Myriam Armant

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.).

Reproducible Isolation of Lymph Node Stromal Cells Reveals Site-Dependent Differences in Fibroblastic Reticular Cells

Within lymph nodes, non-hematopoietic stromal cells organize and interact with leukocytes in an immunologically important manner. In addition to organizing T and B cell segregation and expressing lymphocyte survival factors, several recent studies have shown that lymph node stromal cells shape the naïve T cell repertoire, expressing self-antigens which delete self-reactive T cells in a unique and non-redundant fashion. A fundamental role in peripheral tolerance, in addition to an otherwise extensive functional portfolio, necessitates closer study of lymph node stromal cell subsets using modern immunological techniques; however this has not routinely been possible in the field, due to difficulties reproducibly isolating these rare subsets. Techniques were therefore developed for successful ex vivo and in vitro manipulation and characterization of lymph node stroma. Here we discuss and validate these techniques in mice and humans, and apply them to address several unanswered questions regarding lymph node composition. We explored the steady-state stromal composition of lymph nodes isolated from mice and humans, and found that marginal reticular cells and lymphatic endothelial cells required lymphocytes for their normal maturation in mice. We also report alterations in the proportion and number of fibroblastic reticular cells (FRCs) between skin-draining and mesenteric lymph nodes. Similarly, transcriptional profiling of FRCs revealed changes in cytokine production from these sites. Together, these methods permit highly reproducible stromal cell isolation, sorting, and culture.

Preclinical regulatory validation of a 3-stage amniotic mesenchymal stem cell manufacturing protocol

PURPOSE Because of the 4 to 6-month interval between a diagnostic amniocentesis and birth, clinical application of amniotic mesenchymal stem cell (AMSC)-based therapies demands a 3-stage cell manufacturing process, including isolation/primary expansion, cryopreservation, and thawing/secondary expansion. We sought to determine the feasibility and cell yield of such a staged cell manufacturing process, within regulatory guidelines. METHODS Human AMSCs isolated from diagnostic amniocentesis samples (n = 11) were processed under Food and Drug Administration-accredited good manufacturing practice. Expanded cells were characterized by flow cytometry and cryopreserved for 3 to 5 months. Cell release criteria included more than 90% CD29+, CD73+, and CD44+; less than 5% CD34+ and CD45+; negative mycoplasma quantitative polymerase chain reaction (QPCR) and endotoxin assay; and at least 70% viability. RESULTS Isolation and ample expansion of AMSCs was achieved in 54.5% (6/11) of the samples. Early processing and at least a 2-mL sample were necessary for reliable cell manufacturing. Cell yield before cryopreservation was 223.2 +/- 65.4 x 10(6) cells (44.6-fold expansion), plus a 14.7 x 10(6)-cell backup, after 36.3 +/- 7.8 days. Cell viability postthaw was 88%. Expanded cells maintained a multipotent mesenchymal progenitor profile. CONCLUSIONS Human amniotic mesenchymal stem cells can be manufactured in large numbers from diagnostic amniocentesis, by an accredited staged processing, under definite procurement guidelines. These data further support the viability of clinical trials of amniotic mesenchymal stem cell-based therapies.

Preclinical regulatory validation of an engineered diaphragmatic tendon made with amniotic mesenchymal stem cells

PURPOSE Under a Food and Drug Administration directive, we examined definite long-term safety and efficacy aspects of an engineered diaphragmatic tendon graft as a regulatory prerequisite for clinical trials. METHODS Newborn lambs (N = 27) underwent partial diaphragmatic replacement with a Teflon patch, a composite acellular bioprosthesis, or the same bioprosthesis seeded with autologous amniotic mesenchymal stem cells processed under Good Manufacturing Practice guidelines. Multiple safety and efficacy analyses were performed at different time points up to 14 months of age (ovine adulthood). RESULTS There was no mortality. None of the blood tests or full body autopsy specimens showed any abnormality. There was a significantly higher failure rate in animals that received an acellular bioprosthetic graft vs an engineered graft, with no significant differences between Teflon and acellular bioprosthetic implants. Tensile strength and total collagen levels were significantly higher in engineered grafts than in acellular bioprosthetic grafts. On histology, lysozyme and myeloperoxidase stainings were unremarkable in all grafts. CONCLUSIONS Diaphragmatic repair with a clinically viable autologous tendon engineered with amniotic mesenchymal stem cells leads to improved outcomes when compared with an equivalent acellular bioprosthesis, with no local or systemic adverse effects. Clinical trials of engineered diaphragmatic repair appear practicable within regulatory guidelines.

