Autologous Ex Vivo Lentiviral Gene Therapy for Adenosine Deaminase Deficiency.

BACKGROUND: Severe combined immunodeficiency due to adenosine deaminase (ADA) deficiency (ADA-SCID) is a rare and life-threatening primary immunodeficiency. METHODS: We treated 50 patients with ADA-SCID (30 in the United States and 20 in the United Kingdom) with an investigational gene therapy composed of autologous CD34+ hematopoietic stem and progenitor cells (HSPCs) transduced ex vivo with a self-inactivating lentiviral vector encoding human ADA. Data from the two U.S. studies (in which fresh and cryopreserved formulations were used) at 24 months of follow-up were analyzed alongside data from the U.K. study (in which a fresh formulation was used) at 36 months of follow-up. RESULTS: Overall survival was 100% in all studies up to 24 and 36 months. Event-free survival (in the absence of reinitiation of enzyme-replacement therapy or rescue allogeneic hematopoietic stem-cell transplantation) was 97% (U.S. studies) and 100% (U.K. study) at 12 months; 97% and 95%, respectively, at 24 months; and 95% (U.K. study) at 36 months. Engraftment of genetically modified HSPCs persisted in 29 of 30 patients in the U.S. studies and in 19 of 20 patients in the U.K. study. Patients had sustained metabolic detoxification and normalization of ADA activity levels. Immune reconstitution was robust, with 90% of the patients in the U.S. studies and 100% of those in the U.K. study discontinuing immunoglobulin-replacement therapy by 24 months and 36 months, respectively. No evidence of monoclonal expansion, leukoproliferative complications, or emergence of replication-competent lentivirus was noted, and no events of autoimmunity or graft-versus-host disease occurred. Most adverse events were of low grade. CONCLUSIONS: Treatment of ADA-SCID with ex vivo lentiviral HSPC gene therapy resulted in high overall and event-free survival with sustained ADA expression, metabolic correction, and functional immune reconstitution. (Funded by the National Institutes of Health and others; ClinicalTrials.gov numbers, NCT01852071, NCT02999984, and NCT01380990.).

Lentiviral gene therapy for X-linked chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a rare inherited disorder of phagocytic cells1,2. We report the initial results of nine severely affected X-linked CGD (X-CGD) patients who received ex vivo autologous CD34+ hematopoietic stem and progenitor cell-based lentiviral gene therapy following myeloablative conditioning in first-in-human studies (trial registry nos. NCT02234934 and NCT01855685). The primary objectives were to assess the safety and evaluate the efficacy and stability of biochemical and functional reconstitution in the progeny of engrafted cells at 12 months. The secondary objectives included the evaluation of augmented immunity against bacterial and fungal infection, as well as assessment of hematopoietic stem cell transduction and engraftment. Two enrolled patients died within 3 months of treatment from pre-existing comorbidities. At 12 months, six of the seven surviving patients demonstrated stable vector copy numbers (0.4-1.8 copies per neutrophil) and the persistence of 16-46% oxidase-positive neutrophils. There was no molecular evidence of either clonal dysregulation or transgene silencing. Surviving patients have had no new CGD-related infections, and six have been able to discontinue CGD-related antibiotic prophylaxis. The primary objective was met in six of the nine patients at 12 months follow-up, suggesting that autologous gene therapy is a promising approach for CGD patients.

Enhancing Lentiviral and Alpharetroviral Transduction of Human Hematopoietic Stem Cells for Clinical Application.

Ex vivo retroviral gene transfer into CD34+ hematopoietic stem and progenitor cells (HSPCs) has demonstrated remarkable clinical success in gene therapy for monogenic hematopoietic disorders. However, little attention has been paid to enhancement of culture and transduction conditions to achieve reliable effects across patient and disease contexts and to maximize potential vector usage and reduce treatment cost. We systematically tested three HSPC culture media manufactured to cGMP and eight previously described transduction enhancers (TEs) to develop a state-of-the-art clinically applicable protocol. Six TEs enhanced lentiviral (LV) and five TEs facilitated alpharetroviral (ARV) CD34+ HSPC transduction when used alone. Combinatorial TE application tested with LV vectors yielded more potent effects, with up to a 5.6-fold increase in total expression of a reporter gene and up to a 3.8-fold increase in VCN. Application of one of the most promising combinations, the poloxamer LentiBOOST and protamine sulfate, for GMP-compliant manufacturing of a clinical-grade advanced therapy medicinal product (ATMP) increased total VCN by over 6-fold, with no major changes in global gene expression profiles or inadvertent loss of CD34+CD90+ HSPC populations. Application of these defined culture and transduction conditions is likely to significantly improve ex vivo gene therapy manufacturing protocols for HSPCs and downstream clinical efficacy.

