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

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.

Download Full-text

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.

Download Full-text

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).

Download Full-text

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.

Download Full-text

Co-expression of cytokine and suicide genes to enhance the activity and safety of tumor-specific cytotoxic T lymphocytes

Blood ◽

10.1182/blood-2007-02-072843 ◽

2007 ◽

Vol 110 (8) ◽

pp. 2793-2802 ◽

Cited By ~ 112

Author(s):

Concetta Quintarelli ◽

Juan F. Vera ◽

Barbara Savoldo ◽

Greta M. P. Giordano Attianese ◽

Martin Pule ◽

...

Keyword(s):

T Lymphocytes ◽

Cytotoxic T Lymphocytes ◽

Severe Combined Immunodeficiency ◽

Interleukin 2 ◽

Suicide Gene ◽

Clinical Value ◽

Transgenic Expression ◽

Barr Virus

Abstract The antitumor effect of adoptively transferred tumor-specific cytotoxic T lymphocytes (CTLs) is impaired by the limited capacity of these cells to expand within the tumor microenvironment. Administration of interleukin 2 (IL-2) has been used to overcome this limitation, but the systemic toxicity and the expansion of unwanted cells, including regulatory T cells, limit the clinical value of this strategy. To discover whether transgenic expression of lymphokines by the CTLs themselves might overcome these limitations, we evaluated the effects of transgenic expression of IL-2 and IL-15 in our model of Epstein Barr Virus–specific CTLs (EBV-CTLs). We found that transgenic expression of IL-2 or IL-15 increased the expansion of EBV-CTLs both in vitro and in vivo in a severe combined immunodeficiency disease (SCID) mouse model and enhanced antitumor activity. Although the proliferation of these cytokine genes transduced CTLs remained strictly antigen dependent, clinical application of this approach likely requires the inclusion of a suicide gene to deal with the potential development of T-cell mutants with autonomous growth. We found that the incorporation of an inducible caspase-9 suicide gene allowed efficient elimination of transgenic CTLs after exposure to a chemical inducer of dimerization, thereby increasing the safety and feasibility of the approach.

Download Full-text

Human reconstituting hematopoietic stem cells up-regulate Fas expression upon active cell cycling but remain resistant to Fas-induced suppression

Blood ◽

10.1182/blood-2002-07-2286 ◽

2003 ◽

Vol 102 (1) ◽

pp. 118-126 ◽

Cited By ~ 18

Author(s):

Ingunn Dybedal ◽

Liping Yang ◽

David Bryder ◽

Ingbritt Aastrand-Grundstrom ◽

Karin Leandersson ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Severe Combined Immunodeficiency ◽

Constitutive Expression ◽

Human Cord Blood ◽

Hematopoietic Stem ◽

Nonobese Diabetic

Abstract The Fas receptor and its ligand have been implicated in mediating the bone marrow (BM) suppression observed in graft-versus-host disease and a number of other BM-failure syndromes. However, previous studies have suggested that Fas is probably not expressed on human hematopoietic stem cells (HSCs), but up-regulated as a consequence of their commitment and differentiation, suggesting that progenitors or differentiated blood cells, rather than HSCs, are the targets of Fas-mediated suppression. The present studies confirm that candidate HSCs in human cord blood and BM lack constitutive expression of Fas, but demonstrate that Fas expression on CD34+ progenitor and stem cells is correlated to their cell cycle and activation status. With the use of recently developed in vitro conditions promoting HSC self-renewing divisions, Fas was up-regulated on virtually all HSCs capable of multilineage reconstituting nonobese diabetic/severe combined immunodeficiency (NOD-SCID) mice in vivo, as well as on long-term culture-initiating cells (LTC-ICs). Similarly, in vivo cycling of NOD-SCID repopulating cells upon transplantation, resulted in up-regulation of Fas expression. However, repopulating HSCs expressing high levels of Fas remained highly resistant to Fas-mediated suppression, and HSC function was compromised only upon coactivation with tumor necrosis factor. Thus, reconstituting human HSCs up-regulate Fas expression upon active cycling, demonstrating that HSCs could be targets for Fas-mediated BM suppression. (Blood. 2003;102: 118-126)

Download Full-text

Dynamics of gene-modified progenitor cells analyzed by tracking retroviral integration sites in a human SCID-X1 gene therapy trial

Blood ◽

10.1182/blood-2009-12-257352 ◽

2010 ◽

Vol 115 (22) ◽

pp. 4356-4366 ◽

Cited By ~ 91

Author(s):

Gary P. Wang ◽

Charles C. Berry ◽

Nirav Malani ◽

Philippe Leboulch ◽

Alain Fischer ◽

...

