Sensing antibody functions with a novel CCR8-responsive engineered cell.

Human chemokine receptor 8 (CCR8) is a promising drug target for immunotherapy of cancer and autoimmune diseases. Monoclonal antibody-based CCR8 targeted treatment shows significant inhibition in tumor growth. The inhibition of CCR8 results in the improvement of antitumor immunity and patient survival rates by regulating tumor-resident regulatory T cells. Recently monoclonal antibody drug development targeting CCR8 has become a research hotspot, which also promotes the advancement of antibody evaluation methods. Therefore, we constructed a novel engineered customized cell line HEK293-cAMP-biosensor-CCR8 combined with CCR8 and a cAMP-biosensor reporter. It can be used for the detection of anti-CCR8 antibody functions like specificity and biological activity, in addition to the detection of antibody-dependent cell-mediated cytotoxicity and antibody-dependent-cellular-phagocytosis. We obtained a new CCR8 mAb 22H9 and successfully verified its biological activities with HEK293-cAMP-biosensor-CCR8. Our reporter cell line has high sensitivity and specificity, and also offers a rapid kinetic detection platform for evaluating anti-CCR8 antibody functions.

Direct in vivo CAR T cell engineering.

T cells modified to express intelligently designed chimeric antigen receptors (CARs) are exceptionally powerful therapeutic agents for relapsed and refractory blood cancers and have the potential to revolutionize therapy for many other diseases. To circumvent the complexity and cost associated with broad-scale implementation of ex vivo manufactured adoptive cell therapy products, alternative strategies to generate CAR T cells in vivo by direct infusion of nanoparticle-formulated nucleic acids or engineered viral vectors under development have received a great deal of attention in the past few years. Here, we outline the ex vivo manufacturing process as a motivating framework for direct in vivo strategies and discuss emerging data from preclinical models to highlight the potency of the in vivo approach, the applicability for new disease indications, and the remaining challenges associated with clinical readiness, including delivery specificity, long term efficacy, and safety.

Non-viral delivery of RNA for therapeutic T cell engineering.

Adoptive T cell transfer has shown great success in treating blood cancers, resulting in a growing number of FDA-approved therapies using chimeric antigen receptor (CAR)-engineered T cells. However, the effectiveness of this treatment for solid tumors is still not satisfactory, emphasizing the need for improved T cell engineering strategies and combination approaches. Currently, CAR T cells are mainly manufactured using gammaretroviral and lentiviral vectors due to their high transduction efficiency. However, there are concerns about their safety, the high cost of producing them in compliance with current Good Manufacturing Practices (cGMP), regulatory obstacles, and limited cargo capacity, which limit the broader use of engineered T cell therapies. To overcome these limitations, researchers have explored non-viral approaches, such as membrane permeabilization and carrier-mediated methods, as more versatile and sustainable alternatives for next-generation T cell engineering. Non-viral delivery methods can be designed to transport a wide range of molecules, including RNA, which allows for more controlled and safe modulation of T cell phenotype and function. In this review, we provide an overview of non-viral RNA delivery in adoptive T cell therapy. We first define the different types of RNA therapeutics, highlighting recent advancements in manufacturing for their therapeutic use. We then discuss the challenges associated with achieving effective RNA delivery in T cells. Next, we provide an overview of current and emerging technologies for delivering RNA into T cells. Finally, we discuss ongoing preclinical and clinical studies involving RNA-modified T cells.

Platform-agnostic CellNet enables cross-study analysis of cell fate engineering protocols.

Optimization of cell engineering protocols requires standard, comprehensive quality metrics. We previously developed CellNet, a computational tool to quantitatively assess the transcriptional fidelity of engineered cells compared with their natural counterparts, based on bulk-derived expression profiles. However, this platform and others were limited in their ability to compare data from different sources, and no current tool makes it easy to compare new protocols with existing state-of-the-art protocols in a standardized manner. Here, we utilized our prior application of the top-scoring pair transformation to build a computational platform, platform-agnostic CellNet (PACNet), to address both shortcomings. To demonstrate the utility of PACNet, we applied it to thousands of samples from over 100 studies that describe dozens of protocols designed to produce seven distinct cell types. We performed an in-depth examination of hepatocyte and cardiomyocyte protocols to identify the best-performing methods, characterize the extent of intra-protocol and inter-lab variation, and identify common off-target signatures, including a surprising neural/neuroendocrine signature in primary liver-derived organoids. We have made PACNet available as an easy-to-use web application, allowing users to assess their protocols relative to our database of reference engineered samples, and as open-source, extensible code.

Co-opting signalling molecules enables logic-gated control of CAR T cells.

