A Replicon Platform for Precise Reprogramming of the Tumor Microenvironment

Among immunotherapy treatments under investigation, in situ vaccination with self-amplified RNA derived from alphaviruses (replicon) is a promising strategy, achieving superior efficacy in T-cell activation than conventional RNA and DNA approaches, by coupling persistent expression of therapeutic immunomodulatory genes to the stimulation of innate immunity at the tumor site. Regardless of the therapeutic payload expressed, the nature of the tumor microenvironment (TME) determines the responsiveness of cancer to the replicon vaccine. The TME, can be simply classified into hot or cold based on the levels of proinflammatory cytokines and T cell infiltration, with the former presenting the higher response rate and the latter resistance to cancer immunotherapy. Considering the pivotal role that the nature of the TME plays in determining the effectiveness of immunotherapy, strategies to convert cold tumors into hot ones and thus sensitize tumors to replicon vaccines are of great interest.

We are developing a next generation precision replicon platform to reprogram cold tumors into hot ones via delivery of replicon RNA molecules that selectively induce apoptosis in tumor cells and reprogram key immune infiltrates in the TME. To provide the required selectivity, we implemented a classifier circuit which consists of microRNA (miRNA) sensing modules that by sensing the miRNA profile of the recipient cell, determine the expression of the genetic cargo only in the desired cell type. We show that the presence of miRNA sensing modules aligns the expression of reporter genes to the miRNA profile of tumor and immune cell lines. Replicons equipped with miRNA sensor modules specifically express the encoded payload in primary murine CD8⁺ T cells. Together, these results show the potential application of our replicon platform to precisely deliver synthetic circuits to specific cell types in the TME.