In this issue of the JCI
, Adjali and colleagues (10
) took a different approach to gene therapy for SCID. In this case, the authors targeted T cell precursors instead of HSCs by direct intrathymic injection of a ZAP-70–expressing T cell–specific lentiviral vector into ZAP-70–/–
SCID mice (Figure B). ZAP-70 deficiency is a rare autosomal- recessive inherited subtype of SCID, in which T cell development is blocked at the stage of double-positive cells, resulting in an absence of peripheral CD8+
T cells and the presence of circulating CD4+
T cells that are unresponsive to TCR-mediated stimuli in vitro (11
). The differentation block in this disease is due to the inability of double-positive cells to propagate TCR signals in the absence of ZAP-70 kinase activity despite tyrosine phosphorylation of TCR-associated ZAP-70 molecules (12
). The protein tyrosine kinase ZAP-70 is normally expressed in thymocytes, mature T cells, and natural killer cells (13
Using the T cell–specific lentiviral vector injected directly into the thymus of ZAP-70–/–
mice, Adjali and colleagues demonstrated partial reconstitution of polyclonal and functional T cells in some mice (10
). They chose this approach to avoid transduction of HSCs and precursor cells from other hematopoietic lineages that do not express ZAP-70 and thus do not require correction in this disorder; this potentially reduces the risk of insertional mutagenesis by exposing many fewer precursor cells to insertional events. HSCs may be particularly susceptible to insertional mutagenesis due to their prolonged and increased replicative capacity. Many genes controlling self-renewal and proliferation are expressed in HSCs before being shut off during differentiation, and increased expression of genes has been shown to predispose loci to insertions of retroviral and lentiviral vectors (7
). Insertions at these sites could therefore constitutively activate genes that are normally shut off during hematopoietic development, and thus contribute to leukemogenesis. By the targeting of more mature cells via intrathymic injection, this risk might be decreased.
The in situ approach also avoids ex vivo culture of target cells, which is desirable for a number of reasons. Ex vivo culture of HSCs, progenitors, and even mature T cells has been found to result in impaired homing capabilities and functional deficits (14
). Furthermore, ex vivo culture potentially exposes the cells to infectious agents, serum, and other risks related to complex and prolonged manipulations and removes them from their natural microenvironment. Thus, in situ delivery of a gene transfer vector to T cell precursors without any requirement for ex vivo culture is an inherently attractive approach.
Although the lentiviral vector used in this study (10
) was designed to be T cell–specific, it also transduced thymic epithelial cells (17
). The interaction of thymocytes with thymic epithelial cells is crucial for the development of T cells. Thus the effects on thymic epithelial cell function by ZAP-70 transduction needs to be further evaluated. Changes in the vector design might be necessary to obtain increased T cell specificity. Intrathymic vector injection is technically very challenging both in mice and humans but probably accounted for successful correction in less than a quarter of the mice treated by Adjali et al. (10
). In human subjects suffering from SCID, the need for intrathymic injection will be a real hurdle, since in these patients the thymus is very small, usually less than 1 g, and lacking in thymocytes (18
). Thus, it might be difficult to establish this approach for clinical application, even if the success rate could be significantly improved via sophisticated imaging modalities and optimization in large animal models.