Protein-based bandpass filters for controlling cellular signaling with chemical inputs.

Biological signal processing is vital for cellular function. Similar to electronic circuits, cells process signals via integrated mechanisms. In electronics, bandpass filters transmit frequencies with defined ranges, but protein-based counterparts for controlled responses are lacking in engineered biological systems. Here, we rationally design protein-based, chemically responsive bandpass filters (CBPs) showing OFF-ON-OFF patterns that respond to chemical concentrations within a specific range and reject concentrations outside that range. Employing structure-based strategies, we designed a heterodimeric construct that dimerizes in response to low concentrations of a small molecule (ON), and dissociates at high concentrations of the same molecule (OFF). The CBPs have a multidomain architecture in which we used known drug receptors, a computationally designed protein binder and small-molecule inhibitors. This modular system allows fine-tuning for optimal performance in terms of bandwidth, response, cutoff and fold changes. The CBPs were used to regulate cell surface receptor signaling pathways to control cellular activities in engineered cells.

Rational design of small-molecule responsive protein switches.

Small-molecule responsive protein switches are powerful tools for controlling cellular processes. These switches are designed to respond rapidly and specifically to their inducer. They have been used in numerous applications, including the regulation of gene expression, post-translational protein modification, and signal transduction. Typically, small-molecule responsive protein switches consist of two proteins that interact with each other in the presence or absence of a small molecule. Recent advances in computational protein design already contributed to the development of protein switches with an expanded range of small-molecule inducers and increasingly sophisticated switch mechanisms. Further progress in the engineering of small-molecule responsive switches is fueled by cutting-edge computational design approaches, which will enable more complex and precise control over cellular processes and advance synthetic biology applications in biotechnology and medicine. Here, we discuss recent milestones and how technological advances are impacting the development of chemical switches.

Rational Design of Chemically Controlled Antibodies and Protein Therapeutics.

Protein-based therapeutics, such as monoclonal antibodies and cytokines, are important therapies for various pathophysiological conditions such as oncology, autoimmune disorders, and viral infections. However, the wide application of such protein therapeutics is often hindered by dose-limiting toxicities and adverse effects, namely, cytokine storm syndrome, organ failure, and others. Therefore, spatiotemporal control of the activities of these proteins is crucial to further expand their application. Here, we report the design and application of small-molecule-controlled switchable protein therapeutics by taking advantage of a previously engineered OFF-switch system. We used the Rosetta modeling suite to computationally optimize the affinity between B-cell lymphoma 2 (Bcl-2) protein and a previously developed computationally designed protein partner (LD3) to obtain a fast and efficient heterodimer disruption upon the addition of a competing drug (Venetoclax). The incorporation of the engineered OFF-switch system into anti-CTLA4, anti-HER2 antibodies, or an Fc-fused IL-15 cytokine demonstrated an efficient disruption in vitro, as well as fast clearance in vivo upon the addition of the competing drug Venetoclax. These results provide a proof-of-concept for the rational design of controllable biologics by introducing a drug-induced OFF-switch into existing protein-based therapeutics.

Systematic Investigation of the Effects of Multiple SV40 Nuclear Localization Signal Fusion on the Genome Editing Activity of Purified SpCas9.

The emergence of CRISPR-Cas9 technology has revolutionized both basic and translational biomedical research. For Cas9 nuclease to exert genome editing activity, nuclear localization signal (NLS) derived from simian virus 40 (SV40) T antigen is commonly installed as genetic fusion to direct the intracellular Cas9 proteins to the nucleus of cells. Notably, previous studies have shown that multiple SV40 NLS fusion can improve the targeting activity of Cas9-derived genome-editing and base-editing tools. In addition, the multi-NLS fusion can increase the intracellular activity of Cas9 in the forms of both constitutive expression and directly delivered Cas9-guide RNA ribonucleoprotein (RNP) complex. However, the relationship between NLS fusion and intracellular Cas9 activity has not been fully understood, including the dependency of activity on the number or organization of NLS fusion. In the present study, we constructed and purified a set of Streptococcus pyogenes Cas9 (SpCas9) variants containing one to four NLS repeats at the N- or C-terminus of the proteins and systematically analyzed the effects of multi-NLS fusion on the activity of SpCas9 RNPs. It was found that multi-NLS fusion could improve the intracellular activity as lipofected or nucleofected Cas9 RNPs. Importantly, multi-NLS fusion could enhance the genome-editing activity of SpCas9 RNPs in primary and stem/progenitor cells and mouse embryos.