Viral induction and targeted inhibition of galectin-1 in EBV posttransplant lymphoproliferative disorders

Posttransplant lymphoproliferative disorders (PTLDs) are potentially fatal, EBV-driven B-cell malignancies that develop in immunocompromised solid organ or hematopoietic stem cell recipients. In PTLD, the expression of EBV proteins, including latent membrane protein 1 (LMP1) and LMP2A, viral immune evasion strategies, and impaired host immune surveillance foster the proliferation of EBV-transformed B cells. Current PTLD treatment strategies include reduction of immunosuppression, which increases the risk of graft rejection, anti-CD20 treatment, combination chemotherapy, and administration of EBV-specific cytotoxic T cells. In the present study, we report that EBV-transformed lymphoblastoid B-cell lines (LCLs) and primary PTLDs overexpress galectin-1 (Gal1), a carbohydrate-binding lectin that induces tolerogenic dendritic cells and triggers the selective apoptosis of CD4(+) Th1 and Th17 cells and cytotoxic T cells. In transcriptional reporter assays, LMP2A and LMP1 each increased Gal1-driven luciferase expression, and the combination of LMP2A and LMP1 was additive. In addition, small interfering RNA (siRNA)-mediated depletion of LMP2A decreased Gal1 protein abundance in EBV-transformed LCLs. Gal1 expression in LCLs was dependent on both activating protein 1 (AP-1) and PI3K. A newly developed neutralizing Gal1 mAb selectively inhibited Gal1-mediated apoptosis of EBV-specific CD8(+) T cells. Given the tolerogenic and immunosuppressive function of Gal1, antibody-mediated Gal1 neutralization may represent a novel immunotherapeutic strategy for PTLD and other Gal1-expressing tumors.

Hematopoietic Stem-Cell Gene Therapy for Cerebral Adrenoleukodystrophy

BACKGROUND In X‐linked adrenoleukodystrophy, mutations in ABCD1 lead to loss of function of the ALD protein. Cerebral adrenoleukodystrophy is characterized by demyelination and neurodegeneration. Disease progression, which leads to loss of neurologic function and death, can be halted only with allogeneic hematopoietic stem‐cell transplantation. METHODS We enrolled boys with cerebral adrenoleukodystrophy in a single‐group, open‐label, phase 2–3 safety and efficacy study. Patients were required to have early‐stage disease and gadolinium enhancement on magnetic resonance imaging (MRI) at screening. The investigational therapy involved infusion of autologous CD34+ cells transduced with the elivaldogene tavalentivec (Lenti‐D) lentiviral vector. In this interim analysis, patients were assessed for the occurrence of graft‐versus‐host disease, death, and major functional disabilities, as well as changes in neurologic function and in the extent of lesions on MRI. The primary end point was being alive and having no major functional disability at 24 months after infusion. RESULTS A total of 17 boys received Lenti‐D gene therapy. At the time of the interim analysis, the median follow‐up was 29.4 months (range, 21.6 to 42.0). All the patients had gene‐marked cells after engraftment, with no evidence of preferential integration near known oncogenes or clonal outgrowth. Measurable ALD protein was observed in all the patients. No treatment‐related death or graft‐versus‐host disease had been reported; 15 of the 17 patients (88%) were alive and free of major functional disability, with minimal clinical symptoms. One patient, who had had rapid neurologic deterioration, had died from disease progression. Another patient, who had had evidence of disease progression on MRI, had withdrawn from the study to undergo allogeneic stem‐cell transplantation and later died from transplantation‐related complications. CONCLUSIONS Early results of this study suggest that Lenti‐D gene therapy may be a safe and effective alternative to allogeneic stem‐cell transplantation in boys with early‐stage cerebral adrenoleukodystrophy. Additional follow‐up is needed to fully assess the duration of response and long‐term safety. (Funded by Bluebird Bio and others; STARBEAM ClinicalTrials.gov number, NCT01896102; ClinicalTrialsRegister.eu number, 2011‐001953‐10.)

Concise Review: Guidance in Developing Commercializable Autologous/Patient-Specific Cell Therapy Manufacturing

Cell therapy is poised to play an enormous role in regenerative medicine. However, little guidance is being made available to academic and industrial entities in the start‐up phase. In this technical review, members of the International Society for Cell Therapy provide guidance in developing commercializable autologous and patient‐specific manufacturing strategies from the perspective of process development. Special emphasis is placed on providing guidance to small academic or biotech researchers as to what simple questions can be addressed or answered at the bench in order to make their cell therapy products more feasible for commercial‐scale production. We discuss the processes that are required for scale‐out at the manufacturing level, and how many questions can be addressed at the bench level. The goal of this review is to provide guidance in the form of topics that can be addressed early in the process of development to better the chances of the product being successful for future commercialization.

Enhanced induction of HIV-specific cytotoxic T lymphocytes by dendritic cell-targeted delivery of SOCS-1 siRNA.

