Recurrent retroviral vector integration at the Mds1/Evi1 locus in nonhuman primate hematopoietic cells

Abstract Recent reports linking insertional activation of LMO2 following gene therapy for X-linked severe combined immunodeficiency (X-SCID) have led to a re-evaluation of risks following gene therapy with retroviral vectors. In our analysis of 702 integration sites in rhesus macaques that underwent transplantation up to 7 years earlier with autologous CD34+ cells transduced with amphotropic murine leukemia virus (MLV)-derived retroviral vectors containing marker genes, we detected insertion into one locus, the Mds1/Evi1 region, a total of 14 times in 9 animals. Mds1/Evi1 integrations were observed stably long term, primarily in myeloid cells. We hypothesize that this over-representation likely results from an impact on the self-renewal and engraftment potential of CD34+ progenitor cells via insertional mutagenesis at this specific locus. There is no evidence of ongoing in vivo clonal expansion of the Mds1/Evi1 populations, and all animals are hematologically normal without evidence for leukemia. Characterization of integration sites in this relevant preclinical model provides critical information for gene therapy risk assessment as well as identification of genes controlling hematopoiesis. (Blood. 2005;106:2530-2533)

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High-definition mapping of retroviral integration sites identifies active regulatory elements in human multipotent hematopoietic progenitors

Blood ◽

10.1182/blood-2010-05-283523 ◽

2010 ◽

Vol 116 (25) ◽

pp. 5507-5517 ◽

Cited By ~ 118

Author(s):

Claudia Cattoglio ◽

Danilo Pellin ◽

Ermanno Rizzi ◽

Giulietta Maruggi ◽

Giorgio Corti ◽

...

Keyword(s):

Gene Expression ◽

Gene Therapy ◽

Leukemia Virus ◽

Regulatory Elements ◽

Retroviral Vectors ◽

Alternative Promoters ◽

High Definition ◽

Hematopoietic Stem ◽

Cell Functions ◽

Integration Sites

Abstract Integration of retroviral vectors in the human genome follows nonrandom patterns that favor insertional deregulation of gene expression and increase the risk of their use in clinical gene therapy. The molecular basis of retroviral target site selection is still poorly understood. We used deep sequencing technology to build genomewide, high-definition maps of > 60 000 integration sites of Moloney murine leukemia virus (MLV)– and HIV-based retroviral vectors in the genome of human CD34+ multipotent hematopoietic progenitor cells (HPCs) and used gene expression profiling, chromatin immunoprecipitation, and bioinformatics to associate integration to genetic and epigenetic features of the HPC genome. Clusters of recurrent MLV integrations identify regulatory elements (alternative promoters, enhancers, evolutionarily conserved noncoding regions) within or around protein-coding genes and microRNAs with crucial functions in HPC growth and differentiation, bearing epigenetic marks of active or poised transcription (H3K4me1, H3K4me2, H3K4me3, H3K9Ac, Pol II) and specialized chromatin configurations (H2A.Z). Overall, we mapped 3500 high-frequency integration clusters, which represent a new resource for the identification of transcriptionally active regulatory elements. High-definition MLV integration maps provide a rational basis for predicting genotoxic risks in gene therapy and a new tool for genomewide identification of promoters and regulatory elements controlling hematopoietic stem and progenitor cell functions.

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Profiling Retroviral Integration Sites in Donor T-Lymphocytes for Suicide Gene Therapy.

Blood ◽

10.1182/blood.v104.11.1744.1744 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1744-1744

Author(s):

Frank A. Giordano ◽

Stephanie Laufs ◽

Katalin Z. Nagy ◽

Boris Fehse ◽

Agnes Hotz-Wagenblatt ◽

...

Keyword(s):

