Liliana Wroblewska

Multi-Input RNAi-Based Logic Circuit for Identification of Specific Cancer Cells

A synthetic biomolecular circuit identifies abnormal cell states by the integration of multiple endogenous microRNA inputs. Engineered biological systems that integrate multi-input sensing, sophisticated information processing, and precisely regulated actuation in living cells could be useful in a variety of applications. For example, anticancer therapies could be engineered to detect and respond to complex cellular conditions in individual cells with high specificity. Here, we show a scalable transcriptional/posttranscriptional synthetic regulatory circuit—a cell-type “classifier”—that senses expression levels of a customizable set of endogenous microRNAs and triggers a cellular response only if the expression levels match a predetermined profile of interest. We demonstrate that a HeLa cancer cell classifier selectively identifies HeLa cells and triggers apoptosis without affecting non-HeLa cell types. This approach also provides a general platform for programmed responses to other complex cell states.

Rapid, modular and reliable construction of complex mammalian gene circuits

We developed a framework for quick and reliable construction of complex gene circuits for genetically engineering mammalian cells. Our hierarchical framework is based on a novel nucleotide addressing system for defining the position of each part in an overall circuit. With this framework, we demonstrate construction of synthetic gene circuits of up to 64 kb in size comprising 11 transcription units and 33 basic parts. We show robust gene expression control of multiple transcription units by small molecule inducers in human cells with transient transfection and stable chromosomal integration of these circuits. This framework enables development of complex gene circuits for engineering mammalian cells with unprecedented speed, reliability and scalability and should have broad applicability in a variety of areas including mammalian cell fermentation, cell fate reprogramming and cell-based assays.

Mammalian synthetic circuits with RNA binding proteins for RNA-only delivery.

Synthetic regulatory circuits encoded in RNA rather than DNA could provide a means to control cell behavior while avoiding potentially harmful genomic integration in therapeutic applications. We create post-transcriptional circuits using RNA-binding proteins, which can be wired in a plug-and-play fashion to create networks of higher complexity. We show that the circuits function in mammalian cells when encoded in modified mRNA or self-replicating RNA.

A platform for rapid prototyping of synthetic gene networks in mammalian cells

Mammalian synthetic biology may provide novel therapeutic strategies, help decipher new paths for drug discovery and facilitate synthesis of valuable molecules. Yet, our capacity to genetically program cells is currently hampered by the lack of efficient approaches to streamline the design, construction and screening of synthetic gene networks. To address this problem, here we present a framework for modular and combinatorial assembly of functional (multi)gene expression vectors and their efficient and specific targeted integration into a well-defined chromosomal context in mammalian cells. We demonstrate the potential of this framework by assembling and integrating different functional mammalian regulatory networks including the largest gene circuit built and chromosomally integrated to date (6 transcription units, 27kb) encoding an inducible memory device. Using a library of 18 different circuits as a proof of concept, we also demonstrate that our method enables one-pot/single-flask chromosomal integration and screening of circuit libraries. This rapid and powerful prototyping platform is well suited for comparative studies of genetic regulatory elements, genes and multi-gene circuits as well as facile development of libraries of isogenic engineered cell lines.

Systematic transfer of prokaryotic sensors and circuits to mammalian cells.

Prokaryotic regulatory proteins respond to diverse signals and represent a rich resource for building synthetic sensors and circuits. The TetR family contains >105 members that use a simple mechanism to respond to stimuli and bind distinct DNA operators. We present a platform that enables the transfer of these regulators to mammalian cells, which is demonstrated using human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells. The repressors are modified to include nuclear localization signals (NLS) and responsive promoters are built by incorporating multiple operators. Activators are also constructed by modifying the protein to include a VP16 domain. Together, this approach yields 15 new regulators that demonstrate 19- to 551-fold induction and retain both the low levels of crosstalk in DNA binding specificity observed between the parent regulators in Escherichia coli, as well as their dynamic range of activity. By taking advantage of the DAPG small molecule sensing mediated by the PhlF repressor, we introduce a new inducible system with 50-fold induction and a threshold of 0.9 μM DAPG, which is comparable to the classic Dox-induced TetR system. A set of NOT gates is constructed from the new repressors and their response function quantified. Finally, the Dox- and DAPG- inducible systems and two new activators are used to build a synthetic enhancer (fuzzy AND gate), requiring the coordination of 5 transcription factors organized into two layers. This work introduces a generic approach for the development of mammalian genetic sensors and circuits to populate a toolbox that can be applied to diverse applications from biomanufacturing to living therapeutics.

Mechanical Strain Promotes Oligodendrocyte Differentiation by Global Changes of Gene Expression

