(19) TZZ _ _T (11) EP 2 596 128 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12Q 1/68 (2006.01) G06N 3/00 (2006.01) 22.04.2015 Bulletin 2015/17 (86) International application number: (21) Application number: 11746063.4 PCT/US2011/045038 (22) Date of filing: 22.07.2011 (87) International publication number: WO 2012/012739 (26.01.2012 Gazette 2012/04) (54) MULTIPLE INPUT BIOLOGIC CLASSIFIER CIRCUITS FOR CELLS BIOLOGISCHE KLASSIERSCHALTUNGEN MIT MEHRFACHEINGABE FÜR ZELLEN CIRCUITS CLASSIFICATEURS BIOLOGIQUES À ENTRÉES MULTIPLES POUR CELLULES (84) Designated Contracting States: • WROBLEWSKA, Liliana AL AT BE BG CH CY CZ DE DK EE ES FI FR GB Arlington, Massachusetts 02474 (US) GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO • XIE, Zhen PL PT RO RS SE SI SK SM TR Malden, Massachusetts 02148 (US) (30) Priority: 22.07.2010 US 366787 P (74) Representative: Kasche, André (43) Date of publication of application: Kasche & Partner AG 29.05.2013 Bulletin 2013/22 Resirain 1 8125 Zollikerberg (CH) (73) Proprietors: • President and Fellows of Harvard College (56) References cited: Cambridge, MA 02138 (US) WO-A1-2008/134593 • Massachusetts Institute of Technology Cambridge, MA 02139 (US) • BROWN B D ET AL: "Endogenous microRNA regulation suppresses transgene expression in (72) Inventors: hematopoietic lineages and enables stable gene • BENENSON, Yaakov transfer", NATURE MEDICINE, NATURE CH-4051 Basel (CH) PUBLISHING GROUP, NEW YORK, NY, US, vol. • WEISS, Ron 12, no. 5, 23 April 2006 (2006-04-23) , pages Newton, Massachusetts 02459 (US) 585-591, XP002394629, ISSN: 1078-8956, DOI: 10.1038/NM1398 Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 2 596 128 B1 Printed by Jouve, 75001 PARIS (FR) EP 2 596 128 B1 Description FIELD OF THE INVENTION 5 [0001] The present invention relates to multi-input engineered genetic circuits for classifying cells. BACKGROUND [0002] An important feature of biological pathways is their two-way interaction with the cellular environment in which 10 they operate. Such interaction usually involves (1) sensing of relevant input conditions in the cell, (2) processing those inputs to determine whether and which action to take; and (3) producing a biologically-active output to actuate a phys- iological effect in the cell. Some engineered analogues of natural pathways with sensing, computational and actuation functionalities (1, 2) have been developed that can augment endogenous processes and enable rational manipulation and control ofbiological systems. While reporter constructs (3) that transduce cellular inputs into a detectable output, 15 and tissue-specific transgenes control led transcriptionally and/or posttranscriptionally (4-6) lack complexity, they rep- resent useful components for the development of synthetic circuits. Some synthetic circuits have demonstrated pro- grammed dynamic behavior in cells (oscillators (7-10), memory (11-14), spatial patterns (15), cascades (16) and pulse generators (17)), digital and analog computations (18-20), and complex biosynthetic pathways (21), but the interaction of these circuits with the cellular context has been limited (22, 23). Similarly, molecular network prototypes have dem- 20 onstrated sensing, computation and actuation (24-28) in cell-free environments, but their utility in cellular contexts has been inadequate. WO 2008/134593 discloses promoters operably linked to a repressor sequence encoding a repressor protein and a microRNA target sequence. The repressor is specific (i.e. it has operator sites) in the repressible promoter which is operably linked to a dsRed output sequence. This system can be used for the classification of the status of a cell based on its miRNA expression. 25 [0003] Hence, engineered biological systems described thus far have lacked the necessary complexity, sophistication, and discriminatory capacities tobefunctional and responsive to the multitude of inputs that are found in the normal, unmanipulated cellular millieu. SUMMARY OF THE INVENTION 30 [0004] Described herein are multi-input biological classifier circuits and methods of use thereof developed for process- ing molecular information in mammalian cells. These classifier circuits use transcriptional and posttranscriptional regu- lation in order to classify the status of a cell, i.e., determine whether a cell is in a specific state of interest. The biological classifier circuits described herein implement this task by interrogating the state of the cell through simultaneous as- 35 sessment of multiple inputs, such as the expression levels of a subset of