Transfer of the tumor necrosis factor alpha gene into hematopoietic progenitor cells as a model for site-specific cytokine delivery after marrow transplantation

Relapse of leukemia is the major cause of failure after autologous stem cell transplantation due to reinfusion of residual clonogenic cells and the absence of an immune-mediated graft-versus-leukemia effect. To provide an antileukemia effect, immune-activating cytokines have been given to patients after transplantation. Systemic administration of such cytokines early after transplantation is often accompanied by substantial side effects, and it is unknown whether sufficient concentrations reach the sites of residual disease in the marrow. As a method of site-directed immunotherapy provided early after stem cell transplantation, we have tested in a murine model whether (1) marrow can be retrovirally transduced with the tumor necrosis factor alpha (TNF alpha) gene, (2) local production of TNF alpha by marrow cells after transplantation can be achieved, and (3) adverse effects of TNF alpha occurred. Balb/c mice were treated with 5-fluorouracil and bone marrow (BM) was obtained 4 days later. Whole BM was transduced in the presence of interleukin-3 (IL-3), IL-6, and stem cell factor by coculture with the packaging cell line GP+E-86, producing the cDNA for TNF alpha. Irradiated (1,300 cGy) syngeneic recipient mice were given 10(6) transduced BM cells on day 0. Integration of the TNF alpha gene into the host genome was documented by Southern blotting in spleen and BM cells on days 7 and 12 and in BM on day 40 after marrow infusion, but was no longer found on day 90. Messenger RNA for TNF alpha was present on day 12, but could no longer be shown on day 40 or 90. Although no measurable (L929 bioassay) levels of TNF alpha were found in serum of mice who had received TNF alpha transduced marrow, the supernatant of 10(6) unstimulated BM cells obtained 12 days after marrow infusion was found to have 7 pg of TNF alpha compared with 1.8 pg in nontransduced marrow. Mice that had received TNF alpha transduced marrow showed no side effects suggestive of systemic TNF alpha release, and cellularity of the TNF alpha-transduced marrow was not different from control mice that had received unmanipulated marrow or cells transduced with the neomycin resistance gene only. The studies suggest that gene transfer of immune-activating cytokines into hematopoietic cells could be used as a means to achieve their temporary local production early after transplantation by cells located in the BM.