Strategies of gene and immune therapy for tumors and viral diseases

Abstract: In the treatment of tumors and viral diseases such as HIV-1 infection, immunotherapeutic strategies do not seem to have enough capacity to eradicate an already established disease. However, these strategies could play a unique role as adjuvant treatments of therapies that eliminate the main tumor or viral load in order to avoid the relapse of the disease. Consequently, efforts aimed to improve the current steady state of imunogene-therapy and to find novel therapeutic strategies should be continued. Cytokines are very efficient immunomodulators. Nevertheless, their application is frequently hampered by high toxicity specially, when applied systemically. Cytokines work in a paracrine form and for that reason a highly regulated release of these substances could be desirable for most therapeutic applications. To avoid systemic effects, the target cells can be autoactivated by cytokines produced by the same cells after cytokine gene transfer. However, the uncontrolled cellular secretion of cytokines could even exert adverse bystander effects. Therefore, the development of additional methods for a more restricted administration is still desirable. With the aim of improving the administration and release of cytokines in a strictly localized area, we have designed vectors that express the murine granulocytic macrophage colony stimulating factor (mGM-CSF) with different subcellular localization signals. Using this strategy, we have shown that cytokines can be expressed and targeted to different subcellular compartments as stable and active proteins. We also demonstrated that the mGM-CSF-dependent DA-3 cell line acquires autonomous growth following gene modification with the plasmids encoding either extracellular or intracellular forms of mGM-CSF. In addition, we showed that the autonomous growth induced by the intracellular forms of rnGM-CSF in these cells, is mediated by a mechanism of restricted autocrine stimulation without release of detectable cytokine to the extracellular medium. Our findings support the concept that intracellular cytokines may be used to provide the desired effect of these substances on the target cells, while avoiding the collateral effects of their uncontrolled secretion. When our plasmids were tested as adjuvants of genetic immunizations with HIV-1 genes, it was demonstrated that the Th1 pattern of a primary immune response could be switched toward an enhanced Th2 response, by plasmids expressing the secreted form of mGM-CSF or by recombinant mGM-CSF. On the other hand, plasmids expressing an intracellular form of the mGM-CSF, induced consistently an enhanced primary Th1 response. Currently, chemotherapy is being used with good results for the treatment of some tumors as well as for HIV-1 infection. Nevertheless, resistance and severe side effects have hampered the use of this approach. A new technology with a potential for treatment of tumors and viral diseases is post transcriptional gene silencing induced by RNA interference (RNAi). By using plasmid-mediated expression of small interfering RNAs (siRNA) against either endogenous transcripts for the primary HIV-1 coreceptor, CCR5, or viral transcripts for the Rev gene of HIV-1, we were able to inhibit efficiently both viral infection and replication, respectively. The possibility of inducing a considerable reduction of the viral load or tumor burden using siRNA technology could have a significant impact on the treatment of these diseases in the future.