Keyword(s):

Gene Therapy ◽

Integration Site ◽

Severe Combined Immunodeficiency ◽

Growth Control ◽

Early Time ◽

Cell Populations ◽

Retroviral Integration ◽

Gene Therapy Trial ◽

Vector Integration ◽

Integration Sites

Abstract X-linked severe-combined immunodeficiency (SCID-X1) has been treated by therapeutic gene transfer using gammaretroviral vectors, but insertional activation of proto-oncogenes contributed to leukemia in some patients. Here we report a longitudinal study of gene-corrected progenitor cell populations from 8 patients using 454 pyrosequencing to map vector integration sites, and extensive resampling to allow quantification of clonal abundance. The number of transduced cells infused into patients initially predicted the subsequent diversity of circulating cells. A capture-recapture analysis was used to estimate the size of the gene-corrected cell pool, revealing that less than 1/100th of the infused cells had long-term repopulating activity. Integration sites were clustered even at early time points, often near genes involved in growth control, and several patients harbored expanded cell clones with vectors integrated near the cancer-implicated genes CCND2 and HMGA2, but remain healthy. Integration site tracking also documented that chemotherapy for adverse events resulted in successful control. The longitudinal analysis emphasizes that key features of transduced cell populations—including diversity, integration site clustering, and expansion of some clones—were established early after transplantation. The approaches to sequencing and bioinformatics analysis reported here should be widely useful in assessing the outcome of gene therapy trials.

Download Full-text

Quantifying CRISPR off-target effects

Emerging Topics in Life Sciences ◽

10.1042/etls20180146 ◽

2019 ◽

Vol 3 (3) ◽

pp. 327-334 ◽

Cited By ~ 4

Author(s):

Soragia Athina Gkazi

Keyword(s):

Genome Editing ◽

Viral Vectors ◽

Insertional Mutagenesis ◽

Severe Combined Immunodeficiency ◽

Gene Therapies ◽

Genome Wide ◽

Large Indels ◽

Target Effects

Abstract Recent advances in the era of genetic engineering have significantly improved our ability to make precise changes in the genomes of human cells. Throughout the years, clinical trials based on gene therapies have led to the cure of diseases such as X-linked severe combined immunodeficiency (SCID-X1), adenosine deaminase deficiency (ADA-SCID) and Wiskott–Aldrich syndrome. Despite the success gene therapy has had, there is still the risk of genotoxicity due to the potential oncogenesis introduced by utilising viral vectors. Research has focused on alternative strategies like genome editing without viral vectors as a means to reduce genotoxicity introduced by the viral vectors. Although there is an extensive use of RNA-guided genome editing via the clustered regularly interspaced short palindromic repeats (CRISPR) and associated protein-9 (Cas9) technology for biomedical research, its genome-wide target specificity and its genotoxic side effects remain controversial. There have been reports of on- and off-target effects created by CRISPR–Cas9 that can include small and large indels and inversions, highlighting the potential risk of insertional mutagenesis. In the last few years, a plethora of in silico, in vitro and in vivo genome-wide assays have been introduced with the sole purpose of profiling these effects. Here, we are going to discuss the genotoxic obstacles in gene therapies and give an up-to-date overview of methodologies for quantifying CRISPR–Cas9 effects.

Download Full-text

Altered Proliferative Response by T Lymphocytes of Ly-6A (Sca-1) Null Mice

Journal of Experimental Medicine ◽

10.1084/jem.186.5.705 ◽

1997 ◽

Vol 186 (5) ◽

pp. 705-717 ◽

Cited By ~ 83

Author(s):

William L. Stanford ◽

Salma Haque ◽

Robert Alexander ◽

Xuemei Liu ◽

Anne M. Latour ◽

...

Keyword(s):

T Cell ◽

T Lymphocytes ◽

Proliferative Response ◽

Lymphocyte Activation ◽

Wild Type ◽

Normal Numbers ◽

Hematopoietic Stem ◽

Lymphocyte Responses

Ly-6A is a murine antigen which is implicated in lymphocyte activation and may be involved in activation of hematopoietic stem cells. Antibody cross-linking studies and antisense experiments have suggested that Ly-6A is a lymphocyte coactivation molecule. To better understand the function of Ly-6A, we used gene targeting to produce Ly-6A null mice which are healthy and have normal numbers and percentages of hematopoietic lineages. However, T lymphocytes from Ly-6A–deficient animals proliferate at a significantly higher rate in response to antigens and mitogens than wild-type littermates. In addition, Ly-6A mutant splenocytes generate more cytotoxic T lymphocytes compared to wild-type splenocytes when cocultured with alloantigen. This enhanced proliferation is not due to alterations in kinetics of response, sensitivity to stimulant concentration, or cytokine production by the T cell population, and is manifest in both in vivo and in vitro T cell responses. Moreover, T cells from Ly-6A–deficient animals exhibit a prolonged proliferative response to antigen stimulation, thereby suggesting that Ly-6A acts to downmodulate lymphocyte responses.

