SCID-X1 Gene Therapy Research Articles

299. Insertion Site Analysis and In Vivo Selection

Leukemias arising in 2 patients treated with gene therapy for X-SCID may be viewed to result from a collaboration between a transcription factor, abberantly expressed due to insertional mutagenesis, and a constitutively active, receptor-initiated growth signal. We are undertaking a systematic comparison of retrovirus integration sites in transduced hemopoietic cells prior to and following in vivo selection. The system for selection uses a conditional derivative of the thrombopoietin receptor, F36Vmpl, that transmits a growth signal in the presence of an artificial ligand called a chemical inducer of dimerization (CID). Our initial studies have focused on comparing insertion sites identified by Southern analysis with those identified by LAM-PCR. A cohort of 11 mice was transplanted with marrow cells transduced with a bicistronic vector encoding green fluorescent protein (GFP) and F36Vmpl. Marrow aspirates were obtained at baseline and following administration of 3 monthly cycles of CID (AP20187 10mg/kg IP daily for 3 days). 10 of 11 mice exhibited strong responses to CID treatment, with the percentage of GFP positive red cells rising from <20% at baseline to 90% following CID exposure. Following selection, marrow cells from these mice were then transplanted 1:1 into 11 secondary recipients which in turn received 3 additional monthly courses of CID. No GFP-positive cells were detected in 3 of the secondary recipients, and CID administration in these mice failed to elicit a response. Responses to CID treatment were observed in the remaining 8 mice, and in 3 secondary recipients the responses were the same or better than their donors. We compared retorviral insertion sites in donor-recipient pairs by Southern analysis and LAM-PCR. 4 of 11 donor-recipient pairs displayed a common banding pattern by Southern analysis following HindIII digestion, including the 3 pairs in which the hematological response of the secondary recipient equaled or bettered that of their donors. The same 4 donor-recipient pairs were the only ones found to have common proviral insertion sites as identified by LAM-PCR. However, in only one pair did the number of unique insertions identified by LAM-PCR equal that identified by Southern (4 insertions each). In each of the remaining 3 pairs only a single common insertion was identifed by LAM-PCR, whereas Southern detected 3 shared insertions in two pairs, and 5 shared insertions in the remaining pair. Our findings to date demonstrate that traditional LAM-PCR detects only a fraction of insertion sites that are sufficiently abundant to be detectable by Southern blot. As expected, LAM-PCR also identified multiple unique insertions in animals that had no detectable signal by Southern. In addition to modifying our LAM-PCR method to reveal abundant, yet undetected insertion sites, we are extending our analysis to single progenitor colonies taken from mice prior to and following F36Vmpl-based in vivo selection.

707. Retroviral Insertion Site Analysis in Rhesus Macaques Transplanted with CD34+ Hematopoietic Stem Cells Transduced with a Simian Immunodeficiency Virus-Based Lentiviral Vector

Two children involved in the successful retroviral gene therapy clinical trial of SCID-X1 (2 year survival: 100%, FFTF 60%) developed a malignant T-cell lymphoproliferation 34 months after treatment. In both cases, vector integration into the vicinity of exon 1 of the protooncogene LMO2 occured (Hacein-Bey-Abina, 2003). The potential for unwanted side effects from retroviral insertional mutagenesis is now a focus of biosafety considerations in gene transfer using integrating vector systems. Several groups could show that gene coding areas, in particular transcription start sites, are preferred insertion loci (Schroder, 2002; Wu, 2003). While these studies mapped a great number of integration sites with statistical significance, they were limited to in vitro gene transfer into nonhematopoietic human cell lines. As T cells do not grow in these cases unless they express the gamma c chain, we reasoned that there may be selection pressure on the retrovirus integration site distribution. Using a highly sensitive and specific linear amplification mediated PCR (LAM-PCR), we analyzed DNA samples from 8 patients enrolled in the X-SCID gene therapy trial. We could perform successive insertion site analysis and subsequently identify more than 400 integration sites after transplantation. In all patient samples examined, a stable polyclonal profile demonstrated polyclonal hematopoietic repopulation. To distinguish the influence of required transgene expression on retrovirus integration site distribution, we analyzed insertion sites in patient CD34+ cells prior to autologous transplantation, shortly after ex vivo transduction. More than 100 integration sites present prior to transplantation could be identified for comparative pre-/post-tranplantation analysis. No obvious difference in insertion site distribution could be detected between pre- and post-transplant samples form a first analysis. However, our data indicate that X-SCID correction, which depends on transgene expression, favors chromosomal locations associated with active transciption. We conclude that the distribution of retrovirus integration sites can be assessed by random sampling in a clinical trial setting and may differ significantly from previously published assumptions, especially in settings of selective advantage related to transgene expression.

