Abstract
Contract No.: 601958; EC contribution: € 5,999,997; Total costs: € 7,877,054.80; Starting date: 01/05/2013; Duration: 36 months
Background and Objectives
Whereas there is growing preclinical and clinical evidence of the therapeutic potential of gene therapy for many disabling or fatal diseases, current gene transfer technologies still limit wide application of gene therapy because of partial efficacy and/or safety concerns. Within the field of immunohematology in particular, the use of genetically modified hematopoietic stem cells (HSC) for the treatment of primary immunodeficiencies and the use of receptor-modified T-cells for the treatment of tumors have both shown considerable promise in early phase clinical trials. Yet, clinical testing of these two approaches has also uncovered a number of important roadblocks. The goal of SUPERSIST is to exploit the innovative potential of key technologies based on ex vivo gene targeting by artificial nucleases and gene delivery by lentiviral vectors to address these issues and advance HSC and T-cell gene therapy toward effective and safe application to a range of human diseases. The research and development activities are focused on two diseases chosen as paradigmatic for testing the therapeutic potential of gene editing: X-linked severe combined immunodeficiency (SCID-X1), which is a severe infantile rare inherited disease, and acute myeloid leukemia, a disease affecting adults and children.
Approach and Methodology
HSC-based gene therapy of SCID-X1
For inherited disorders, stem cells are the ideal cell target for gene transfer, because they retain the ability to renew themselves through mitotic cell division (self-renewal) and can differentiate into a diverse range of specialized cell types (multilineage differentiation). Indeed, their self-renewal capacity allows lifelong persistence of the corrected cells and maintenance of their genetic modifications in all the derived progeny. Moreover, gene transfer into the stem cells of patients with congenital deficiencies, followed by their autologous transplantation, could provide similar benefits as allogenic transplantation without the immunological complications of graft rejection and posttransplant immunosuppressive therapy. Indeed, toxicity risks and the limited numbers of appropriate donors still remain sufficiently problematic and, for many monogenic diseases, no drugs or valid therapeutic interventions are available. Thus, gene therapy represents a valid alternative solution for the cure of these diseases.
HSC-based gene therapy has indeed already provided substantial therapeutic benefits in several patients affected by primary immunodeficiencies, thalassemia, and leukodystrophies. However, to fully exploit the therapeutic potential of HSC gene therapy and broaden its application to other diseases, the adverse consequences of insertional mutagenesis and unregulated or ectopic transgene expression associated with the use of semirandomly integrating vectors must be abrogated. These adverse effects may trigger oncogenesis, toxicity, or elimination of the gene-modified cells, thus jeopardizing the efficacy and safety of the therapy. In the SUPERSIST project, the consortium aims to overcome both these hurdles by exploiting the targeted gene-editing strategies for direct correction of the disease-causing mutation. Thus, transferable-to-the-clinic protocols will be established for the genetic correction of disease-causing mutations in human HSC. Engineered nucleases and integrase-defective lentiviral vectors will be used to knock-in a functional cDNA into an inherited defective gene, downstream its own promoter, thus restoring both function and endogenous expression control of the mutant gene, without the risk of random insertional mutagenesis. This strategy will be applied to correct mutations in the interleukin 2 receptor common gamma chain (IL2RG) gene that are responsible for SCID-X1.
T-cell-based gene therapy of cancer
Cancer immunotherapy aims at harnessing the exquisite power and specificity of the immune system for the treatment of malignancies. In principle, the aim of this attractive approach is to translate the potency and specificity of immunity, well documented by its ability to combat infectious pathogens, toward tumors. This objective has been chased for decades by researchers, who clearly identified the potential benefits of a “living therapy,” able to persist, expand, and shrink in vivo to face specifically, proportionally, and persistently the multifaceted tumors. Cancer immunotherapy has been limited, for several years, by a combination of biological and technological factors, including immune escape mechanisms adopted by tumors, the resulting immunosuppressive tumor microenvironment, immune defects secondary to patient treatments, the lower immunogenicity of cancer cells compared with infectious pathogens, and preexisting tolerance toward tumor-associated, but often not tumor-specific, antigens. Today, the identification and characterization of these factors and of their relative contribution to tumor growth has met innovative technological solutions, raising highly and reasonably the hopes and leading the field of cancer immunotherapy to become the breakthrough of the year for 2013. The SUPERSIST consortium aims at generating and validating tumor-redirected T-cell receptor (TCR) gene-edited lymphocytes. This approach is based on the permanent substitution of T-cell specificity achieved in GMP-compliant conditions designed to promote the generation of memory T-lymphocytes able to recognize with high avidity and exquisite specificity cancer cells and to persist long-term in treated patients.
The SUPERSIST consortium is composed of five partners from four European countries and gathers the following leading expertise: development and application of new gene transfer technologies based on artificial nucleases, HSC-based gene therapy of rare genetic diseases of the hematopoietic system, and adoptive T-cell therapy for the treatment of leukemia (USR); cutting-edge technologies to identify tumor-reactive T-cell populations (NKI); high-throughput PCR-based technologies for genome-wide retrieval of vector–genomic junctions, next-generation sequencing technologies, and bioinformatics analysis (DKFZ); and SCID-X1 animal model and validation of gene and cell therapy products (UCL). The consortium also includes an industrial partner (MOLMED) that holds a unique expertise in translating research project into products approved for clinical trials. To achieve its objectives, the project is structured in three scientific and technological work packages, plus exploitation and management activities.
