 0.05 in peripheral blood leukocytes as determined by real-time PCR. Vector insertion site analysis demonstrated polyclonal reconstitution with vector-containing cells. SIV vectors appear promising for evaluating gene therapy approaches in nonhuman primate models.
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Defects in T-cell-mediated immunity to influenza virus in murine Wiskott-Aldrich syndrome are corrected by oncoretroviral vector-mediated gene transfer into repopulating hematopoietic cells.
The Wiskott-Aldrich syndrome (WAS) is an X-linked disorder characterized by immune dysfunction, thrombocytopenia, and eczema. We used a murine model created by knockout of the WAS protein gene (WASP) to evaluate the potential of gene therapy for WAS. Lethally irradiated, male WASP- animals that received transplants of mixtures of wild type (WT) and WASP- bone marrow cells demonstrated enrichment of WT cells in the lymphoid and myeloid lineages with a progressive increase in the proportion of WT T-lymphoid and B-lymphoid cells. WASP- mice had a defective secondary T-cell response to influenza virus which was normalized in animals that received transplants of 35% or more WT cells. The WASP gene was inserted into WASP- bone marrow cells with a bicistronic oncoretroviral vector also encoding green fluorescent protein (GFP), followed by transplantation into irradiated male WASP- recipients. There was a selective advantage for gene-corrected cells in multiple lineages. Animals with higher proportions of GFP+ T cells showed normalization of their lymphocyte counts. Gene-corrected, blood T cells exhibited full and partial correction, respectively, of their defective proliferative and cytokine secretory responses to in vitro T-cell-receptor stimulation. The defective secondary T-cell response to influenza virus was also improved in gene-corrected animals.
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Successful treatment of murine beta-thalassemia using in vivo selection of genetically modified, drug-resistant hematopoietic stem cells.
Successful gene therapy of beta-thalassemia will require replacement of the abnormal erythroid compartment with erythropoiesis derived from genetically corrected, autologous hematopoietic stem cells (HSCs). However, currently attainable gene transfer efficiencies into human HSCs are unlikely to yield sufficient numbers of corrected cells for a clinical benefit. Here, using a murine model of beta-thalassemia, we demonstrate for the first time that selective enrichment in vivo of transplanted, drug-resistant HSCs can be used therapeutically and may therefore be a useful approach to overcome limiting gene transfer. We used an oncoretroviral vector to transfer a methylguanine methyltransferase (MGMT) drug-resistance gene into normal bone marrow cells. These cells were transplanted into beta-thalassemic mice given nonmyeloablative pretransplantation conditioning with temozolomide (TMZ) and O6-benzylguanine (BG). A majority of mice receiving 2 additional courses of TMZ/BG demonstrated in vivo selection of the drug-resistant cells and amelioration of anemia, compared with untreated control animals. These results were extended using a novel gamma-globin/MGMT dual gene lentiviral vector. Following drug treatment, normal mice that received transduced cells had an average 67-fold increase in gamma-globin expressing red cells. These studies demonstrate that MGMT-based in vivo selection may be useful to increase genetically corrected cells to therapeutic levels in patients with beta-thalassemia.
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Development of gene therapy for hemoglobin disorders.
The hemoglobin disorders, severe beta-thalassemia and sickle cell anemia, are prevalent monogenetic disorders which cause severe morbidity and mortality worldwide. Gene therapy approaches to these disorders envision stem cell targeted gene transfer, autologous transplantation of gene-corrected stem cells, and functional, phenotypically corrective globin gene expression in developing erythroid cells. Lentiviral vector systems potentially appear to afford adequately efficient gene transfer into stem cells and are capable, with appropriate genetic engineering, of transferring a globin gene with the regulatory elements required to achieve high-level, erythroid-specific expression. Herein are results obtained in use of lentiviral vectors to insert a gamma-globin gene into murine stem cells with phenotypic correction of the thalassemia phenotype. Further, we have developed a drug-selection system for genetically modified stem cells based on a mutant form of methylguanine, methyltransferase, which allows selective amplification of genetically modified stem cells with phenotypic correction even in the absence of myeloablation prior to stem cell transplantation. These advances provide essential preclinical data which build toward the development of effective gene therapy for the severe hemoglobin disorders.
Anemia, Sickle Cell↗
Current status of gene therapy for hemophilia.