A modified γ-retrovirus vector for X-linked severe combined immunodeficiency.

BACKGROUND: In previous clinical trials involving children with X-linked severe combined immunodeficiency (SCID-X1), a Moloney murine leukemia virus-based γ-retrovirus vector expressing interleukin-2 receptor γ-chain (γc) complementary DNA successfully restored immunity in most patients but resulted in vector-induced leukemia through enhancer-mediated mutagenesis in 25% of patients. We assessed the efficacy and safety of a self-inactivating retrovirus for the treatment of SCID-X1. METHODS: We enrolled nine boys with SCID-X1 in parallel trials in Europe and the United States to evaluate treatment with a self-inactivating (SIN) γ-retrovirus vector containing deletions in viral enhancer sequences expressing γc (SIN-γc). RESULTS: All patients received bone marrow-derived CD34+ cells transduced with the SIN-γc vector, without preparative conditioning. After 12.1 to 38.7 months of follow-up, eight of the nine children were still alive. One patient died from an overwhelming adenoviral infection before reconstitution with genetically modified T cells. Of the remaining eight patients, seven had recovery of peripheral-blood T cells that were functional and led to resolution of infections. The patients remained healthy thereafter. The kinetics of CD3+ T-cell recovery was not significantly different from that observed in previous trials. Assessment of insertion sites in peripheral blood from patients in the current trial as compared with those in previous trials revealed significantly less clustering of insertion sites within LMO2, MECOM, and other lymphoid proto-oncogenes in our patients. CONCLUSIONS: This modified γ-retrovirus vector was found to retain efficacy in the treatment of SCID-X1. The long-term effect of this therapy on leukemogenesis remains unknown. (Funded by the National Institutes of Health and others; ClinicalTrials.gov numbers, NCT01410019, NCT01175239, and NCT01129544.).

Gene and cell therapy for children--new medicines, new challenges?

The range of possible gene and cell therapy applications is expanding at an extremely rapid rate and advanced therapy medicinal products (ATMPs) are currently the hottest topic in novel medicines, particularly for inherited diseases. Paediatric patients stand to gain enormously from these novel therapies as it now seems plausible to develop a gene or cell therapy for a vast number of inherited diseases. There are a wide variety of potential gene and cell therapies in various stages of development. Patients who received first gene therapy treatments for primary immune deficiencies (PIDs) are reaching 10 and 15 years post-treatment, with robust and sustained immune recovery. Cell therapy clinical trials are underway for a variety of tissues including corneal, retinal and muscle repair and islet cell transplantation. Various cell therapy approaches are also being trialled to enhance the safety of bone marrow transplants, which should improve survival rates in childhood cancers and PIDs. Progress in genetic engineering of lymphocyte populations to target and kill cancerous cells is also described. If successful these ATMPs may enhance or replace the existing chemo-ablative therapy for several paediatric cancers. Emerging applications of gene therapy now include skin and neurological disorders such as epidermolysis bullosa, epilepsy and leukodystrophy. Gene therapy trials for haemophilia, muscular dystrophy and a range of metabolic disorders are underway. There is a vast array of potential advanced therapy medicinal products (ATMPs), and these are likely to be more cost effective than existing medicines. However, the first clinical trials have not been without setbacks and some of the key adverse events are discussed. Furthermore, the arrival of this novel class of therapies brings many new challenges for the healthcare industry. We present a summary of the key non-clinical factors required for successful delivery of these potential treatments. Technological advances are needed in vector design, raw material manufacture, cell culture and transduction methodology, and particularly in making all these technologies readily scalable.