Main Findings
The recent development of engineered endonucleases, such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), has brought the possibility of gene targeting within the reach of cell and gene therapy. These artificial proteins comprise a nuclease domain derived from the FokI endonuclease and a DNA binding domain whose sequence specificity can be engineered. They are used to target a DNA double-strand break (DSB) with high efficiency and specificity to a preselected sequence within the genome. According to the repair process that seals the break, the outcome can be (1) mutagenesis at the nucleases target sites introduced by the error-prone nonhomologous end joining pathway, giving rise to a somatic gene knockout if essential coding sequences are targeted, and (2) faithful reconstitution of the original sequence by homology-driven repair, and (3) if an exogenous donor template is provided that contains sequence homology to each side of the DSB, the targeted sequence can be edited and novel sequences introduced at the site according to the design of the donor.
USR first showed that gene targeting can be used to correct genetic mutations by inserting a functional cDNA copy of the gene downstream its own promoter. This approach has the advantage that most disease-causing mutations affecting the locus, including deletions, can be treated with the same engineered nuclease(s). Importantly, gene correction, as opposed to gene replacement, restores not only the function of the gene but also its physiological expression control, coming close to the long-sought holy grail of gene therapy. Despite the great promise of these technologies, some challenges remain to be addressed to fully exploit their potential for cell and gene therapy. Delivery of nucleases and cognate donor template DNA is challenging in primary cells. USR was also the first to report a delivery strategy based on integrase-defective LV to achieve significant levels of gene targeting in several human primary cell types, but the demonstration of successful targeting in primitive long-term repopulating HSC was for long time only elusive. Within the SUPERSIST project, we found that poor permissiveness to gene transfer and limited proficiency of the homology-directed DNA repair pathway both constrain gene targeting in human HSCs. By tailoring delivery platforms and culture conditions, they successfully overcame these barriers and provided stringent evidence of targeted integration in human HSCs by long-term multilineage repopulation of transplanted mice. The therapeutic potential of this strategy was demonstrated by targeting a corrective cDNA into the IL2RG gene of HSCs. Gene-edited HSCs sustained normal hematopoiesis and gave rise to functional lymphoid cells that possess a marked selective growth advantage over those carrying disruptive IL2RG mutations. These results represent a milestone achievement for the project, were published in a top scientific journal, and received as a breakthrough from the scientific community and open the way to the exploitation of targeted genome-editing strategies in HSC-based gene therapy.
Although gene therapy was initially designed to correct genetic defects, it was readily adapted for treating cancer. The possibility to tame toxicity, increase effector activity, and ultimately redirect T-cells to kill cancer cells by genetic engineering approaches, such as the transfer of TCR, allows deploying all the firing power of immunity against cancer. However, despite that TCR gene transfer is a promising approach of adoptive immunotherapy for cancer patients, T-lymphocytes transduced with an exogenous TCR have proved less effective than naturally occurring tumor-specific T-cells, and led to suboptimal clinical results. This could be because of the dilution of the tumor-specific TCR that competes with endogenous TCRs for its expression on the T-cell surface. Moreover, random mispairing between endogenous and exogenous TCR α and β chains could generate new TCRs with unpredictable and potentially harmful specificities. To overcome these issues, USR developed the TCR gene-editing procedure, based on the knockout of the endogenous TCR genes by transient exposure to α and β chain-specific ZFNs, followed by the introduction of tumor-specific TCR genes by lentiviral vectors. The original approach comprises four sequential steps of genetic manipulation and selection of engineered T-cells, and required approximately 40 days of culture. Within the SUPERSIST project and in view of a prompt clinical translation, we have recently simplified the editing protocol to retain the benefits of the original TCR gene-editing strategy while increasing its feasibility. This optimized gene-editing protocol can now be combined to T-cell activation and culture procedures that allow generating gene-edited T-cells with an early memory T-cell phenotype, which is associated to long-term persistence of the cells in several mouse models. These advances are instrumental to pave the way toward the development of a feasible and cost-efficient process supporting clinical testing of this new technology.
In another fundamental advance made within the project, NKI has developed highly innovative TCR capture techniques that allow identifying novel tumor-reactive TCRs from primary clinical specimens. These studies have provided major new insights in the antigenic space targeted by the immune system in human tumors. These findings were reported in a top scientific journal and granted great attention by the scientific community, as they have substantial implications for understanding the process of cancer–directed immune response as well as improving the design and application of gene-based immunotherapy. Within the SUPERSIST summary, we will now exploit these technologies to widen the reach of TCR gene editing toward a broader range of neoplastic diseases.
With the support of the SME partner MOLMED, the consortium will continue to perform the scale-up and optimization of gene-targeting protocols for both HSC and T-cells for the two gene therapy applications being developed. These processes will enable clinical testing of the new strategies. We are also designing an exploitation roadmap for SUPERSIST results, taking advantage of partnerships already established by consortium partners with leading biotech companies in the field, such as Sangamo BioSciences, for the development of ZFNs, and other big pharmas.
Expected Outcome
HSC gene therapy and cancer adoptive immunotherapy are facing an accelerated phase of development, largely because of the first therapeutic successes obtained with genetically modified cells in clinical trials of inherited diseases and lymphoid malignancies. We can envisage that adoptive T-cell therapy will soon become a major pillar in cancer treatment. The expected outcome of SUPERSIST will be the next generation of HSC- and T-cell-based gene therapy, in which conventional gene addition is replaced by the more precise and powerful approach of targeted gene editing, achieved through a combination of state-of-the-art gene transfer vectors and artificial nucleases. While two diseases have been selected for investigation within the project for their paradigmatic features, the strategies being developed are expected to be potentially applicable to several additional human diseases. Thus, the outcome of SUPERSIST should benefit the European population by providing a long-sought perspective of cure for a number of severe diseases, leading to improvement in population health and reduction in healthcare costs, while at the same time increasing the competitiveness of European biomedical and pharmaceutical industry through the generation of new scientific knowledge, processes, and technologies.
