Primary immune deficiencies comprise a group of inherited genetic disorders caused by interruption of normal lymphoid development. These diseases are considered prime candidates for gene therapy by introduction of the missing gene into hematopoietic stem cells that can then differentiate into lymphoid cells, and thus restore immunity. X-linked severe combined immunodeficiency (X-SCID), caused by a mutation in the gene encoding the common ( chain gene ((c) of the receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-213, is one of the more common of the primary immunodeficiencies. Clinical gene therapy trials for X-SCID have focused on the use of retroviruses as an integrating viral vector for introduction of the (c gene, resulting in restoration of immunity in patients that have engrafted with transduced, autologous hematopoietic stem cells. However, serious adverse events have emerged in these clinical trials, namely a T-cell leukemia-like syndrome developing in 5 of 21 treated patients. Therefore, safer, alterative therapies are needed. At Discovery Genomics, Inc. (DGI), we are working on developing the Sleeping Beauty (SB) transposon system for gene therapy, including the targeting of hematopoietic stem cells for treatment of primary immunodeficiencies. The two-component SB system consists of a transposon (inverted repeats (IR's) flanking a therapeutic gene of interest) and a transposase that catalyzes excision of the transposon at the ends of the IRs and then integration into host cell chromosomal sequence. As a lead immunodeficiency, here we propose that X-SCID may be treatable without the use of a virus by combining the power of electroporation for introduction of DNA into cells, along with use of DGI's Sleeping Beauty transposon system to achieve integration and long-term (c gene expression. In this Phase I application, the overall goal is to establish conditions for SB-mediated gene therapy for X-SCID. In this regard, there are two key questions that will need to be addressed: (i) Will SB-mediated transposition of the (c gene allow functional correction of lymphoid cells? (ii) What is the effectiveness of SB-mediated (c gene insertion in the treatment of an animal model of X-SCID? There are two Specific Aims:
Aim 1. Evaluate the effectiveness of Sleeping Beauty transposons for correction of (c chain deficiency in a lymphoblastoid cell line derived from an X-linked SCID patient.
Aim 2. Evaluate Sleeping Beauty-mediated transposition and long term expression of (c gene in hematopoietic stem cells derived of X-SCID mice, as a model for SB-mediated gene therapy of X-SCID. Successful accomplishment of these goals will provide evidence for the effectiveness of the SB system to mediate non-viral gene transfer in murine hematopoietic stem cells (previously undemonstrated), as well as provide key preclinical data for the development of SB transposons for X-SCID as a lead primary immunodeficiency.
Results from these experiments will provide the technical basis for achieving transposon-mediated integration and long-term expression in hematopoietic stem cells, the cellular target inr gene therapy of primary immunodeficiencies. In addition, it will provide the basis for the development of the SB transposon system for treatment of other hematologic diseases.