Triggering receptor expressed on myeloid cells-1 (TREM-1) modulates immune responses to Aspergillus fumigatus during fungal asthma in mice.

Triggering receptor expressed on myeloid cells-1 (TREM-1) expression is increased during pulmonary fungal infection suggesting that this receptor might be involved in anti-fungal immune responses. To address the role of TREM-1 in a murine model of fungal allergic airway disease, A. fumigatus-sensitized CBA/J mice received by intratracheal injection a mixture of live A. fumigatus conidia and one of a control adenovirus vector (Ad70), an adenovirus containing a gene encoding for the extracellular domain of mouse TREM-1 and the F(c) portion of human IgG (AdTREM-1Ig; a soluble inhibitor of TREM-1 function), or an adenovirus containing mouse DAP12 (AdDAP12; DAP12 is an intracellular adaptor protein required for TREM-1 signaling), and examined at various days after challenge. Whole lung TREM-1 levels peaked at day 3 whereas circulating TREM-1 levels peaked at day 30 in this fungal asthma model. AdTREM-1Ig-treated mice exhibited significantly higher airway hyperresponsiveness following methacholine challenge compared with Ad70- and AdDAP12-treated mice. Whole lung analysis of AdTREM-1Ig treated mice revealed markedly higher amounts of fungal material compared with the other groups. ELISA analysis of whole lung and bronchoalveolar lavage samples indicated that several pro-allergic cytokine and chemokines including CCL17 and CCL22 were significantly increased in the AdTREM-1Ig group compared with the other groups. Finally, Pam3Cys and soluble Aspergillus antigens induced TREM-1 transcript expression in macrophages in a TLR2 dependent manner. In conclusion, TREM-1 modulates the immune response directed against A. fumigatus during experimental fungal asthma.

The C76R transmembrane activator and calcium modulator cyclophilin ligand interactor mutation disrupts antibody production and B-cell homeostasis in heterozygous and homozygous mice.

BACKGROUND: Mutations in TNFRSF13B, the gene encoding transmembrane activator and calcium modulator cyclophilin ligand interactor (TACI), are found in 10% of patients with common variable immunodeficiency. However, the most commonly detected mutation is the heterozygous change C104R, which is also found in 0.5% to 1% of healthy subjects. The contribution of the C104R mutation to the B-cell defects observed in patients with common variable immunodeficiency therefore remains unclear. OBJECTIVE: We sought to define the functional consequences of the C104R mutation on B-cell function. METHODS: We performed in vitro studies of TACI C104R expression and signaling. A knock-in mouse with the equivalent mutation murine TACI (mTACI) C76R was generated as a physiologically relevant model of human disease. We examined homozygous and heterozygous C76R mutant mice alongside wild-type littermates and studied specific B-cell lineages and antibody responses to T cell-independent and T cell-dependent challenge. RESULTS: C104R expression and ligand binding are significantly diminished when the mutant protein is expressed in 293T cells or in patients' cell lines. This leads to defective nuclear factor κB activation, which is proportionally restored by reintroduction of wild-type TACI. Mice heterozygous and homozygous for mTACI C76R exhibit significant B-cell dysfunction with splenomegaly, marginal zone B-cell expansion, diminished immunoglobulin production and serological responses to T cell-independent antigen, and abnormal immunoglobulin synthesis. CONCLUSIONS: These data show that the C104R mutation and its murine equivalent, C76R, can significantly disrupt TACI function, probably through haploinsufficiency. Furthermore, the heterozygous C76R mutation alone is sufficient to disturb B-cell function with lymphoproliferation and immunoglobulin production defects.

Peptide immunotherapy in allergic asthma generates IL-10-dependent immunological tolerance associated with linked epitope suppression.