Dendritic cells (DCs) are potent antigen-presenting cells that play a critical role in the activation of T cells. RNA interference (RNAi)-mediated silencing of negative immunoregulatory molecules expressed by DCs may provide a strategy to enhance the potency of DC-based vaccines and immunotherapy. Ablation of suppressor of cytokine signaling-1 (SOCS-1) in antigen-presenting cells has been shown to enhance cellular immune response in mice. Here, we used a previously reported DC-targeting approach to deliver small interfering RNA (siRNA) against SOCS-1 to human myeloid-derived DCs (MDDCs). SOCS1-silencing in MDDCs resulted in enhanced cytokine responses to lipopolysaccharide (LPS) and a strong mixed-lymphocyte reaction. Moreover, only DCs treated with SOCS-1 siRNA, and not controls, elicited strong primary in vitro responses to well-characterized HLA-A*0201-restricted Melan-A/MART-1 and human immunodeficiency virus (HIV) Gag epitopes in naive CD8(+) T cells from healthy donors. Finally, stimulation of CD8(+) T cells from HIV-seropositive subjects with SOCS1-silenced DCs resulted in an augmented polyfunctional cytotoxic T-lymphocyte (CTL) response, suggesting that SOCS-1 silencing can restore functionally compromised T cells in HIV infection. Collectively, these results demonstrate the feasibility of DC3-9dR-mediated manipulation of DC function to enhance DC immunogenicity for potential vaccine or immunotherapeutic applications.Dendritic cells (DCs) are potent antigen-presenting cells that play a critical role in the activation of T cells. RNA interference (RNAi)-mediated silencing of negative immunoregulatory molecules expressed by DCs may provide a strategy to enhance the potency of DC-based vaccines and immunotherapy. Ablation of suppressor of cytokine signaling-1 (SOCS-1) in antigen-presenting cells has been shown to enhance cellular immune response in mice. Here, we used a previously reported DC-targeting approach to deliver small interfering RNA (siRNA) against SOCS-1 to human myeloid-derived DCs (MDDCs). SOCS1-silencing in MDDCs resulted in enhanced cytokine responses to lipopolysaccharide (LPS) and a strong mixed-lymphocyte reaction. Moreover, only DCs treated with SOCS-1 siRNA, and not controls, elicited strong primary in vitro responses to well-characterized HLA-A*0201-restricted Melan-A/MART-1 and human immunodeficiency virus (HIV) Gag epitopes in naive CD8+ T cells from healthy donors. Finally, stimulation of CD8+ T cells from HIV-seropositive subjects with SOCS1-silenced DCs resulted in an augmented polyfunctional cytotoxic T-lymphocyte (CTL) response, suggesting that SOCS-1 silencing can restore functionally compromised T cells in HIV infection. Collectively, these results demonstrate the feasibility of DC3-9dR-mediated manipulation of DC function to enhance DC immunogenicity for potential vaccine or immunotherapeutic applications.

Manufacturing Differences Affect Human Bone Marrow Stromal Cell Characteristics and Function: Comparison of Production Methods and Products from Multiple Centers

Human bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) are manufactured using many different methods, but little is known about the spectrum of manufacturing methods used and their effects on BMSC characteristics and function. Seven centers using, and one developing, Good Manufacturing Practices (GMP) processes were surveyed as to their production methods. Among the seven centers, all used marrow aspirates as the starting material, but no two centers used the same manufacturing methods. Two to four BMSC lots from each center were compared using global gene expression. Among the twenty-four BMSC lots from the eight centers intra-center transcriptome variability was low and similar among centers. Principal component analysis and unsupervised hierarchical clustering analysis separated all the lots from five centers into five distinct clusters. BMSCs from six of the eight centers were tested for their ability to form bone and support hematopoiesis by in vivo transplantation (defining features of BMSCs). Those from all six centers tested formed bone, but the quantity formed was highly variable and BMSCs from only three centers supported hematopoiesis. These results show that differences in manufacturing resulted in variable BMSC characteristics including their ability to form bone and support hematopoiesis.

Mast Cell-Activated Bone Marrow Mesenchymal Stromal Cells Regulate Proliferation and Lineage Commitment of CD34+ Progenitor Cells

Background: Shortly after allergen exposure, the number of bone marrow (BM) and circulating CD34+ progenitors increases. We aim to analyze the possible mechanism whereby the allergic reaction stimulates BM to release these effector cells in increased numbers. We hypothesize that mast cells (MCs) may play a predominant role in this process. Objective: To examine the effect of IgE-activated MCs on BM mesenchymal stromal cells which regulate proliferation and differentiation of CD34+ progenitors. Methods: Primary MCs were derived from CD34+ precursors and activated with IgE/anti-IgE. BM mesenchymal stromal cells were co-cultured with CD34+ progenitor cells and stimulated with IL-1/TNF or IgE/anti-IgE-activated MCs in Transwell system. Results: BM mesenchymal stromal cells produce low level of thymic stromal lymphopoietin (TSLP) under steady state conditions, which is markedly increased by stimulation with proinflammatory cytokines IL-1 and TNF or IgE-activated MCs. The latter also triggers bone marrow-derived mesenchymal stromal cells production of G-CSF, and GM-CSF while inhibiting SDF-1. MC-activated mesenchymal stromal cells stimulate CD34+ cells to proliferate and to regulate their expression of early allergy-associated genes. Conclusion and Clinical Relevance: This in vitro study indicates that IgE-activated MCs trigger BM mesenchymal stromal cells to release TSLP and hematopoietic growth factors and to regulate the proliferation and lineage commitment of CD34+ precursor cells. The data predict that the effective inhibition of MCs should impair mobilization and accumulation of allergic effector cells and thereby reduce the severity of allergic diseases.