This project involves the conduct of diagnostic, natural history assessment and therapeutic clinical trials for inherited immune deficiencies. This project specifically include studies of the diagnostic procedures (including genetic diagnosis), and development of treatment modalities that are alternatives to current standard treatment, such as novel allogeneic transplantation regimen and gene therapy (translation of such treatment modalities that are the subject of companion projects with the same types of patients). Patients with X-linked severe combined immunodeficiency (X-SCID/SCID-X1) caused by mutations in the IL2RG gene encoding the common gamma chain (gc) of receptors for interleukins (IL)-2, -4, -7, -9, -15 and -21 often are treated as infants. We evaluate X-SCID patients who have failed to achieve or maintain immune reconstitution despite prior allogeneic bone marrow transplants. Such patients often have waning immunity near the end of their first decade of life, often associated with immune dysregulation, failure to thrive, malnutrition from gastrointestinal malabsorption, pulmonary dysfunction, chronic sinusitis, chronic norovirus and protein-losing enteropathy. We previously noted a defect in response to growth hormone in such patients. In our small clinical trial to treat short stature in a few pre-adolescent XSCID children with Increlex, improved growth was observed in some patients and no adverse effects were observed. For correction of the disease, we currently have a protocol to treat older patients with X-SCID using lentiviral gene therapy. To date, we have treated 8 patients and preliminary data demonstrate promising immune correction and clinical benefits. We follow many patients with both autosomal and X-linked forms of chronic granulomatous disease (AR or X-CGD). Patients with CGD have defective circulating blood neutrophils that fail to produce microbicidal hydrogen peroxide. They suffer from recurrent life threatening infections and premature mortality. Some of these emerging infections are first diagnosed in CGD patients, for example, geosmithia argillacea as an emerging fungus infection in CGD. In addition to recurrent infections including many kinds of difficult to treat fungus infections, CGD patients often have a variety of autoinflammatory syndromes. In addition, we have also noted a high incidence of actual well-defined autoimmune disorders in CGD such as Crohns disease of the gastrointestinal system, systemic lupus erythematosis, sarcoidosis, IgA nephropathy, anti-phospholipid syndrome, and other syndromes of autoimmunity. Autoimmune problems can affect patients with a variety of primary immune deficiencies (PID), so that many types of PID are more aptly characterized as diseases of immune dysregulation rather than just as immune deficiency with recurrent infections. We have described in CGD autoimmune manifestations such as sarcoidosis, Crohns disease, discoid lupus erythematosis and juvenile idiopathic arthritis, and proposed an important new paradigm in understanding CGD, suggesting that the immune dysregulation associated with CGD may trigger autoimmune diseases in a subset of patients, where the specific autoimmune disease triggered likely related to an individual patients genetic predisposition to a particular autoimmune disease. This has important therapeutic management implications in that specific therapies proven to be effective for the specific autoimmune disease triggered by CGD must be used in such patients rather than just the general clinical management modalities designed to prevent infections, or control the general inflammation common to most CGD patients. We have successfully created iPS cells from all forms of CGD, which provide important models for functional studies and for evaluating treatment approaches. Using X-CGD iPSCs derived from X-CGD and AR-CGD, we demonstrated correction of CGD at the safe harbor site as well as at the constitutive genetic loci using gene editing approaches. Moreover,our group has also recently demonstrated that sufficient number of CD34+ stem cells can be obtained from a small quantity of peripheral blood for reprogramming to generate adequate numbers of iPS cells, providing a great resource for investigators in general. Using Zinc finger nuclease targeting the safe harbor AAVS1 site, we demonstrated correction of X-CGD patient CD34+ HSCs. More recently, we explored the use of CRISPR/Cas9 system for gene correction. Using a single strand short 100 basepair-oligonucleotide, we demonstrated efficient correction of a relatively common missense mutation in CYBB Exon 7. We also demonstrated efficient gene correction using Adeno-associated virus to deliver larger cargoes such as partial or whole ORF. We have also in collaboration with Dr. Philip Murphy and Dr. David McDermott in the Lab of Molecular Immunity, NIAID, been studying the problems that affect patients with WHIM syndrome noting severe neutopenia, increased incidence of human papilloma related cancers and other problems such as chronic pulmonary disease. Studies are in progress to determine better treatments for this disorder. Study of WHIM also interfaced with our related project that seeks to understand the role of CXCR4 (defective in WHIM) in trafficking of hematopoietic cells, including CD34 stem cells into and out of the bone marrow. More recently our project has extended to the study of adenosine deaminase deficient SCID as well as Wiskott-Aldrich syndrome. Development of novel improved therapy for these subjects include improved versions of lentivector for gene therapy for these subjects. Gene therapy has been shown to provide unequivocal clinical benefit to patients with a variety of underlying immune diseases. However, insertional mutagenesis remains a concern. To address concerns regarding random insertion-related complications, our project has focused on developing targeted integration or correction of the human hematopoietic stem cells using zinc finger nucleases, TALEN or CRISPR-mediated DNA breaks. We are working on optimizing the delivery of designer nucleases and correction templates for targeted genome editing using a clinical-scalable system which is also highly efficient in human CD34+ hematopoietic stem cells. Our hope is in generating a universal platform for providing novel therapeutic modalities for many rare immune-deficient genetic disorders. For cellular therapy, we have successfully corrected circulating primary immune cells, including terminally differentiated granulocytes, for the purpose of using corrected autologous immune cells for treatment of infections.
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