Download Full-text

Distinct hematopoietic progenitor compartments are delineated by the expression of aldehyde dehydrogenase and CD34

Blood ◽

10.1182/blood-2004-09-3652 ◽

2005 ◽

Vol 106 (1) ◽

pp. 95-102 ◽

Cited By ~ 91

Author(s):

Robert W. Storms ◽

Patrick D. Green ◽

Kristine M. Safford ◽

Donna Niedzwiecki ◽

Christopher R. Cogle ◽

...

Keyword(s):

Aldehyde Dehydrogenase ◽

Nk Cell ◽

Severe Combined Immunodeficiency ◽

In Vitro Assays ◽

Short Term ◽

Hematopoietic Stem ◽

Developmental Potential ◽

Development In Vitro

A broad range of hematopoietic stem cells and progenitors reside within a fraction of umbilical cord blood (UCB) that exhibits low light scatter properties (SSClo) and high expression of aldehyde dehydrogenase (ALDHbr). Many SSClo ALDHbr cells coexpress CD34; however, other cells express either ALDH or CD34. To investigate the developmental potential of these cell subsets, purified ALDHbr CD34+, ALDHneg CD34+, and ALDHbr CD34neg UCB cells were characterized within a variety of in vivo and in vitro assays. Primitive progenitors capable of multilineage development were monitored in long- and short-term repopulation assays performed on nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and in primary and secondary long-term culture assays. These progenitors were highly enriched within the ALDHbr CD34+ fraction. This cell fraction also enriched short-term myeloid progenitors that were detected in vitro. By comparison, ALDHneg CD34+ cells contained few primitive progenitors and had diminished short-term myeloid potential but exhibited enhanced short-term natural killer (NK) cell development in vitro. The ALDHbr CD34neg cells were not efficiently supported by any of the assays used. These studies suggested that in particular the expression of ALDH delineated distinct CD34+ stem cell and progenitor compartments. The differential expression of ALDH may provide a means to explore normal and malignant processes associated with myeloid and lymphoid development.

Download Full-text

Jak3 negatively regulates dendritic-cell cytokine production and survival

Blood ◽

10.1182/blood-2005-02-0769 ◽

2005 ◽

Vol 106 (9) ◽

pp. 3227-3233 ◽

Cited By ~ 39

Author(s):

Kunihiro Yamaoka ◽

Booki Min ◽

Yong-Jie Zhou ◽

William E. Paul ◽

John J. O'Shea

Keyword(s):

Severe Combined Immunodeficiency ◽

Interleukin 2 ◽

Janus Kinase ◽

Negative Regulator ◽

T Helper 1 ◽

Gamma Chain ◽

Antigen Uptake ◽

Hematopoietic Stem

AbstractCytokines are critical in regulating the development and function of diverse cells. Janus kinase 3 (Jak3) is a tyrosine kinase expressed in hematopoietic cells that associates with the common gamma chain (γc) and is required for signaling for a family of cytokines including interleukin-2 (IL-2), IL-4, IL-7, IL-9, IL-15, and IL-21; deficiency of either Jak3 or γc results in severe combined immunodeficiency (SCID). While Jak3 is essential for lymphoid-cell development, the potential roles for Jak3 in regulating dendritic cells (DCs) were unclear. Herein, we show that although CD8+CD11c+ splenic DCs are absent in Jak3-/- mice, bone marrow–derived DCs developed normally in vitro from Jak3-/- precursor cells. In fact, the survival of Jak3-/- DCs was enhanced, and they expressed lower levels of proapoptotic proteins. Jak3-/- DCs exhibited normal antigen uptake and up-regulation of costimulatory molecules. However, Jak3-/- DCs produced more IL-12 and IL-10 in response to Toll-like receptor ligands, which correlated with enhanced T helper 1 (Th1) differentiation in vivo. In summary, Jak3 is not essential for DC development but unexpectedly appears to be an important negative regulator. These results may be relevant clinically for patients with SCID who have undergone hematopoietic stem cell transplantation and for patients who might be treated with a Jak3 inhibitor.

Download Full-text