Long-Term Clinical and Molecular Follow-up of Large Animals Receiving Retrovirally Transduced Stem and Progenitor Cells: No Progression to Clonal Hematopoiesis or Leukemia

There has been significant progress toward clinically relevant levels of retroviral gene transfer into hematopoietic stem cells (HSC), and the therapeutic potential of HSC-based gene transfer has been convincingly demonstrated in children with severe combined immunodeficiency syndrome (SCID). However, the subsequent development of leukemia in two children with X-linked SCID who were apparently cured after transplantation of retrovirally corrected CD34+ cells has raised concerns regarding the safety of gene therapy approaches utilizing integrating vectors. Nonhuman primates and dogs represent the best available models for gene transfer safety and efficacy and are particularly valuable for evaluation of long-term effects. We have followed 42 rhesus macaques, 23 baboons, and 17 dogs with significant levels of gene transfer for a median of 3.5 years (range 1–7) after infusion of CD34+ cells transduced with retroviral vectors expressing marker or drug-resistance genes. None developed abnormal hematopoiesis or leukemia. Integration site analysis confirmed stable, polyclonal retrovirally marked hematopoiesis, without progression toward mono- or oligoclonality over time. These results suggest that retroviral integrations using replication-incompetent vectors, at copy numbers achieved using standard protocols, are unlikely to result in leukemogenesis and that patient- or transgene-specific factors most likely contributed to the occurrence of leukemia in the X-SCID gene therapy trial.

Corrections and Clarifications

Research Articles: “LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1” by S. Hacein-Bey-Abina et al. (17 Oct., p. 415). The second and third authors, C. Von Kalle and M. Schmidt, should have had asterisks after their names, to indicate shared first authorship with S. Hacein-Bey-Abina. The asterisks were inadvertently omitted because of an editorial error.

LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1.

We have previously shown correction of X-linked severe combined immunodeficiency [SCID-X1, also known as gamma chain (gamma(c)) deficiency] in 9 out of 10 patients by retrovirus-mediated gamma(c) gene transfer into autologous CD34 bone marrow cells. However, almost 3 years after gene therapy, uncontrolled exponential clonal proliferation of mature T cells (with gammadelta+ or alphabeta+ T cell receptors) has occurred in the two youngest patients. Both patients' clones showed retrovirus vector integration in proximity to the LMO2 proto-oncogene promoter, leading to aberrant transcription and expression of LMO2. Thus, retrovirus vector insertion can trigger deregulated premalignant cell proliferation with unexpected frequency, most likely driven by retrovirus enhancer activity on the LMO2 gene promoter.

The Sixth Annual Meeting of the American Society of Gene Therapy

thought to be a multistep process of sequential genetic lesions that take a cell from a normal phenotype through various stages of hyperplasia to a blatantly malignant phenotype—at least for solid tumor development. The development of leukemia may not necessarily follow such a path, and the adverse event in the French trial may eventually provide valuable information on the mechanism of leukemogenesis. Robert Weinberg presented this year’s Presidential Symposium, outlining work in his lab and others to understand the diversity and idiosyncratic nature of the genetic roadmap leading to cancer development in specific tissues, as well as the effects of surrounding tissues on the cancer cell. The Corporate Symposium this year focused on oncolytic viruses for cancer therapy, many of which have made their way to the clinic. While promising results have been obtained in many cases, a familiar theme was the need for better systems to evaluate these antitumor agents and the need to develop viruses that are more robust in their effects upon tumor cells relative to other cells of the host. Nevertheless, the symposium highlighted the slow but inexorable march of gene therapeutics out of the lab and into the complex world of clinical medicine. It would be impossible here to give more than a bird’s eye view of the work presented at this year’s meeting. With over 1200 abstracts submitted from laboratories around the world, the meeting represented the breadth of research into molecular and cellular therapies based on gene transfer and gene modification technology. Some may eventually work in patients, and many will not. However, as outgoing ASGT President Joe Glorioso stated in his farewell address to the attendees, “On balance, we are pioneering a new wave of genetic medicines. Progress is not without setbacks, and the path requires faith, innovation, stamina, and reason.”

Efficient characterization of retro-, lenti-, and foamyvector-transduced cell populations by high-accuracy insertion site sequencing.