A rational blueprint for the design of chemically-controlled protein switches.

Small-molecule responsive protein switches are crucial components to control synthetic cellular activities. However, the repertoire of small-molecule protein switches is insufficient for many applications, including those in the translational spaces, where properties such as safety, immunogenicity, drug half-life, and drug side-effects are critical. Here, we present a computational protein design strategy to repurpose drug-inhibited protein-protein interactions as OFF- and ON-switches. The designed binders and drug-receptors form chemically-disruptable heterodimers (CDH) which dissociate in the presence of small molecules. To design ON-switches, we converted the CDHs into a multi-domain architecture which we refer to as activation by inhibitor release switches (AIR) that incorporate a rationally designed drug-insensitive receptor protein. CDHs and AIRs showed excellent performance as drug responsive switches to control combinations of synthetic circuits in mammalian cells. This approach effectively expands the chemical space and logic responses in living cells and provides a blueprint to develop new ON- and OFF-switches.

Author Correction: A computationally designed chimeric antigen receptor provides a small-molecule safety switch for T-cell therapy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A computationally designed chimeric antigen receptor provides a small-molecule safety switch for T-cell therapy.

Approaches to increase the activity of chimeric antigen receptor (CAR)-T cells against solid tumors may also increase the risk of toxicity and other side effects. To improve the safety of CAR-T-cell therapy, we computationally designed a chemically disruptable heterodimer (CDH) based on the binding of two human proteins. The CDH self-assembles, can be disrupted by a small-molecule drug and has a high-affinity protein interface with minimal amino acid deviation from wild-type human proteins. We incorporated the CDH into a synthetic heterodimeric CAR, called STOP-CAR, that has an antigen-recognition chain and a CD3ζ- and CD28-containing endodomain signaling chain. We tested STOP-CAR-T cells specific for two antigens in vitro and in vivo and found similar antitumor activity compared to second-generation (2G) CAR-T cells. Timed administration of the small-molecule drug dynamically inactivated the activity of STOP-CAR-T cells. Our work highlights the potential for structure-based design to add controllable elements to synthetic cellular therapies.

AXL promotes Zika virus infection in astrocytes by antagonizing type I interferon signalling.

Zika virus (ZIKV) is associated with neonatal microcephaly and Guillain-Barré syndrome1,2. While progress has been made in understanding the causal link between ZIKV infection and microcephaly3-9, the life cycle and pathogenesis of ZIKV are less well understood. In particular, there are conflicting reports on the role of AXL, a TAM family kinase receptor that was initially described as the entry receptor for ZIKV10-22. Here, we show that while genetic ablation of AXL protected primary human astrocytes and astrocytoma cell lines from ZIKV infection, AXL knockout did not block the entry of ZIKV. We found, instead, that the presence of AXL attenuated the ZIKV-induced activation of type I interferon (IFN) signalling genes, including several type I IFNs and IFN-stimulating genes. Knocking out type I IFN receptor α chain (IFNAR1) restored the vulnerability of AXL knockout astrocytes to ZIKV infection. Further experiments suggested that AXL regulates the expression of SOCS1, a known type I IFN signalling suppressor, in a STAT1/STAT2-dependent manner. Collectively, our results demonstrate that AXL is unlikely to function as an entry receptor for ZIKV and may instead promote ZIKV infection in human astrocytes by antagonizing type I IFN signalling.