Signal Transduction ◽

Gene Therapy ◽

T Cells ◽

T Lymphocytes ◽

Homo Sapiens ◽

Severe Combined Immunodeficiency ◽

Retroviral Vectors ◽

Hematopoietic Stem ◽

Retroviral Integration ◽

Integration Sites

Abstract Retroviral vectors encoding the herpes simplex thymidine kinase gene (HSV-Tk) have been employed to render T-lymphocytes (TLCs) sensitive to the prodrug ganciclovir. Such genetically modified T cells have been used for adoptive immunotherapy in the context of allogeneic hematopoietic stem cell transplantation (SCT). Infused T cells have been shown to be susceptible to elimination through exposure to ganciclovir in the event of graft-versus-host disease (GvHD). Recent reports on insertional “genotoxicity” in a mouse gene marking study and a clinical gene therapy trial for X-chromosomal severe combined immunodeficiency (X-SCID) reminded us the actual risk of insertional oncogene activation and subsequent leukemia development. Here we investigated retroviral integration sites in donor TLCs transduced with the MoLV-based TK/neoR vector Mo3TIN of four donors in a clinical HSV-Tk study. A total of 114 retroviral integration sites were detected using highly sensitive and specific ligation-mediated PCR (LM-PCR). 41.2% of all integrations appeared near the transcription start regions (+/−10kb) of genes. Further analysis showed that 57 (50%) of all integrations targeted RefSeq genes. 24 of those appeared in intron 1 (42% of all integrations into genes) while 18% (10/57) of all integrations into genes landed in exon sequences whereas 6 hit the first exon. 18 of the targeted genes (15.8% of all integrations) could be at last assigned to signal transduction pathways, whereas the transcription factor family was afflicted 13 times (11.4% of all integrations). The zinc ion binding group makes up 4 (resp. 6, minding that GTF2HII contains a C2H2 type and KIAA0427 a C-x8-C-x5-C-x3-H-type zinc finger) of them. Among the targeted genes we found integrations into the CD8, CD100, CD44, CX3CR1, HLA-DMP and IL10-receptor genes. Within at a range of 5kb up- and 5kb downstream of vector integrations 15 genes were located that were not hit. 5 are known as transcription factors, whereas two of those are involved in leukemia, namely the homo sapiens myeloid/lymphoid or mixed-lineage leukemia 5 gene (MLL5) and the homo sapiens ALL1 fused gene from 5q31 (AF5Q31). Hit genes are currently examined more systematically in terms of function, e.g. involvement in signal transduction and transcription promoting processes. RISC (Retroviral Insertion estimate of Chromosomal Integration) scores and integration specific data will be submitted to a data warehouse, the collaborative RISC score database (CRSD). Such a systematic data collection similar to the IBMTR or EBMT databases will allow to recognize factors contributing to the safety, optimal transgene expression and persistence of transduced cells in the setting of allogenic matched donor transplantation.

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Canine model for gene therapy: inefficient gene expression in dogs reconstituted with autologous marrow infected with retroviral vectors

Blood ◽

10.1182/blood.v71.3.742.bloodjournal713742 ◽

1988 ◽

Vol 71 (3) ◽

pp. 742-747 ◽

Cited By ~ 1

Author(s):

RB Stead ◽

WW Kwok ◽

R Storb ◽

AD Miller

Keyword(s):

Gene Expression ◽

Gene Therapy ◽

Gene Transfer ◽

Transient Gene Expression ◽

Retroviral Vectors ◽

Canine Model ◽

Preclinical Model ◽

Hematopoietic Stem ◽

In Vivo Selection

Successful retroviral gene transfer into murine hematopoietic stem cells indicates the potential for somatic gene therapy in the treatment of certain human hereditary diseases. We developed a canine model to test the applicability of these techniques to a preclinical model of human marrow transplantation. Previously we reported that canine CFU-GM could be infected with retroviral vectors carrying either the gene for a mutant dihydrofolate reductase (DHFR) or neomycin phosphotransferase (NEO). This study reports six lethally irradiated dogs transplanted with autologous marrow cocultivated with retroviral vector-producing cells. This procedure conferred drug resistance to 3% to 13% of the CFU- GM. Three dogs infected with either the NEO or DHFR virus engrafted, but we detected no drug-resistant CFU-GM. Three dogs were given marrow infected with a DHFR virus and received methotrexate (MTX) as in vivo selection; all three had evidence of engraftment. In the surviving dog, we detected 0.03% to 0.1% MTX-resistant CFU-GM at 3 to 5 weeks posttransplant during in vivo selection. These results indicate that we can reconstitute lethally irradiated dogs with autologous marrow exposed to retroviral vectors and suggest that gene transfer into hematopoietic cells is feasible on a large scale. However, the low- level transient gene expression indicates that considerable obstacles remain before human gene therapy can be considered.