The hemophilias are an attractive model for gene therapy because their clinical manifestations are attributable to the lack of a single protein that circulates in minute amounts in the plasma. Sustained therapeutic expression of factors VIII and IX has been achieved in preclinical studies using a wide range of gene transfer technologies targeted at different tissues. This achievement has led to six different phase I/II clinical trials that resulted in limited efficacy but minimal toxicity. Recombinant adeno-associated viral vectors appear most promising for hemophilia gene therapy; however, this review summarizes all the major gene therapy approaches used and outlines the future challenges.
Factor IX↗
Gene therapy for the hemoglobin disorders.
The hematologic disorders of beta-thalassemia and sickle cell disease are the most prevalent of human genetic diseases. Although palliative therapies and curative stem cell transplantation therapy have been developed for these disorders, treatment still remains suboptimal and many patients suffer significant morbidity and early mortality. Therefore, development of a gene therapy approach has been sought for many years. Major progress in the globin gene therapy field has been achieved by several laboratories. Using lentiviral vectors to obtain high-level expression of complex globin gene cassettes, therapeutic correction of several murine models of beta-thalassemia, and sickle cell disease was recently reported. This progress, coupled with developments in the ability to select and expand genetically modified stem cells in vivo, has advanced the possibility of gene therapy for the hemoglobin disorders in the near future. We review the developments in several areas that are critical for successful gene therapy of the hemoglobin disorders.
Anemia, Sickle Cell↗
High-level erythroid lineage-directed gene expression using globin gene regulatory elements after lentiviral vector-mediated gene transfer into primitive human and murine hematopoietic cells.
Lentiviral vectors efficiently transduce primitive human hematopoietic cells and are capable of transferring complex genomes. Vectors were designed with hypersensitive sites containing regulatory elements from the beta-globin locus control region linked to the beta-globin gene promoter to drive expression of the enhanced green fluorescent protein marker to facilitate analysis of the pattern of gene expression in various hematopoietic lineages. Such vectors gave higher level, induced expression in mouse erythroleukemia cells than a previously described vector that utilized an enhancer from the alpha locus and the ankyrin-1 promoter [Moreau-Gaudry, F., Xia, P., Jiang, G., Perelman, N.P., Bauer, G., Ellis, J., Surinya, K.H., Mavilio, F., Shen, C.K., and Malik, P. (2001). Blood 98, 2664-2672]. The addition of gamma-globin intron sequences further augmented vector expression. Expression was also effectively targeted to the erythroid lineage in cultured human cells from peripheral blood and in mouse red blood cells in vivo, although lower levels of expression were also observed in other lineages. Thus, these newly described vectors provide a means to achieve high-level gene expression, predominantly in erythroid cells, an outcome that may have potential therapeutic application.
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The degree of phenotypic correction of murine beta -thalassemia intermedia following lentiviral-mediated transfer of a human gamma-globin gene is influenced by chromosomal position effects and vector copy number.
Increased fetal hemoglobin (HbF) levels diminish the clinical severity of beta-thalassemia and sickle cell anemia. A treatment strategy using autologous stem cell-targeted gene transfer of a gamma-globin gene may therefore have therapeutic potential. We evaluated oncoretroviral- and lentiviral-based gamma-globin vectors for expression in transduced erythroid cell lines. Compared with gamma-globin, oncoretroviral vectors containing either a beta-spectrin or beta-globin promoter and the alpha-globin HS40 element, a gamma-globin lentiviral vector utilizing the beta-globin promoter and elements from the beta-globin locus control region demonstrated a higher probability of expression. This lentiviral vector design was evaluated in lethally irradiated mice that received transplants of transduced bone marrow cells. Long-term, stable erythroid expression of human gamma-globin was observed with levels of vector-encoded gamma-globin mRNA ranging from 9% to 19% of total murine alpha-globin mRNA. The therapeutic efficacy of the vector was subsequently evaluated in a murine model of beta-thalassemia intermedia. The majority of mice that underwent transplantation expressed significant levels of chimeric m(alpha)(2)h(gamma)(2) molecules (termed HbF), the amount of which correlated with the degree of phenotypic improvement. A group of animals with a mean HbF level of 21% displayed a 2.5 g/dL (25 g/L) improvement in Hb concentration and normalization of erythrocyte morphology relative to control animals. gamma-Globin expression and phenotypic improvement was variably lower in other animals due to differences in vector copy number and chromosomal position effects. These data establish the potential of using a gamma-globin lentiviral vector for gene therapy of beta-thalassemia.
