19 research outputs found
Efficient delivery of nuclease proteins for genome editing in human stem cells and primary cells
No full textTargeted 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.N
Rational design of protein switches: applications in synthetic biology and cancer immunotherapy
No full textSynthetic biology aims to engineer cells as miniature biological devices to sense, process, and respond to exogenous stimuli. Protein switches are designed to sense and respond to various molecular queues in a fast and specific manner, which fits the requirements of mammalian cell engineering. Especially, small-molecule responsive protein switches are particularly suitable for remotely controlling engineered cell functions, and could be significantly important in the context of therapeutic applications.
My thesis leverages computational tools for the design of chemically responsive protein switches. We developed a strategy to repurpose drug-inhibited protein-protein interactions into OFF- and ON-switches controlled by preclinical or clinically approved drugs. The designed binders and drug-receptors form chemically-disruptable heterodimers (CDH) which dissociate in the presence of small molecules. Moreover, we converted the CDH 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 have been applied in regulating gene expression, protein degradation and signal transduction, and they showed excellent performance as drug responsive switches to control combinations of synthetic circuits in mammalian cells. Moreover, we anticipate that the CDHs and AIRs are highly modular for many other proximity-dependent applications. I further elaborated these designs to create a bifunctional switch to sense and respond to two chemicals, which is referred to as small-molecule controlled activation and repression integrator (sm-ART). sm-ART consists of a bifunctional scaffold that binds to two drug-receptor proteins in a mutually exclusive manner, representing two steady and opposing states (e.g., ON vs OFF) that can be switched by the corresponding drug-receptor inhibitor. The sm-ART regulated by ON- and OFF- signals have been engineered with cell surface receptors to reversibly control the signaling transduction.
In a different line of research we discovered that the human Bcl2 protein homodimerize in the presence of its inhibitors, which led to the construction of three chemically inducible gene expression circuits assembling different chimeric transcription systems. Furthermore, the truncated human Bcl2 proteins were incorporated into a split heterodimeric CAR construct (ON-CAR) which can be switched ON in the presence of Bcl2's FDA-approved inhibitor, Venetoclax. The ON-CAR showed comparable killing activities to the standard second-generation CARs in the presence of Venetoclax in vitro and in vivo. We demonstrated that truncated human Bcl2 dimerize under the effect of its clinically approved drug, which opens up exciting possibilities for the engineering of safe and controllable CARs to meet many of the persistent challenges in clinical development of cell-based therapies.
Altogether, my thesis presented a computational protein design blueprint to rationally design chemically responsive protein switches controlled by preclinical or clinically validated drugs. These protein switches are also useful tools in synthetic biology and cancer immunotherapy.LPD
Protein-based bandpass filters for controlling cellular signaling with chemical inputs
No full textBiological 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.|Development of chemically responsive bandpass filters mimics the signal-processing abilities of electronic circuits in mammalian cells by responding to chemical concentrations within a specific range and rejecting ones outside that range.LPD
Systematic Investigation of the Effects of Multiple SV40 Nuclear Localization Signal Fusion on the Genome Editing Activity of Purified SpCas9
No full textThe 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
Novel chemically controlled cellular switches
No full textThe invention relates to activation by inhibitor release (AIR) switches and chemically disruptable heterodimers (CDH), their use in controlling cell signaling components, and their use for treatments and therapies.LPDIAVP-R-TTOAlternative title(s) : (fr) Nouveaux commutateurs cellulaires chimiquement régulé
Rational design of small-molecule responsive protein switches
No full textSmall-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.LPD
Computational design of bioactive protein switches with multi-logics for cell-based therapeutics
No full textLPD
Chemically disruptable molecule switch and use thereof
No full textThe invention relates to chemically disruptable molecule (CDM) switch, their use in controlling cell signaling components, and their use for treatments and therapies. Further disclosed are methods of treating and/or preventing a disease, such as e.g. a cancer, an inflammatory disease, a genetic disorder, an infectious disease, and a degenerative diseaseAVP-R-TTOLPDIAlternative title(s) :
(fr) Commutateur moléculaire pouvant être perturbé chimiquement et son utilisatio
Rational Design of Chemically Controlled Antibodies and Protein Therapeutics
No full textProtein-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.LPDILB