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Canine model for gene therapy: inefficient gene expression in dogs reconstituted with autologous marrow infected with retroviral vectors

Blood ◽

10.1182/blood.v71.3.742.742 ◽

1988 ◽

Vol 71 (3) ◽

pp. 742-747 ◽

Cited By ~ 62

Author(s):

RB Stead ◽

WW Kwok ◽

R Storb ◽

AD Miller

Keyword(s):

Gene Expression ◽

Gene Therapy ◽

Gene Transfer ◽

Transient Gene Expression ◽

Retroviral Vectors ◽

Canine Model ◽

Preclinical Model ◽

Hematopoietic Stem ◽

In Vivo Selection

Abstract Successful retroviral gene transfer into murine hematopoietic stem cells indicates the potential for somatic gene therapy in the treatment of certain human hereditary diseases. We developed a canine model to test the applicability of these techniques to a preclinical model of human marrow transplantation. Previously we reported that canine CFU-GM could be infected with retroviral vectors carrying either the gene for a mutant dihydrofolate reductase (DHFR) or neomycin phosphotransferase (NEO). This study reports six lethally irradiated dogs transplanted with autologous marrow cocultivated with retroviral vector-producing cells. This procedure conferred drug resistance to 3% to 13% of the CFU- GM. Three dogs infected with either the NEO or DHFR virus engrafted, but we detected no drug-resistant CFU-GM. Three dogs were given marrow infected with a DHFR virus and received methotrexate (MTX) as in vivo selection; all three had evidence of engraftment. In the surviving dog, we detected 0.03% to 0.1% MTX-resistant CFU-GM at 3 to 5 weeks posttransplant during in vivo selection. These results indicate that we can reconstitute lethally irradiated dogs with autologous marrow exposed to retroviral vectors and suggest that gene transfer into hematopoietic cells is feasible on a large scale. However, the low- level transient gene expression indicates that considerable obstacles remain before human gene therapy can be considered.

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Lentiviral Vectors for the Treatment and Prevention of Cystic Fibrosis Lung Disease

Genes ◽

10.3390/genes10030218 ◽

2019 ◽

Vol 10 (3) ◽

pp. 218 ◽

Cited By ~ 16

Author(s):

Laura Marquez Loza ◽

Eric Yuen ◽

Paul McCray

Keyword(s):

Cystic Fibrosis ◽

Gene Therapy ◽

Clinical Trials ◽

Insertional Mutagenesis ◽

Primary Cultures ◽

Severe Combined Immunodeficiency ◽

Lentiviral Vectors ◽

Retroviral Vectors ◽

Airway Epithelia

Despite the continued development of cystic fibrosis transmembrane conductance regulator (CFTR) modulator drugs for the treatment of cystic fibrosis (CF), the need for mutation agnostic treatments remains. In a sub-group of CF individuals with mutations that may not respond to modulators, such as those with nonsense mutations, CFTR gene transfer to airway epithelia offers the potential for an effective treatment. Lentiviral vectors are well-suited for this purpose because they transduce nondividing cells, and provide long-term transgene expression. Studies in primary cultures of human CF airway epithelia and CF animal models demonstrate the long-term correction of CF phenotypes and low immunogenicity using lentiviral vectors. Further development of CF gene therapy requires the investigation of optimal CFTR expression in the airways. Lentiviral vectors with improved safety features have minimized insertional mutagenesis safety concerns raised in early clinical trials for severe combined immunodeficiency using γ-retroviral vectors. Recent clinical trials using improved lentiviral vectors support the feasibility and safety of lentiviral gene therapy for monogenetic diseases. While work remains to be done before CF gene therapy reaches the bedside, recent advances in lentiviral vector development reviewed here are encouraging and suggest it could be tested in clinical studies in the near future.

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A ComParison of Retroviral Integration Sites in Donor T-Lymphocytes In Vivo and In Vitro.

Blood ◽

10.1182/blood.v106.11.1403.1403 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1403-1403

Author(s):

Frank A. Giordano ◽

Stephanie Laufs ◽

Boris Fehse ◽

K. Zsuzsanna Nagy ◽

Agnes Hotz-Wagenblatt ◽

...

Keyword(s):