The identification of unknown genomic flanking DNA sequences can be used for the molecular monitoring of retro-, lenti- and foamyviral integration, transgenes in early embryogenesis, insertional mutagenesis, cell fate, and stem cell plasticity. Most existing methods reflect shortcomings in sensitivity and or specificity, thus limiting genomic sequencing of unknown flanking DNA to clonal preparations. The application of linear amplification-mediated PCR (LAM-PCR), a recently developed direct sequencing technique for flanking DNA, should circumvent current limitations in different research fields. This technique combines preamplification of target DNA with a unique succession of enzymatic reactions on solid-phase. Using LAM-PCR, we show the previously unfeasible in vivo retro-, lenti- and foamyvirus integration site analysis in primate peripheral blood hematopoietic cells and human xenograft hematopoiesis. In light of two severe adverse events that occurred in a clinical SCID-X1 gene therapy trial, in vivo monitoring of the reinfused transduced cell pool by integration site analysis will be an important component of each gene transfer and therapy study aimed at clinical use.

Gene therapy of severe combined immunodeficiencies.

Recent advances in gene transfer in human hematopoietic cells, combined with a better understanding of the genetic aspects of several immunodeficiencies, has offered new opportunities in the domain of gene therapy. Severe combined immunodeficiency (SCID) appear to represent a good model for the application of gene therapy, combining an expected selective advantage for transduced cells, an absence of immunological response to the vector and/or the therapeutic transgene, together with accessibility to hematopoietic stem cells (HSC). Ex vivo retroviral transduction of a therapeutic transgene in HSC prior to transplantation appears to be a particularly effective and long-lasting means of restoring the expression of a mutated gene in the lymphoid lineage. Furthermore, encouraging therapeutic benefits as a result of a gene therapy protocol for the treatment of X-linked severe combined immunodeficiencies (SCID-X1) invites many questions as to the reasons for this therapeutic benefit. This review outlines the results that have been achieved in gene therapy for SCID-X1, ADA-SCID as well as other types of SCID, and discusses the possible relationship between the physiopathology of each disease and the success of relevant trials.

Is gene therapy for SCID X1 all it claims to be?

Is gene therapy for SCID X1 all it claims to be?

Defective lymphoid development in mice lacking expression of the common cytokine receptor γ chain

The common γ chain ( γ c) of the IL-2, IL-4, IL-7, IL-9, and IL-15 receptors is defective in humans with XSCID. Mice lacking γ c expression had poplastic thymuses; the thymocytes responded to γ c-independent mitogens, but not γ c-dependent stimuli. Splenic T cells were diminished at 3 weeks of age, but CD4 + T cells markedly increased by 4 weeks. B cells were greatly diminished, in contrast with the situation in XSCID. NK cells, γδ intestinal intraepithelial lymphocytes, dendritic epidermal T cells, peripheral lymph nodes, and gut-associated lymphoid tissue were absent. These findings underscore the Importance of γ c in lymphoid development. Moreover, differences in humans and mice lacking γ c expression indicate species-specific differences in the roles of γ c-dependent cytokines or in the existence of redundant pathways. These mice provide an important model for studying the pathophysiology of and gene therapy for human XSCID.

The Molecular Basis of X‐Linked Severe Combined Immunodeficiency: The Role of the Interleukin‐2 Receptor γ Chain as a Common γ Chain, γc

X-linked severe combined immunodeficiency is characterized by severe and persistent infections from early life resulting from profound impairment of both cellular and humoral immune function. XSCID is characterized by an absence or diminished number of T cells and histologic evidence of hypoplastic and abnormal differention of the thymic epithelium. The discovery that this disease results from the mutations of the IL-2R gamma chain was surprising since IL-2-deficient mice and human SCID patients had milder phenotypes. This led to the speculation that IL-2R gamma would prove to be a common gamma chain, gamma c, which would play important roles in other cytokine receptors in addition to the IL-2 receptor. There is now compelling evidence to support a role in at least two other cytokine receptors, namely the IL-4 and IL-7 receptors. Thus, with inactivation of gamma c, multiple cytokine systems are simultaneously affected, resulting in the profoundly impaired phenotype of XSCID. It is possible and even likely that gamma c will be found to be a functional component of additional receptors as well. These findings have resulted in a significant improvement in our understanding of the pathophysiologic development of the defects in XSCID and also have important ramifications for prenatal and postnatal diagnosis, carrier female identification, and gene therapy for XSCID.