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Sustained high-level expression of human factor IX (hFIX) after liver-targeted delivery of recombinant adeno-associated virus encoding the hFIX gene in rhesus macaques.
The feasibility, safety, and efficacy of liver-directed gene transfer was evaluated in 5 male macaques (aged 2.5 to 6.5 years) by using a recombinant adeno-associated viral (rAAV) vector (rAAV-2 CAGG-hFIX) that had previously mediated persistent therapeutic expression of human factor IX (hFIX; 6%-10% of physiologic levels) in murine models. A dose of 4 x 10(12) vector genomes (vgs)/kg of body weight was administered through the hepatic artery or portal vein. Persistence of the rAAV vgs as circular monomers and dimers and high-molecular-weight concatamers was documented in liver tissue by Southern blot analysis for periods of up to 1 year. Vector particles were present in plasma, urine, or saliva for several days after infusion (as shown by polymerase chain reaction analysis), and the vgs were detected in spleen tissue at low copy numbers. An enzyme-linked immunosorption assay capable of detecting between 1% and 25% of normal levels of hFIX in rhesus plasma was developed by using hyperimmune serum from a rhesus monkey that had received an adenoviral vector encoding hFIX. Two macaques having 3 and 40 rAAV genome equivalents/cell, respectively, in liver tissue had 4% and 8% of normal physiologic plasma levels of hFIX, respectively. A level of hFIX that was 3% of normal levels was transiently detected in one other macaque, which had a genome copy number of 25 before abrogation by a neutralizing antibody (inhibitor) to hFIX. This nonhuman-primate model will be useful in further evaluation and development of rAAV vectors for gene therapy of hemophilia B.
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Correction of the murine Wiskott-Aldrich syndrome phenotype by hematopoietic stem cell transplantation.
Allogeneic hematopoietic stem cell transplantation (HSCT) corrects the Wiskott-Aldrich syndrome (WAS) phenotype. However, the toxicity and mortality frequently associated with this approach warrant the exploration of new therapeutic strategies. Transplantation studies of a murine model of WAS deficiency have been limited by the occurrence of a radiation-induced fatal exacerbation of a pre-existing colitis in the peritransplantation period. Here we demonstrate that when crossed to a C57/B6 background, WAS-deficient males show little if any colitis and reliably survive HSCT. We show that HSCT corrects the hematologic and functional deficiencies of WAS knockout mice. These results strengthen the analogy between murine and human WAS and provide a basis for the use of WAS-deficient mice to explore novel approaches for correction of the disease phenotype.
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Functional amelioration of murine galactosialidosis by genetically modified bone marrow hematopoietic progenitor cells.
Protective protein/cathepsin A (PPCA), a lysosomal carboxypeptidase, is deficient in the neurodegenerative lysosomal disorder galactosialidosis (GS). PPCA(-/-) mice display a disease course similar to that of severe human GS, resulting in nephropathy, ataxia, and premature death. Bone marrow transplantation (BMT) in mutant animals using transgenic BM overexpressing the corrective enzyme in either erythroid cells or monocytes/macrophages has proven effective for the improvement of the phenotype, and encouraged the use of genetically modified BM cells for ex vivo gene therapy of GS. Here, we established stable donor hematopoiesis in PPCA(-/-) mice that received hematopoietic progenitors transduced with a murine stem cell virus (MSCV)-based, bicistronic retroviral vector overexpressing PPCA and the green fluorescent protein (GFP) marker. We observed complete correction of the disease phenotype in the systemic organs up to 10 months after transplantation. PPCA(+) BM-derived cells were detected in all tissues, with the highest expression in liver, spleen, BM, thymus, and lung. In addition, a lysosomal immunostaining was seen in nonhematopoietic cells, indicating efficient uptake of the corrective protein by these cells and cross-correction. Expression in the brain occurred throughout the parenchyma but was mainly localized on perivascular areas. However, PPCA expression in the central nervous system was apparently sufficient to delay the onset of Purkinje cell degeneration and to correct the ataxia. The long-term expression and internalization of the PPCA by cells of systemic organs and the clear improvement of the neurologic phenotype support the use of this approach for the treatment of GS in humans. (Blood. 2002;99:3169-3178)
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