Transfer of the ADA Gene into Bone Marrow Cells and Peripheral Blood Lymphocytes for the Treatment of Patients Affected by ADA-Deficient SCID

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Abstract
Severe combined immunodeficiency (SCID) caused by deficiency of the enzyme adenosine deaminase (ADA) is the first genetic disorder considered for human somatic cell gene therapy. ADA ¯SCID patients can be cured by HLA-matched sibling donor bone marrow transplantation. Alternative transplantation strategies as well as enzyme replacement are being tested in those patients who do not have a suitable matched sibling donor. Some ADA ¯SCID patients may not be candidates for cytoablation due to infectious damage to the lung or liver, or may have a milder phenotype that does not justify the risks associated with haploidentical bone marrow transplantation. Replacement therapy with PEG-ADA has resulted in improvement in growth, a variable increase in the number of peripheral blood lymphocytes, and a decrease in the incidence of severe infections. Another approach to the treatment of severe genetic diseases is now represented by somatic cell gene therapy. We and others have conducted experiments in vitro and in vivo that have documented that T-lymphocytes are suitable vehicles for gene transfer. Although the pluripotent stem cell remains the ideal target cell for somatic cell gene therapy of disorders of the hematopoietic system, the use of T-lymphocytes as gene therapy vehicles is specifically indicated for ADA-deficient patients where they represent the affected cells. Furthermore, the selective engraftment of T-cells only, following bone marrow transplantation, has resulted in reconstitution of cellular and humoral immunity. A model for the functional analysis in vivo of the human immune system has been utilized for the preclinical evaluation of this approach. In this model, immunodeficient mice (SCID or bg/nu/xid) were reconstituted by human peripheral blood lymphocytes obtained from a SCID patient after ADA gene transfer. We made the following preclinical observations: 1) in vivo high levels of gene transfer and expression; 2) successful transduction of lymphopoietic progenitors capable of undergoing TCR genes rearrangement and generation of a new immune repertoire; 3) strong selective advantage of transduced cells over non-transduced cells. The observation that freshly obtained ADA deficient PBL transduced to express the hADA gene had a significant survival and functional advantage over non-transduced ADA deficient lymphocytes when transplanted into immunodeficient (but ADA-normal) BNX mice strongly suggests that intracellular ADA is more efficacious than extracellular enzyme alone. Based on these results, the clinical application of gene therapy for the treatment of ADA ¯SCID patients who previously failed replacement treatment therapy with bovine pegilated enzyme is proposed. The aim of this study is to evaluate safety and efficacy of the procedure, and to identify the relative role of peripheral blood lymphocytes, and hematopoietic stem cells and progenitor cells in the long term reconstitution of immune functions after retroviral vector-mediated ADA gene transfer. For this purpose, two vectors will be utilized for gene transfer into peripheral blood lymphocytes (PBL) and bone marrow cells (BM), DCA1 and DCAm respectively. The two vectors are identical in the construction design, the packaging cell line utilized (AM-12), and viral titer (1–2 × 105). However, they differ for a restriction site in the viral LTR that allows one to distinguish the progeny of cells transduced with the two vectors.