T Lymphocytes ◽

Insertional Mutagenesis ◽

Homo Sapiens ◽

Severe Combined Immunodeficiency ◽

Hematopoietic Stem ◽

Retroviral Integration ◽

Gene Therapy Trial ◽

Integration Sites

Abstract Genetically modified T-Lymphocytes (TLCs) have been used for adoptive immunotherapy in the context of allogeneic hematopoietic stem cell transplantation (SCT). Infused TLCs have been shown to be susceptible to elimination through exposure to ganciclovir in the event of graft-versus-host disease (GvHD). Yet, reports on insertional mutagenesis in a mouse gene marking study and a clinical gene therapy trial for X-chromosomal severe combined immunodeficiency (X-SCID) reminded us the actual risk of insertional oncogene activation and subsequent leukemia development. We investigated retroviral integration sites in vitro and in vivo. Therefore, donor TLCs transduced with the MoLV-based TK/neoR vector Mo3TIN for a clinical HSV-Tk study were examined. TLCs of four different donors as well as whole blood samples of two patients transplanted with donor TLCs were analyzed either using highly sensitive and specific ligation-mediated PCR (LM-PCR). A total of 114 retroviral integration sites were detected in vitro. 41.2% of all integrations appeared near the transcription start regions (+/−10kb) of genes. Further analysis showed that 57 (50%) of all integrations targeted RefSeq genes. 24 of those appeared in intron 1 (42% of all integrations into genes) while 18% (10/57) of all integrations into genes landed in exon sequences whereas 6 hit the first exon. 18 of the targeted genes (15.8% of all integrations) could be at last assigned to signal transduction pathways, whereas the transcription factor family was afflicted 13 times (11.4% of all integrations). Among the targeted genes we found integrations into the CD8, CD100, CD44, CX3CR1, HLA-DMP and IL10-receptor genes. Within at a range of 5kb up- and 5kb downstream of vector integrations 15 genes were located that were not hit. 5 are known as transcription factors, whereas two of those are involved in leukemia, namely the homo sapiens myeloid/lymphoid or mixed-lineage leukemia 5 gene (MLL5) and the homo sapiens ALL1 fused gene from 5q31 (AF5Q31). Current analyses are focusing at the in vivo pattern of retroviral integration in DNA of TLCs obtained from transplanted patient’s TLCs. Therefore we developed a new high sensitive PCR method (HS-PCR), an improved LM-PCR to even detect minimal quantities of transduced DNA.

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Preclinical Development of Autologous Hematopoietic Stem Cell-Based Gene Therapy for Immune Deficiencies: A Journey from Mouse Cage to Bed Side

Pharmaceutics ◽

10.3390/pharmaceutics12060549 ◽

2020 ◽

Vol 12 (6) ◽

pp. 549

Author(s):

Laura Garcia-Perez ◽

Anita Ordas ◽

Kirsten Canté-Barrett ◽

Pauline Meij ◽

Karin Pike-Overzet ◽

...

Keyword(s):

Gene Therapy ◽

Severe Combined Immunodeficiency ◽

Good Manufacturing Practice ◽

Proof Of Concept ◽

Preclinical Development ◽

Hematopoietic Stem ◽

Immune Deficiencies ◽

Suitable Vector

Recent clinical trials using patient’s own corrected hematopoietic stem cells (HSCs), such as for primary immunodeficiencies (Adenosine deaminase (ADA) deficiency, X-linked Severe Combined Immunodeficiency (SCID), X-linked chronic granulomatous disease (CGD), Wiskott–Aldrich Syndrome (WAS)), have yielded promising results in the clinic; endorsing gene therapy to become standard therapy for a number of diseases. However, the journey to achieve such a successful therapy is not easy, and several challenges have to be overcome. In this review, we will address several different challenges in the development of gene therapy for immune deficiencies using our own experience with Recombinase-activating gene 1 (RAG1) SCID as an example. We will discuss product development (targeting of the therapeutic cells and choice of a suitable vector and delivery method), the proof-of-concept (in vitro and in vivo efficacy, toxicology, and safety), and the final release steps to the clinic (scaling up, good manufacturing practice (GMP) procedures/protocols and regulatory hurdles).

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Adaptation of Chimeric Retroviruses In Vitro and In Vivo: Isolation of Avian Retroviral Vectors with Extended Host Range

Journal of Virology ◽

10.1128/jvi.75.11.4973-4983.2001 ◽

2001 ◽

Vol 75 (11) ◽

pp. 4973-4983 ◽

Cited By ~ 20

Author(s):

Eugene V. Barsov ◽

William S. Payne ◽

Stephen H. Hughes

Keyword(s):

Host Range ◽

Mammalian Cells ◽

Leukemia Virus ◽

Retroviral Vectors ◽

Retroviral Vector ◽

General Strategy ◽

Coding Region ◽

Chimeric Viruses

ABSTRACT We have designed and characterized two new replication-competent avian sarcoma/leukosis virus-based retroviral vectors with amphotropic and ecotropic host ranges. The amphotropic vector RCASBP-M2C(797-8), was obtained by passaging the chimeric retroviral vector RCASBP-M2C(4070A) (6) in chicken embryos. The ecotropic vector, RCASBP(Eco), was created by replacing theenv-coding region in the retroviral vector RCASBP(A) with the env region from an ecotropic murine leukemia virus. It replicates efficiently in avian DFJ8 cells that express murine ecotropic receptor. For both vectors, permanent cell lines that produce viral stocks with titers of about 5 × 106 CFU/ml on mammalian cells can be easily established by passaging transfected avian cells. Some chimeric viruses, for example, RCASBP(Eco), replicate efficiently without modifications. For those chimeric viruses that do require modification, adaptation by passage in vitro or in vivo is a general strategy. This strategy has been used to prepare vectors with altered host range and could potentially be used to develop vectors that would be useful for targeted gene delivery.

