Gene therapy for hematologic disease: Don’t throw the baby out with the bathwater!

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The 21st century thus far has been a roller coaster ride for gene therapies targeting hematopoietic stem cells or hematologic diseases such as hemophilia. Disappointing results from pioneering clinical trials during the 1990s stimulated a re-examination of the entire field, with both scientists and funding agencies stressing the need to expand knowledge regarding vector systems, preclinical models, and the pathophysiology of target diseases, before returning to the clinic. Significant progress was made in improving the efficiency of hematopoietic cell gene transfer using novel cytokines, transduction conditions, and appropriate large animal or immunodeficient murine xenograft models to reach levels of gene transfer efficiency likely to be therapeutic for some disorders. Sufficient and persistent expression of factor IX at levels adequate to prevent bleeding in dogs with hemophilia B was achieved using adeno-associated virus vectors injected into skeletal muscle. The tragic death of Jesse Gelsinger in 1999 focused attention on appropriate conduct of clinical trials and regulatory oversight, but this event did not impact on the optimism and determination of investigators and patients to develop gene therapy for serious hematologic diseases such as severe combined immunodeficiencies (SCID) or hemophilia.

In 2000, all these efforts came to fruition with the initial report of unequivocal immune reconstitution following transplantation of retrovirally transduced CD34⁺ cells in two children with X-linked severe combined immunodeficiency, in a study from France led by Alain Fischer. Two years later follow-up showed persistent benefit in four of five patients, and eventually 11 patients were enrolled in this trial. Soon thereafter, the Italian group led by Claudio Bordignon reported positive results in a second variety of SCID, adenosine deaminase deficiency (ADA-SCID). But the euphoria was short-lived, when two cases of T-cell leukemias consisting of clonal vector-containing blasts were reported, first in August 2002 and then in December 2002, in the two youngest children enrolled in the X-SCID trial. With the news of the second case, most regulatory agencies worldwide suspended ongoing clinical trials of integrating retroviral vectors to transduce hematopoietic targets. A multinational cooperative effort rapidly ensued to understand the events, with full cooperation and disclosure of data to interested parties worldwide by the investigators conducting the clinical trial. Successful application of the powerful linear amplification-mediated polymerase chain reaction (LAM-PCR) technique by its developers, Christof von Kalle and Manfred Schimdt, showed that both children had retroviral insertions activating the LMO-2 transcription factor gene, a locus previously implicated in spontaneous T-cell leukemias via chromosomal translocations.

This molecular finding has stimulated a complete re-evaluation of the mechanisms and pattern of retroviral integration events, and serendipitously at about the same time, new information regarding integration events was available through rapid site identification techniques and the draft complete human genome sequence. Patients enrolled in other gene therapy trials, concurrent and previous, were closely examined for any signs of malignancy or progression towards clonal hematopoiesis, thankfully with no evidence for additional cases. The remaining children in the X-SCID trial in France and additional patients treated on a similar study in England remain well. Long-term follow-up studies in relevant large animals transplanted with CD34⁺ cells transduced with marking vectors also have failed to reveal any worrisome clinical or molecular events. Many questions remain, but attention has focused on the possible interaction of the gamma c transgene and integration events, possibly favored in an expanded population of lymphoid progenitors in the X-SCID patients.

During the past 2 years, the gene therapy community and regulatory agencies have worked together to respond appropriately to these events. Despite predictions of the immediate demise of gene therapy in the press, optimism remains, tempered by the realization that there are real dangers and that further clinical protocols must carefully weigh risks and benefits while ongoing research refines techniques and vectors to reduce risk. Regulatory agencies have allowed trials for patients with life-threatening disorders to proceed, given the clear benefit for the majority of patients even in the X-SCID trial.

Now is an opportune moment in which to summarize the status of gene therapies for hematologic diseases, in particular to update hematologists and investigators outside the field regarding our current understanding of the potential risks and benefits, as well as ongoing research to improve safety and expand applications to more common hematologic disorders. First, LaRochelle and myself summarize the concepts and techniques central to gene therapy directed at primitive hematopoietic stem and progenitor cells, focusing on the factors allowing marked improvement in gene transfer efficiency to primate and human hematopoietic stem cells which have been developed over the past 7 or 8 years. We then summarize the many ongoing challenges, such as control of the level and lineage specificity of transgene expression, requirements for conditioning therapy and high doses of appropriate target cells to approach disorders without an inherent in vivo selective advantage for corrected cells, and highlight a number of recent advances in vector derivation and in vivo or ex vivo selection that could overcome problems such as in vivo selection.

Hacien-Bey-Albina, Cavazzano-Calvo and Fischer, the lead investigators in the French X-SCID clinical trial, summarize both the triumphs and the recent setbacks in gene therapy for X-SCID, along with the status of gene therapy for other immunodeficiency disorders, including protocols already in the clinic and others in preclinical development. The immunodeficiency disorder ADA-SCID was the target of the first clinical gene therapy trial more than 10 years ago, and these rare disorders continue to serve as paradigms for development of better and safer approaches.

Next, Persons and Tisdale describe and comment on the current status and future challenges for moving stem cell gene therapy of hemoglobinopathies finally into the clinic. Sickle cell anemia and the thalassemias were historically considered prime targets for gene therapy, given decades of molecular understanding of their pathophysiology, their relative frequency in the world population, and a lack of other effective or widely available treatment options. It became apparent in the early 1990s that, despite the many dedicated and creative investigators placing a priority on developing gene therapy approaches to these diseases, high-level and erythroid lineage-specific expression of hemoglobin genes was impossible using standard retroviral vectors. Thus little progress occurred until the introduction of lentiviral vectors in the late 1990s, finally allowing high-level expression without vector instability—and cure of animal models of both sickle cell anemia and thalassemia.

Progress in the gene therapy of hemophilias is summarized by Lozier. Over the past 5 years very promising results in murine and canine hemophilia models using adenoviral and then adenoassociated viral vectors generated excitement, predicting therapeutic success in carefully designed and performed clinical trials. Unfortunately, these trials have not produced unequivocal evidence of benefit. Lozier explains some of the possible reasons for these difficulties, as well as ongoing and future preclinical and clinical attempts to overcome these obstacles and result in a safer and more tolerable treatment than life-long factor infusions.

Walsh discusses very exciting new approaches to correct genetic or acquired disorders by RNA correction or modulation, also in the context of hemophilia. Trans-splicing technologies can correct mutant genes while retaining endogenous control of expression, promising in particular for both efficacy and safety in disorders where exact regulation of gene product level is required.

Finally Baum, Felse, Kelly, and von Kalle examine the issue of gene therapy toxicity, in particular insertional mutagenesis associated with the use of integrating vectors. Their groups have been central to the development of techniques for insertion site identification and tracking and to the rapid molecular dissection of the events resulting in leukemia in the two children enrolled in the X-SCID trial. They summarize active ongoing work in understanding the scope of the problem, summarize multiple potential approaches to decreasing risks in the future, and provide concrete examples and classifications of risk and benefit, based on underlying disease and the type of transgene. Their careful assessments should serve as a roadmap for criteria to guide gene therapy using integrating vectors in clinical trials.