Researchers have developed an RNAi-based synthetic circuit known as a cell-type classifier, which they claim can accurately distinguish between cancerous and noncancerous cells, and trigger apoptosis specifically in the cancer cells.
Developed by an international team led by scientists at the ETH in Zurich, Switzerland, and the Massachusetts Institute of Technology (MIT), the logic circuit is transiently expressed in a cell, where it uses Boolean operators to effectively assess the presence of a predefined set of miRNA factors using logic operations such as “and” and “not”. Cell death is triggered only when all the cell type-specific factors are present at the correct concentrations.
The ETH’s Yaakov Benenson, Ph.D., MIT’s Ron Weiss, and colleagues report in Science on a classifier that can specifically identify and kill HeLa cells. In their paper titled “Multi-Input RNAi-Based Logic Circuit for Identification of Specific Cancer Cells”, the team describes how the classifier was engineered to assess the combined presence (at specified expression levels) of five miRNAs in cells within a population, in order to selectively identify HeLa cells and trigger their apoptosis without affecting non-HeLa cell types.
The concept of sensing cancer-specific markers and signals in order to control therapeutic agents is not new, the authors report. Previous work has used a combination of up to two tissue-specific signals, including promoter and/or microRNA (miRNA) activity, mRNA, and protein levels, to partially restrict therapeutic activity to cancer cells. In parallel, research in the field of synthetic biology has demonstrated the ability to effect multi-information processing in living cells. What has been limited is the interaction between these systems, in a cellular context, involving the integration of multiple input signals to detect cell state and precisely regulate therapeutic activity.
To try and achieve this goal, the ETH and MIT researchers set out to design a classifier gene circuit that could integrate sensory information from a large number of molecular markers to identify whether its host cell is an HeLa cell, and, if so, produce a biologically active protein output to trigger apoptosis. It was a task of two halves, requiring both the identification of a set of HeLa-specific, high- and low-expression miRNAs, and the synthetic gene-based circuitry that could systematically and categorically identify and react to their presence.
Building on the team’s previous work, the concept included a sensor motif for HeLa-high markers comprising a “double-inversion” module that allows output expression only if the marker is present at or above its level in HeLa cells, but efficiently represses the output if the marker’s level is low. This design was based on Dr. Weiss et al’s previously described module consisting of the small interfering RNA (siRNA)–targeted transcriptional Lac repressor (LacI) and a LacI-controlled promoter CAGop (CAG promoter followed by an intron with two LacO sites), but with modifications to improve the ON:Off ratio. A HeLa-low marker sensor was implemented by fusing four repeats of fully complementary target sites into the output’s 3′-untranslated region (3′-UTR). The complete classifier circuit thus comprised a set of HeLa-high and HeLa-low marker sensors that were all arranged to target the same output. The overall network was designed to implement a multi-input, AND-like logic function for identification and selective killing of HeLa cells through regulated expression of hBax.
The team tested both the subcircuits and complete circuitry in HeLa cells and control cell lines, and made some subtle modifications to improve specificity. When transfected into populations of HeLa and normal cells, the introduced classifier correctly identified the cancer cells and expressed hBax, triggering apoptosis. Transfected non-HeLa cells, which didn’t fulfill the search criteria, were largely unaffected. In fact, the authors state, “among transfected cells, apoptosis-inducing classifier circuit inflicts almost the same degree of cell death in HeLa cells as does constitutively expressed hBax.”
The potential therapeutic applications of classifier circuits are evident, provided that challenges such as efficient in vivo DNA delivery to cells can be met. Moreover, the resarchers suggest, classifiers could also be used for in vitro applications such a drug screening or monitoring developmental processes.