DESCRIPTION (provided by applicant): Reticular Dysgenesis (RD) is one of the most serious forms of severe combined immunodeficiency (SCID) because it affects both innate and adaptive immunity. The disease is characterized by arrested neutrophil maturation, profound lymphopenia, and sensorineural hearing loss. It is invariably fatal early in life unless immune reconstitution is achieved by hematopoietic stem cell transplantation (HSCT). The simultaneous occurrence of severe neutropenia and lymphopenia is responsible for a high risk of death in infancy, and the predominance of bacterial and fungal infections supports neutropenia as the prevailing cause of death. RD is caused by mutations in the mitochondrial enzyme Adenylate Kinase 2 (AK2), however, how AK2 defects translate into disease pathology is largely unknown. Overall, transplant outcomes in RD are significantly worse compared to any other form of SCID, suggesting that the nature of the genetic defect may directly impact the poor prognosis. Therefore, elucidating the mechanistic basis of RD is critical in order to target the underlying problem and develop additional therapeutic options. Our prior work in induced pluripotent stem cell and zebrafish models of RD has shown that hematopoietic stem and progenitor cell differentiation is compromised but can be rescued by treatment with antioxidant agents (Rissone, Weinacht et al., J Exp Med, 2015, in press). These data led to the hypothesis that AK2 defects impair mitochondrial metabolism and increase oxidative stress, and that antioxidant agents improve mitochondrial function and represent a targeted supportive therapy to treat the neutropenia and overall constitution of patients with RD. To test this hypothesis I will dissect th molecular mechanisms underlying RD and their responsiveness to antioxidants by defining mitochondrial metabolism, oxidative stress and antioxidant reserve in different iPSC-models of RD before and after treatment with antioxidants. To demonstrate that the in vitro findings can be translated to patients, I will develop a xenograft model of RD based on transplantation of AK2-deficient iPSC-derived respecified multipotent hematopoietic progenitors into NSG-mice, and examine how antioxidant treatment affects engraftment, differentiation potential and mitochondrial function. If my preliminary findings that antioxidants improve mitochondrial function are supported by the proposed research, the therapeutic potential of these agents could be exploited in a much wider range of diseases, in which mitochondrial pathology and oxidative stress are at play. I am a pediatric hematologist with focus on immune and immune-mediated diseases and substantial prior research experience in molecular microbiology, stem cell differentiation and reprogramming, who is seeking K08 support for mentored research under the guidance of Dr. Luigi Notarangelo, Division of Immunology, Boston Children's Hospital, with Dr. George Daley, Division of Hematology/Oncology, Boston Children's Hospital/Dana-Farber Cancer Institute, as co-mentor. My long-term career objective is to obtain a tenure-track position as a physician-scientist at an academic center with special commitment to the study, diagnosis and treatment of primary immunodeficiencies. The K08 award will provide the protected time I need to advance my training in stem cell biology, metabolism, xenografting, in situ gene editing and translational research. I will devote a minimum of 80% of my time to a focused research project investigating the molecular mechanisms underlying RD and its responsiveness to antioxidants, and will complement this with 20% of my effort dedicated to clinical care of children with hematologic diseases. Boston Children's Hospital, Dana-Farber Cancer Institute and Harvard University are internationally recognized research institutions with renowned expert researchers in the areas of stem cell biology, hematopoiesis, metabolism, in situ gene editing, and development of xenograft-models. Furthermore, the Divisions of Pediatric Hematology/Oncology and Immunology have a distinguished record of training successful physician-scientists. I have assembled a mentoring and advisory committee, consisting of Drs. Raif Geha, David Williams, Alan Beggs, and Suneet Agarwal, who will guide my research and training experience. The expertise of my advisory committee will be complemented by a set of additional collaborators who are experts in their respective fields (Dr. Kiran Musunuru, gene editing with CRISPR/Cas9; Dr. Marcia Haigis, mitochondrial biology; and Dr. Giancarlo la Marca, Tandem Mass Spectrometry). This research proposal is part of a structured plan with scientific, technical, clinical training and career development components. The career development plan builds upon my prior research and clinical experiences with the goal of ensuring that I acquire the expertise required to become a successful, independent investigator whose focus is on translating disease mechanisms underlying immunodeficiencies into targeted therapies for patients.

PUBLIC HEALTH RELEVANCE: Reticular Dysgenesis is one of the most serious forms of severe combined immunodeficiency, in which mutations in the mitochondrial enzyme Adenylate Kinase 2 (AK2) lead to neutropenia, lymphopenia and sensorineural hearing loss; however, the disease mechanism is poorly understood. I used induced pluripotent stem cells (iPSCs) from a patient with Reticular Dysgenesis to study this disease in a tissue culture dish and found that AK2 deficiency impairs hematopoietic stem and progenitor cell differentiation, which can be rescued by adding the antioxidant agent glutathione to the culture. In this proposal, I will investigate the molecular mechanisms underlying Reticular Dysgenesis and its responsiveness to antioxidants in iPSCs and a humanized mouse model of this disease to show that antioxidants have a therapeutic role in treating patients with Reticular Dysgenesis.