Treatment of patients with allergic asthma using low doses of peptides containing T cell epitopes from Fel d 1, the major cat allergen, reduces allergic sensitization and improves surrogate markers of disease. Here, we demonstrate a key immunological mechanism, linked epitope suppression, associated with this therapeutic effect. Treatment with selected epitopes from a single allergen resulted in suppression of responses to other ("linked") epitopes within the same molecule. This phenomenon was induced after peptide immunotherapy in human asthmatic subjects and in a novel HLA-DR1 transgenic mouse model of asthma. Tracking of allergen-specific T cells using DR1 tetramers determined that suppression was associated with the induction of interleukin (IL)-10(+) T cells that were more abundant than T cells specific for the single-treatment peptide and was reversed by anti-IL-10 receptor administration. Resolution of airway pathophysiology in this model was associated with reduced recruitment, proliferation, and effector function of allergen-specific Th2 cells. Our results provide, for the first time, in vivo evidence of linked epitope suppression and IL-10 induction in both human allergic disease and a mouse model designed to closely mimic peptide therapy in humans.

Resolution of allergic inflammation and airway hyperreactivity is dependent upon disruption of the T1/ST2-IL-33 pathway.

RATIONALE: Although there have been numerous studies on the development of allergen-induced inflammation, the mechanisms leading to resolution of inflammation remain poorly understood. This represents an important consideration because failure to resolve allergen driven inflammation potentially leads to irreversible airway remodeling, characteristic of chronic asthma. OBJECTIVES: We investigated the resolution of allergic inflammation and identified the factors responsible. METHODS: BALB/c and C57BL/6 mice were sensitized to ovalbumin and challenged through the airways to induce allergic inflammation. Mice were analyzed at 24 hours and 7 days after the final challenge. MEASUREMENTS AND MAIN RESULTS: Airway hyperreactivity (AHR) and increased mucus production were present 7 days after the cessation of allergen challenge in BALB/c mice. Persisting AHR correlated with the continued presence of Th2 cells but not eosinophils in the lungs. The role of Th2 cells in maintaining AHR was confirmed using blocking antibodies against T1/ST2, IL-4, and IL-13 during the resolution period. Moreover, AHR in the "Th1 type" C57BL/6 mouse strain was resolved 1 week after allergen challenge, concomitant with clearance of Th2 cells from the lung. Expression of the T1/ST2 ligand, IL-33, also correlated with maintenance of AHR. CONCLUSIONS: We have used blockade of Th2 function and strain differences to show for the first time that resolution of allergic inflammation and AHR may be dependent on the T1/ST2-IL-33 pathway and the presence of Th2 cells, suggesting they are necessary not only for the development of an allergic response but also for its maintenance.

Remission of chronic fungal asthma in the absence of CCR8.

BACKGROUND: Experimental studies have generated conflicting data regarding the role of CCR8 in antigen-driven allergic airway disease models, thereby dampening enthusiasm for further exploration of the targeting of CCR8 in asthma. OBJECTIVE: Recent data show that the absence of CCR8 leads to a marked amplification of the innate immune response, and these data provided impetus for the current study, which addressed the role of this chemokine receptor in a model of fungal asthma. METHODS: Wild-type (CCR8(+/+)) and CCR8-deficient (CCR8(-/-)) mice were sensitized to Aspergillus fumigatus antigens and challenged via intra-tracheal injection with live fungal conidia, and parameters of airway hyperresponsiveness, inflammation, and remodeling were examined. RESULTS: At day 7 after conidia challenge in wild-type (CCR8(+/+)) and CCR8-deficient (CCR8(-/-)) mice sensitized to A. fumigatus antigens, markedly less fungal material was present in the lungs of the CCR8(-/-) group compared with the CCR8(+/+) group. At day 14 after conidia challenge, all characteristic airway physiology, inflammatory, and remodeling parameters of fungal asthma were significantly decreased or abolished in the CCR8(-/-) group relative to the CCR8(+/+) group. CONCLUSION: Together these data show that an enhanced innate immune response in the absence of CCR8 promotes the rapid clearance of fungal material from the lung, thereby facilitating the remission of fungal asthma. CLINICAL IMPLICATIONS: This study shows that the clearance of fungal material from the lung was enhanced in the absence of CCR8, which suggests that this receptor may be an attractive target in fungal-allergic asthma and other fungal-associated pulmonary diseases.

Chemokines in allergic aspergillosis--from animal models to human lung diseases.