Differentiation of oligodendrocyte progenitor cells (OPC) to oligodendrocytes and subsequent axon myelination are critical steps in vertebrate central nervous system (CNS) development and regeneration. Growing evidence supports the significance of mechanical factors in oligodendrocyte biology. Here, we explore the effect of mechanical strains within physiological range on OPC proliferation and differentiation, and strain-associated changes in chromatin structure, epigenetics, and gene expression. Sustained tensile strain of 10–15% inhibited OPC proliferation and promoted differentiation into oligodendrocytes. This response to strain required specific interactions of OPCs with extracellular matrix ligands. Applied strain induced changes in nuclear shape, chromatin organization, and resulted in enhanced histone deacetylation, consistent with increased oligodendrocyte differentiation. This response was concurrent with increased mRNA levels of the epigenetic modifier histone deacetylase Hdac11. Inhibition of HDAC proteins eliminated the strain-mediated increase of OPC differentiation, demonstrating a role of HDACs in mechanotransduction of strain to chromatin. RNA sequencing revealed global changes in gene expression associated with strain. Specifically, expression of multiple genes associated with oligodendrocyte differentiation and axon-oligodendrocyte interactions was increased, including cell surface ligands (Ncam, ephrins), cyto- and nucleo-skeleton genes (Fyn, actinins, myosin, nesprin, Sun1), transcription factors (Sox10, Zfp191, Nkx2.2), and myelin genes (Cnp, Plp, Mag). These findings show how mechanical strain can be transmitted to the nucleus to promote oligodendrocyte differentiation, and identify the global landscape of signaling pathways involved in mechanotransduction. These data provide a source of potential new therapeutic avenues to enhance OPC differentiation in vivo.

A novel Bxb1 integrase RMCE system for high fidelity site-specific integration of mAb expression cassette in CHO Cells

As CHO cell line development for biotherapeutic production becomes more sophisticated through the availability of the CHO genome sequence, the ability to accurately and reproducibly engineer the host cell genome has become increasingly important. Multiple well characterized systems for site‐specific integration will enable more complex cell line engineering to generate cell lines with desirable attributes. We built and characterized a novel recombinase mediated cassette exchange (RMCE) system using Bxb1 integrase and compared it to the commonly used Flp/FRT RMCE system. We first integrated a DNA construct flanked by either Bxb1 attachment sites or FRT sequences (referred to as a landing pad) into the Fer1L4 genomic locus of CHO‐S cells using CRISPR/Cas9 mediated homologous recombination. We characterized the resulting clones harboring either the Bxb1 or Flp/FRT landing pad using whole genome resequencing to compare their genomes with the parental host cell line. We determined that each landing pad was specifically integrated into the Fer1L4 locus in the selected clones and observed no major structural changes in the genome or variations in copy number as a result of CRISPR/Cas9 modification. We subsequently tested the ability of the Bxb1 and Flp/FRT landing pad clones to perform proper RMCE with donor vectors containing identical mAb expression cassettes flanked by either Bxb1 attachment sites or FRT sites. We demonstrated that both RMCE systems were able to generate stable pools in a similar time frame with comparable mAb expression. Through genetic characterization of up to 24 clones derived from either system, we determined that the BxB1 RMCE system yielded higher fidelity RMCE events than the Flp/FRT system as evidenced by a higher percentage of clones with expected integration of the mAb cassette into the landing pad in the respective cell lines. We conclude that Bxb1 RMCE is an excellent alternative to Flp/FRT RMCE and valuable addition to our toolbox enabling the engineering of more sophisticated cell lines for biotherapeutic production. Biotechnol. Bioeng. 2017;114: 1837–1846.

A multi-landing pad DNA integration platform for mammalian cell engineering

Abstract Engineering mammalian cell lines that stably express many transgenes requires the precise insertion of large amounts of heterologous DNA into well-characterized genomic loci, but current methods are limited. To facilitate reliable large-scale engineering of CHO cells, we identified 21 novel genomic sites that supported stable long-term expression of transgenes, and then constructed cell lines containing one, two or three ‘landing pad’ recombination sites at selected loci. By using a highly efficient BxB1 recombinase along with different selection markers at each site, we directed recombinase-mediated insertion of heterologous DNA to selected sites, including targeting all three with a single transfection. We used this method to controllably integrate up to nine copies of a monoclonal antibody, representing about 100 kb of heterologous DNA in 21 transcriptional units. Because the integration was targeted to pre-validated loci, recombinant protein expression remained stable for weeks and additional copies of the antibody cassette in the integrated payload resulted in a linear increase in antibody expression. Overall, this multi-copy site-specific integration platform allows for controllable and reproducible insertion of large amounts of DNA into stable genomic sites, which has broad applications for mammalian synthetic biology, recombinant protein production and biomanufacturing.

Engineering protein-protein devices for multilayered regulation of mRNA translation using orthogonal proteases in mammalian cells

The development of RNA-encoded regulatory circuits relying on RNA-binding proteins (RBPs) has enhanced the applicability and prospects of post-transcriptional synthetic network for reprogramming cellular functions. However, the construction of RNA-encoded multilayer networks is still limited by the availability of composable and orthogonal regulatory devices. Here, we report on control of mRNA translation with newly engineered RBPs regulated by viral proteases in mammalian cells. By combining post-transcriptional and post-translational control, we expand the operational landscape of RNA-encoded genetic circuits with a set of regulatory devices including: i) RBP-protease, ii) protease-RBP, iii) protease–protease, iv) protein sensor protease-RBP, and v) miRNA-protease/RBP interactions. The rational design of protease-regulated proteins provides a diverse toolbox for synthetic circuit regulation that enhances multi-input information processing-actuation of cellular responses. Our approach enables design of artificial circuits that can reprogram cellular function with potential benefits as research tools and for future in vivo therapeutics and biotechnological applications.RNA-encoded regulatory circuits are desirable because they do not integrate in the host and are less immunogenic, but the availability of regulatory devices is limited. Here the authors develop viral protease RNA-binding proteins and protease–protease genetic circuits that ultimately regulate mRNA translation.