Routes of administration cell targets for gene therapy vectors

Intrathymic administration
The application of the process of intrathymic T-cell development to transplantation and tolerance induction was first described by Posselt and colleagues.Self tolerance (the failure to respond to antigen borne on self tissue) develops as T-lymphocyte precursor cells that are CD4^- and CD8^- (‘double negative’) pass through the thymus. Because the T cells are exposed to antigen on thymic epithelial cells, any T cells that have a high-affinity interaction with antigen in the thymus, and are therefore potentially autoreactive cells, are ‘negatively selected’ by the process of clonal deletion. Cells that have TCRs that have no (or an extremely low) affinity for intrathymic antigen, yet still have a high-affinity interaction with self MHC, are ‘positively selected’; they can thus mature and go on to populate and expand into larger clonal populations in the periphery. For a recent review of the mechanisms of the induction of intrathymic tolerance, see Turvey and colleagues .

Knechtle and colleagues .showed it was possible to induce tolerance using a gene therapy strategy in a rat model. First, they took syngeneic recipient muscle cells and then transfected them in vitro with an MHC class I gene derived from the donor. These cells were then injected into the thymus of the recipient. Next, the peripheral immune system of the recipient was depleted of potentially alloreactive T cells using anti-lymphocyte serum. This was then followed by a liver transplant from the donor, to which the recipient was found to be unresponsive. In a subsequent study (Ref. 38), the MHC class I complementary DNA (cDNA) from the donor strain rat was introduced directly into the thymus to transfect recipient thymic cells in situ; analysis using the polymerase chain reaction (PCR) detected the transient expression of donor DNA in the thymus (and later in the spleen, which was probably due to the export of transfected thymocytes out of the thymus).

The above approaches have used either live cells transfected with DNA or naked DNA itself to deliver donor MHC genes. Adenovirus could be used to improve the efficiency of gene therapy that has been achieved using DNA transfection. Adenovirus vectors (as discussed later in the section entitled ‘Adenovirus’) are ideal for use in intrathymic applications because they can be generated at high titres and can transduce a range of cell types. Not only can genes be transferred to the antigen-presenting thymic epithelial cells and possibly to the developing thymocytes, but central tolerance (tolerance that is established in lymphocytes developing in central lymphoid organs such as the thymus, spleen and bone marrow) to the immunogenic adenoviral antigens can be induced, as shown by Ilan and colleagues Their work demonstrated that intrathymic inoculation of the recombinant adenovirus inhibited the appearance of neutralising antibodies and CTLs against the recombinant adenovirus.