Genome-Editing Technologies: Principles and Applications.

Targeted nucleases have provided researchers with the ability to manipulate virtually any genomic sequence, enabling the facile creation of isogenic cell lines and animal models for the study of human disease, and promoting exciting new possibilities for human gene therapy. Here we review three foundational technologies-clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9), transcription activator-like effector nucleases (TALENs), and zinc-finger nucleases (ZFNs). We discuss the engineering advances that facilitated their development and highlight several achievements in genome engineering that were made possible by these tools. We also consider artificial transcription factors, illustrating how this technology can complement targeted nucleases for synthetic biology and gene therapy.

Delivery methods for site-specific nucleases: Achieving the full potential of therapeutic gene editing.

The advent of site-specific nucleases, particularly CRISPR/Cas9, provides researchers with the unprecedented ability to manipulate genomic sequences. These nucleases are used to create model cell lines, engineer metabolic pathways, produce transgenic animals and plants, perform genome-wide functional screen and, most importantly, treat human diseases that are difficult to tackle by traditional medications. Considerable efforts have been devoted to improving the efficiency and specificity of nucleases for clinical applications. However, safe and efficient delivery methods remain the major obstacle for therapeutic gene editing. In this review, we summarize the recent progress on nuclease delivery methods, highlight their impact on the outcomes of gene editing and discuss the potential of different delivery approaches for therapeutic gene editing.

Efficient delivery of nuclease proteins for genome editing in human stem cells and primary cells.

Targeted nucleases, including zinc-finger nucleases (ZFNs), transcription activator-like (TAL) effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9), have provided researchers with the ability to manipulate nearly any genomic sequence in human cells and model organisms. However, realizing the full potential of these genome-modifying technologies requires their safe and efficient delivery into relevant cell types. Unlike methods that rely on expression from nucleic acids, the direct delivery of nuclease proteins to cells provides rapid action and fast turnover, leading to fewer off-target effects while maintaining high rates of targeted modification. These features make nuclease protein delivery particularly well suited for precision genome engineering. Here we describe procedures for implementing protein-based genome editing in human embryonic stem cells and primary cells. Protocols for the expression, purification and delivery of ZFN proteins, which are intrinsically cell-permeable; TALEN proteins, which can be internalized via conjugation with cell-penetrating peptide moieties; and Cas9 ribonucleoprotein, whose nucleofection into cells facilitates rapid induction of multiplexed modifications, are described, along with procedures for evaluating nuclease protein activity. Once they are constructed, nuclease proteins can be expressed and purified within 6 d, and they can be used to induce genomic modifications in human cells within 2 d.

A biomimetic enzyme modified electrode for H2O2 highly sensitive detection.

An efficient catalyst based on artificial bionic peroxidase was synthesized for electrocatalysis. A poly(ethyleneimine)/Au nanoparticle composite (PEI-AuNP) was prepared and it was then linked to hemin via a coupling reaction between carboxyl groups in hemin and amino groups in PEI without the activation of a carboxyl group by carbodiimide. Fourier transform infrared (FTIR) spectroscopy verified the formation of amido bonds within the structure. The presence of AuNPs contributed greatly in establishing the amido bonds within the composite. Transmission electron microscopy (TEM) and UV-visible spectroscopy were also used to characterize the PEI-AuNP-hemin catalyst. PEI-AuNP-hemin exhibited intrinsic peroxidase-like catalytic activities. The PEI-AuNP-hemin deposited on a glass carbon electrode had strong sensing for H2O2 with a well-defined linear relationship between the amperometric response and H2O2 concentration in the range from 1 µM to 0.25 mM. The detection limit was 0.247 nM with a high sensitivity of 0.347 mA mM(-1) cm(-2). The peroxidase-like catalytic activity of PEI-AuNP-hemin is discussed in relation to its microstructure. The study suggests that PEI-AuNP-hemin may have promising application prospects in biocatalysis and bioelectronics.