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Gene therapy may provide a cure for hemophilia and overcome the limitations of protein replacement therapy. Increasing the potency of gene transfer vectors may allow improvement of their therapeutic index, as lower doses can be administered to achieve therapeutic benefit, reducing toxicity of in vivo administration. Here we generated codon-usage optimized and hyperfunctional factor IX (FIX) transgenes carrying an R338L amino acid substitution (FIX Padua), previously associated with clotting hyperactivity and thrombophilia. We delivered these transgenes to hemophilia B mice by hepatocyte-targeted integration-competent and -defective lentiviral vectors. The hyperfunctional FIX transgenes increased FIX activity reconstituted in the plasma without detectable adverse effects, allowing correction of the disease phenotype at lower vector doses and resulting in improved hemostasis in vivo. The combined effect of codon optimization with the hyperactivating FIX-R338L mutation resulted in a robust 15-fold gain in potency and therefore provides a promising strategy to improve the efficacy, feasibility, and safety of hemophilia gene therapy.

Adenosine acts as anti-inflammatory mediator on the immune system and has been described in regulatory T cell (Treg)-mediated suppression. In the absence of adenosine deaminase (ADA), adenosine and other purine metabolites accumulate, leading to severe immunodeficiency with recurrent infections (ADA-SCID). Particularly ADA-deficient patients with late-onset forms and after enzyme replacement therapy (PEG-ADA) are known to manifest immune dysregulation. Herein we provide evidence that alterations in the purine metabolism interfere with Treg function, thereby contributing to autoimmune manifestations in ADA deficiency. Tregs isolated from PEG-ADA-treated patients are reduced in number and show decreased suppressive activity, whereas they are corrected after gene therapy. Untreated murine ADA(-/-) Tregs show alterations in the plasma membrane CD39/CD73 ectonucleotidase machinery and limited suppressive activity via extracellular adenosine. PEG-ADA-treated mice developed multiple autoantibodies and hypothyroidism in contrast to mice treated with bone marrow transplantation or gene therapy. Tregs isolated from PEG-ADA-treated mice lacked suppressive activity, suggesting that this treatment interferes with Treg functionality. The alterations in the CD39/CD73 adenosinergic machinery and loss of function in ADA-deficient Tregs provide new insights into a predisposition to autoimmunity and the underlying mechanisms causing defective peripheral tolerance in ADA-SCID.

Type I mucopolysaccharidosis (MPS I) is a lysosomal storage disorder caused by the deficiency of α-L-iduronidase, which results in glycosaminoglycan accumulation in tissues. Clinical manifestations include skeletal dysplasia, joint stiffness, visual and auditory defects, cardiac insufficiency, hepatosplenomegaly, and mental retardation (the last being present exclusively in the severe Hurler variant). The available treatments, enzyme-replacement therapy and hematopoietic stem cell (HSC) transplantation, can ameliorate most disease manifestations, but their outcome on skeletal and brain disease could be further improved. We demonstrate here that HSC gene therapy, based on lentiviral vectors, completely corrects disease manifestations in the mouse model. Of note, the therapeutic benefit provided by gene therapy on critical MPS I manifestations, such as neurologic and skeletal disease, greatly exceeds that exerted by HSC transplantation, the standard of care treatment for Hurler patients. Interestingly, therapeutic efficacy of HSC gene therapy is strictly dependent on the achievement of supranormal enzyme activity in the hematopoietic system of transplanted mice, which allows enzyme delivery to the brain and skeleton for disease correction. Overall, our data provide evidence of an efficacious treatment for MPS I Hurler patients, warranting future development toward clinical testing.

Long-term clinical remissions of leukemia, after allogeneic hematopoietic stem cell transplantation, depend on alloreactive memory T cells able to self-renew and differentiate into antileukemia effectors. This is counterbalanced by detrimental graft-versus-host disease (GVHD). Induction of a selective suicide in donor T cells is a current gene therapy approach to abrogate GVHD. Unfortunately, genetic modification reduces alloreactivity of lymphocytes. This associates with an effector memory (T(EM)) phenotype of gene-modified lymphocytes and may limit antileukemia effect. We hypothesized that alloreactivity of gene-modified lymphocytes segregates with the central memory (T(CM)) phenotype. To this, we generated suicide gene-modified T(CM) lymphocytes with a retroviral vector after CD28 costimulation and culture with IL-2, IL-7, or a combination of IL-7 and IL-15. In vitro, suicide gene-modified T(CM) cells self-renewed upon alloantigen stimulation and resisted activation-induced cell death. In a humanized mouse model, only suicide gene-modified T cells cultured with IL-7 and IL-15 persisted, differentiated in T(EM) cells, and were as potent as unmanipulated lymphocytes in causing GVHD. GVHD was halted through the activation of the suicide gene machinery. These results warrant the use of suicide gene-modified T(CM) cells cultured with IL-7 and IL-15 for the safe exploitation of the alloreactive response against cancer.

Adenosine deaminase (ADA) deficiency is caused by a purine metabolic dysfunction, leading to severe combined immunodeficiency (SCID) and multiple organ damage. To investigate the efficacy of ex vivo gene therapy with self-inactivating lentiviral vectors (LVs) in correcting this complex phenotype, we used an ADA(-/-) mouse model characterized by early postnatal lethality. LV-mediated ADA gene transfer into bone marrow cells combined with low-dose irradiation rescued mice from lethality and restored their growth, as did transplantation of wild-type bone marrow. Mixed chimerism with multilineage engraftment of transduced cells was detected in the long term in animals that underwent transplantation. ADA activity was normalized in lymphocytes and partially corrected in red blood cells (RBCs), resulting in full metabolic detoxification and prevention of severe pulmonary insufficiency. Moreover, gene therapy restored normal lymphoid differentiation and immune functions, including antigen-specific antibody production. Similar degrees of detoxification and immune reconstitution were obtained in mice treated early after birth or after 1 month of enzyme-replacement therapy, mimicking 2 potential applications for ADA-SCID. Overall, this study demonstrates the efficacy of LV gene transfer in correcting both the immunological and metabolic phenotypes of ADA-SCID and supports the future clinical use of this approach.

In allogeneic hematopoietic cell transplantation (allo-HCT), the immune recognition of host antigens by donor T lymphocytes leads to a beneficial graft-versus-leukemia (GvL) effect as well as to life-threatening graft-versus-host disease (GvHD). Genetic modification of T lymphocytes with a retroviral vector (RV) expressing the herpes simplex virus-thymidine kinase (TK) suicide gene confers selective sensitivity to the prodrug ganciclovir (GCV). In patients, the infusion of TK+ lymphocytes and the subsequent administration of GCV resulted in a time-wise modulation of antihost reactivity for a GvL effect, while controlling GvHD. Because activation required for genetic modification with RV may reduce antihost reactivity, we investigated the requirements for maximizing the potency of human TK+ lymphocytes. Whereas T-cell receptor triggering alone led to effector memory (EM) TK+ lymphocytes, the addition of CD28 costimulation through cell-sized beads resulted in the generation of central memory (CM) TK+ lymphocytes. In a quantitative model for GvHD using nonobese diabetic/severely combined immunodeficient mice, CM TK+ lymphocytes were more potent than EM TK+ lymphocytes. GCV administration efficiently controlled GvHD induced by CM TK+ lymphocytes. These results warrant the clinical investigation of CM suicide gene-modified human T lymphocytes for safe and effective allo-HCT.