Correction of Wiskott-Aldrich Syndrome by Hematopoietic Stem Cell Gene Therapy
Authors: Boztug Kaan , Schmidt Manfred , Schwarzer Adrian , Banerjee Pinaki , Böhm Marie , Beier Rita , Díez Inés Avedillo , Dewey Ricardo , Ball Claudia R , Nowrouzi Ali , Naundorf Sonja , Küuhlcke Klaus , Blasczyk Rainer , Rose Martina , Fraser Christopher , Liesl Mathias , Ferrari Rudolf , Abboud Miguel R. , Al-Herz Waleed , Kondratenko Irina , Maródi László , Orange Jordan , Christof von Kalle , Klein Christoph 
Published: October 2019

Abstract
Abstract 5
Wiskott Aldrich Syndrome is a life-threatening immune-disorder characterized by bleeding secondary to microthrombocytopenia, immunodeficiency, autoimmunity, and susceptibility to lymphoma. A clinical gene therapy protocol using a GALV pseudotyped MLV-derived retroviral vector was developed at Hannover Medical School. We here present an analysis of ten patients treated in this trial between 2006 and 2009. All patients had evidence of engraftment of WASP-positive hematopoietic progenitor cells except for one patient in whom an insufficient number of CD34+ cells could be harvested. WASP expression was determined in myeloid and lymphoid cells as well as in CD34+ hematopoietic progenitor cells using Western Blot and FACS analysis, respectively. While the percentage of WASP-positive myeloid cells was relatively stable over time (range 10 to 60%), a marked increase over time in the percentage of WASP-positive T lymphocytes and NK cells was observed, resulting in more than 80% of WASP-positive lymphoid cells 12 months after gene therapy.
Early after gene therapy, an increase in thrombocyte counts was observed in all patients. Furthermore, the majority of peripheral thrombocytes showed evidence of WASP expression, detectable as early as 3 months after gene therapy. Functional immune reconstitution was documented in dendritic cells (podosome formation), T cells (proliferation in response to CD3-signaling), and NK cells (formation of immunological synapse and NK cell killing activity). TCR Vb spectratyping analyses showed in improvement of receptor skewing upon gene therapy in some patients. A clinical benefit was notable in all patients with the exception of the patients with insufficient engraftment of genetically corrected progenitor cells: eczema, bleeding diathesis and immunodeficiency resolved. Strikingly, the various autoimmune phenomena which the patients in this trial had shown prior to gene therapy resolved when the majority of T lymphocytes were positive for WASP expression, and a particular advantage of WASP-positive regulatory T lymphocytes (Tregs) could be observed at this point, suggesting a crucial role in WASP reconstitution in Tregs for autoimmnity resolution. Repetitive bone marrow examinations did not reveal morphological or cytogenetic alterations. Comprehensive insertion site analysis using 454 pyrosequencing demonstrated vector integration that targeted multiple genes controlling growth, development and immunological responses in a persistently polyclonal hematopoiesis.
In sum, hematopoietic stem cell gene therapy for Wiskott-Aldrich Syndrome is feasible and effective at correcting the various cellular defects implicated in this disease. Our trial provides the first proof-of-principle for gene therapy in Wiskott-Aldrich syndrome and provides evidence that gene therapy may be effective at correcting disorders involving autoimmunity and/or platelets. Prospective monitoring is used in this trial to determine the long-term efficacy and safety profile of this experimental therapeutic approach in Wiskott-Aldrich syndrome.

Disclosures:
No relevant conflicts of interest to declare.