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ADA-deficient SCID is associated with a specific microenvironment and bone phenotype characterized by RANKL/OPG imbalance and osteoblast insufficiency

Blood ◽

10.1182/blood-2009-03-209221 ◽

2009 ◽

Vol 114 (15) ◽

pp. 3216-3226 ◽

Cited By ~ 45

Author(s):

Aisha V. Sauer ◽

Emanuela Mrak ◽

Raisa Jofra Hernandez ◽

Elena Zacchi ◽

Francesco Cavani ◽

...

Keyword(s):

Gene Therapy ◽

Bone Marrow ◽

Severe Combined Immunodeficiency ◽

Intrinsic Defect ◽

Combined Immunodeficiency ◽

Hematopoietic Stem ◽

Enzyme Replacement ◽

Bone Phenotype

Abstract Adenosine deaminase (ADA) deficiency is a disorder of the purine metabolism leading to combined immunodeficiency and systemic alterations, including skeletal abnormalities. We report that ADA deficiency in mice causes a specific bone phenotype characterized by alterations of structural properties and impaired mechanical competence. These alterations are the combined result of an imbalanced receptor activator of nuclear factor-κB ligand (RANKL)/osteoprotegerin axis, causing decreased osteoclastogenesis and an intrinsic defect of osteoblast function with subsequent low bone formation. In vitro, osteoblasts lacking ADA displayed an altered transcriptional profile and growth reduction. Furthermore, the bone marrow microenvironment of ADA-deficient mice showed a reduced capacity to support in vitro and in vivo hematopoiesis. Treatment of ADA-deficient neonatal mice with enzyme replacement therapy, bone marrow transplantation, or gene therapy resulted in full recovery of the altered bone parameters. Remarkably, untreated ADA–severe combined immunodeficiency patients showed a similar imbalance in RANKL/osteoprotegerin levels alongside severe growth retardation. Gene therapy with ADA-transduced hematopoietic stem cells increased serum RANKL levels and children's growth. Our results indicate that the ADA metabolism represents a crucial modulatory factor of bone cell activities and remodeling. The trials were registered at www.clinicaltrials.gov as #NCT00598481 and #NCT00599781.

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Bromo- and Extraterminal Domain Chromatin Regulators Serve as Cofactors for Murine Leukemia Virus Integration

Journal of Virology ◽

10.1128/jvi.01942-13 ◽

2013 ◽

Vol 87 (23) ◽

pp. 12721-12736 ◽

Cited By ~ 87

Author(s):

Saumya Shree Gupta ◽

Tobias Maetzig ◽

Goedele N. Maertens ◽

Azar Sharif ◽

Michael Rothe ◽

...

Keyword(s):

Murine Leukemia Virus ◽

Leukemia Virus ◽

Retroviral Vectors ◽

Transcription Start ◽

Preintegration Complex ◽

Transcription Start Sites ◽

Bet Inhibitors ◽

Murine Leukemia

Retroviral integrase (IN) proteins catalyze the permanent integration of proviral genomes into host DNA with the help of cellular cofactors. Lens epithelium-derived growth factor (LEDGF) is a cofactor for lentiviruses, including human immunodeficiency virus type 1 (HIV-1), and targets lentiviral integration toward active transcription units in the host genome. In contrast to lentiviruses, murine leukemia virus (MLV), a gammaretrovirus, tends to integrate near transcription start sites. Here, we show that the bromodomain and extraterminal domain (BET) proteins BRD2, BRD3, and BRD4 interact with gammaretroviral INs and stimulate the catalytic activity of MLV INin vitro. We mapped the interaction site to a characteristic structural feature within the BET protein extraterminal (ET) domain and to three amino acids in MLV IN. The ET domains of different BET proteins stimulate MLV integrationin vitroand, in the case of BRD2, alsoin vivo. Furthermore, two small-molecule BET inhibitors, JQ1 and I-BET, decrease MLV integration and shift it away from transcription start sites. Our data suggest that BET proteins might act as chromatin-bound acceptors for the MLV preintegration complex. These results could pave a way to redirecting MLV DNA integration as a basis for creating safer retroviral vectors.

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