Allergic bronchopulmonary aspergillosis (ABPA) is a frequent syndrome in patients with cystic fibrosis (CF) or asthma. Animal models revealed distinct roles for the chemokines CCL2, CCL3, CCL5, CCL6, CCL17 and CCL22 and their receptors in the pathogenesis of allergic aspergillosis. In humans, serum levels of the CCR4 ligand CCL17 identified ABPA in patients with CF or asthma, suggesting CCL17 as novel diagnostic marker and future therapeutical target in ABPA. This review illustrates the manifold role of chemokines in animal models of allergic aspergillosis and translates these findings to human lung diseases.

Intrapulmonary, adenovirus-mediated overexpression of KARAP/DAP12 enhances fungal clearance during invasive aspergillosis.

Herein, we report that the intrapulmonary delivery of an adenovirus vector expressing KARAP/DAP12, an adaptor protein expressed in granulocytes and mononuclear cells, enhanced fungal clearance during experimental invasive pulmonary aspergillosis in neutropenic mice.

The therapeutic potential in targeting CCR5 and CXCR4 receptors in infectious and allergic pulmonary disease.

Targeting chemokines and chemokine receptors in various acute and chronic pulmonary diseases remains a vibrant area of basic and clinical research despite major hurdles including cross-species barriers, toxicity, and redundancy. In this review, we draw upon our basic research with a murine model in which innate and acquired immunity are linked in the development and maintenance of chronic asthma due to Aspergillus fumigatus. Using intact and genetically altered mice, studies have also been undertaken to elucidate safe and effective therapeutic strategies that interrupt the initiation and amplification of inflammatory and immune events that follow the intrapulmonary introduction of Aspergillus into A. fumigatus-sensitized mice. These events include resident immune cell activation, immune and inflammatory cell recruitment to the airways, changes in lung physiology, and profound changes in the architecture of the airway due to the activation of lung resident cells. The expression of 2 major chemokine receptors, namely, CC chemokine receptor (CCR) 5 and CXC chemokine receptor (CXCR) 4, has been identified and their roles in innate and acquired immune events during fungal asthma have been explored. CCR5 and CXCR4 are best known for their roles in human immunodeficiency virus-1 (HIV-1) infection, but both are attractive targets in the context of overt inflammatory and remodeling responses in the lung. This avenue of research is markedly enhanced by the existence of numerous small molecule antagonists that are available to selectively target these receptors.

Histamine induces cytoskeletal changes in human eosinophils via the H(4) receptor.

1. Histamine (0.004-2 microm) induced a concentration-dependent shape change of human eosinophils, but not of neutrophils or basophils, detected as an increase in forward scatter (FSC) in the gated autofluorescence/forward scatter (GAFS) assay. 2. The histamine-induced eosinophil shape change was completely abolished by thioperamide (10 microm), an H3/H4 receptor antagonist, but was not inhibited by pyrilamine or cimetidine (10 microm), H1 and H2 receptor antagonists, respectively. The H4 receptor agonists, clobenpropit and clozapine (0.004-2 microm), which are also H3 receptor antagonists, both induced eosinophil shape change, which was inhibited by thioperamide (10 microm). The H3/H4 receptor agonists, imetit, R-alpha-methyl histamine and N-alpha-methyl histamine (0.004-2 microm) also induced eosinophil shape change. 3. Histamine induced actin polymerisation (0.015-10 microm), intracellular calcium mobilisation (10-100 microm) and a significant upregulation of expression of the cell adhesion molecule CD11b (0.004-10 microm) in eosinophils, all of which were inhibited by thioperamide (10-100 microm). In addition, the H4 receptor agonist/H3 receptor antagonist clozapine (20 microm) stimulated a rise in intracellular calcium in eosinophils. 4. Activation of H4 receptors by histamine (1 microm) primed eosinophils for increased chemotactic responses to eotaxin, but histamine (0.1-10 microm) did not directly induce chemotaxis of eosinophils. 5. Pertussis toxin (1 microg ml-1) inhibited shape change and actin polymerisation responses induced by histamine showing that these effects are mediated by coupling to a Galphai/o G-protein. 6. This study demonstrates that human eosinophils express functional H4 receptors and may provide a novel target for allergic disease therapy.