predefined markers, for example, endogenous, mature microRNAs. The classifier circuits described herein are designed to ’compute’ whether the expression profile of the markers matches a pre-determined reference profile that characterizes the specific cell state that the classifier circuits are intended to detect. If so, the classifier circuits produce a biological response, such as expression of a reporter molecule. These biological circuits are termed herein as ’classifiers’ because they classify individual cells into a number 40 of categories based on processing a multitude of inputs indicative of the cells’ internal states, in a manner similar to current practices for characterizing bulk tissue ( e.g., biopsy samples) using gene array analysis and computer algorithms (31). [0005] The biological classifier circuits described herein can be used in a variety of applications, such as those requiring precise classification and identification of cell types. In some aspects, described herein are biological classifier circuits 45 for use as therapeutic agents, for example, in highly precise and selective cancer therapy. Many mainstream and experimental drugs exhibit a degree of selectivity toward cancer cells by relying on individual cancer markers (32). However, cancer cells exhibit a complex set of conditions deviating from the normal state of their progenitor tissue (33, 34), and using a single marker to distinguish them from healthy cells is rarely sufficient and often results in harmful side- effects (35). Therefore, sensing and integration of information from multiple markers by a therapeutic agent is crucial 50 for creating next-generation treatments, and for use in a variety of applications, which can include, but are not limited to identification, sorting, or targeting of stem cells from heterogenous populations of differentiated cells; identification, sorting, or targeting of specific cell types for the treatments of various diseases, such as cancer; identification, sorting, targeting, or detection of cell types at various developmental stages; drug screening assays; and identification, sorting, targeting, or detection of cell types in experimental models to be used in tracking therapuetic treatment responses to a 55 drug or other molecule, such as during a tumor treatment. For example, described herein is an exemplary biological classifier circuit tested in human cell culture that acts as a programmed therapeutic agent that, via identification and processing of a combination of input markers, selectively identifies and triggers apoptosis in a cancer cell line, but not in healthy cells. 2 EP 2 596 128 B1 [0006] Accordingly, provided herein are high-input detector modules for classifying a cell status based on detecting whether an input microRNA is expressed at a specific level or higher than a reference level. Such high-input detector modules comprise an inducible promoter sequence operably linked to: (i) a repressor sequence, which encodes a repressor product, and (ii) a sequence which encodes one or more microRNA target sequences, such that the one or 5 more microRNA target sequences comprise target sequences of the one or more input microRNAs the module is designed to detect. In some embodiments, such high-input detector modules can further comprise a repressible promoter sequence operably linked to an output sequence encoding an ouput product, wherein the repressor product is specific for the repressible promoter sequence. [0007] In some embodiments of the high-input detector modules described herein, the high-input detector module can 10 further comprise one or more regulatory units. Such regulatory units comprise a constitutive or inducible promoter sequence operably linked to: (i) a sequence that encodes for a transcriptional activator product, and (ii) a sequence encoding one or more microRNA target sequences, such that the transcriptional activator product activates the inducible promoter sequence operably linked to the repressor sequence and the sequence encoding the one or more microRNA target sequences. In such embodiments, the sequences encoding one or more microRNA target sequences are the 15 same throughout all the units and components of the high-input detector module, i.e., each unit and component of the high-input detector module detects the same input microRNA(s). In some embodiments, the inducible promoter of a second regulatory unit is activated by the transcriptional activator encoded by a first regulatory unit, such that the represso r product of the high-input detector module is expressed