Although chimeric antigen receptor (CAR) T cells have altered the treatment landscape for B cell malignancies, the risk of on-target, off-tumour toxicity has hampered their development for solid tumours because most target antigens are shared with normal cells1,2. Researchers have attempted to apply Boolean-logic gating to CAR T cells to prevent toxicity3-5; however, a truly safe and effective logic-gated CAR has remained elusive6. Here we describe an approach to CAR engineering in which we replace traditional CD3ζ domains with intracellular proximal T cell signalling molecules. We show that certain proximal signalling CARs, such as a ZAP-70 CAR, can activate T cells and eradicate tumours in vivo while bypassing upstream signalling proteins, including CD3ζ. The primary role of ZAP-70 is to phosphorylate LAT and SLP-76, which form a scaffold for signal propagation. We exploited the cooperative role of LAT and SLP-76 to engineer logic-gated intracellular network (LINK) CAR, a rapid and reversible Boolean-logic AND-gated CAR T cell platform that outperforms other systems in both efficacy and prevention of on-target, off-tumour toxicity. LINK CAR will expand the range of molecules that can be targeted with CAR T cells, and will enable these powerful therapeutic agents to be used for solid tumours and diverse diseases such as autoimmunity7 and fibrosis8. In addition, this work shows that the internal signalling machinery of cells can be repurposed into surface receptors, which could open new avenues for cellular engineering.

Preclinical proof of concept for VivoVec, a lentiviral-based platform for in vivo CAR T-cell engineering.

BACKGROUND: Chimeric antigen receptor (CAR) T-cell therapies have demonstrated transformational outcomes in the treatment of B-cell malignancies, but their widespread use is hindered by technical and logistical challenges associated with ex vivo cell manufacturing. To overcome these challenges, we developed VivoVec, a lentiviral vector-based platform for in vivo engineering of T cells. UB-VV100, a VivoVec clinical candidate for the treatment of B-cell malignancies, displays an anti-CD3 single-chain variable fragment (scFv) on the surface and delivers a genetic payload that encodes a second-generation CD19-targeted CAR along with a rapamycin-activated cytokine receptor (RACR) system designed to overcome the need for lymphodepleting chemotherapy in supporting successful CAR T-cell expansion and persistence. In the presence of exogenous rapamycin, non-transduced immune cells are suppressed, while the RACR system in transduced cells converts rapamycin binding to an interleukin (IL)-2/IL-15 signal to promote proliferation. METHODS: UB-VV100 was administered to peripheral blood mononuclear cells (PBMCs) from healthy donors and from patients with B-cell malignancy without additional stimulation. Cultures were assessed for CAR T-cell transduction and function. Biodistribution was evaluated in CD34-humanized mice and in canines. In vivo efficacy was evaluated against normal B cells in CD34-humanized mice and against systemic tumor xenografts in PBMC-humanized mice. RESULTS: In vitro, administration of UB-VV100 resulted in dose-dependent and anti-CD3 scFv-dependent T-cell activation and CAR T-cell transduction. The resulting CAR T cells exhibited selective expansion in rapamycin and antigen-dependent activity against malignant B-cell targets. In humanized mouse and canine studies, UB-VV100 demonstrated a favorable biodistribution profile, with transduction events limited to the immune compartment after intranodal or intraperitoneal administration. Administration of UB-VV100 to humanized mice engrafted with B-cell tumors resulted in CAR T-cell transduction, expansion, and elimination of systemic malignancy. CONCLUSIONS: These findings demonstrate that UB-VV100 generates functional CAR T cells in vivo, which could expand patient access to CAR T technology in both hematological and solid tumors without the need for ex vivo cell manufacturing.

Engineered exosome-mediated messenger RNA and single-chain variable fragment delivery for human chimeric antigen receptor T-cell engineering.

BACKGROUND AIMS: Most current chimeric antigen receptor (CAR) T cells are generated by viral transduction, which induces persistent expression of CARs and may cause serious undesirable effects. Messenger RNA (mRNA)-based approaches in manufacturing CAR T cells are being developed to overcome these challenges. However, the most common method of delivering mRNA to T cells is electroporation, which can be toxic to cells. METHODS: The authors designed and engineered an exosome delivery platform using the bacteriophage MS2 system in combination with the highly expressed protein lysosome-associated membrane protein 2 isoform B on exosomes. RESULTS: The authors' delivery platform achieved specific loading and delivery of mRNA into target cells and achieved expression of specific proteins, and anti-CD3/CD28 single-chain variable fragments (scFvs) expressed outside the exosomal membrane effectively activated primary T cells in a similar way to commercial magnetic beads. CONCLUSIONS: The delivery of CAR mRNA and anti-CD3/CD28 scFvs via designed exosomes can be used for ex vivo production of CAR T cells with cancer cell killing capacity. The authors' results indicate the potential applications of the engineered exosome delivery platform for direct conversion of primary T cells to CAR T cells while providing a novel strategy for producing CAR T cells in vivo.

Engineered Bone Marrow Stem Cell-Sheets Alleviate Renal Damage in a Rat Chronic Glomerulonephritis Model.

Although mesenchymal stem cell (MSC)-based regenerative therapy is being developed for the treatment of kidney diseases, cell delivery and engraftment still need to be improved. Cell sheet technology has been developed as a new cell delivery method, to recover cells as a sheet form retaining intrinsic cell adhesion proteins, which promotes its transplantation efficiency to the target tissue. We thus hypothesized that MSC sheets would therapeutically reduce kidney disease with high transplantation efficiency. When the chronic glomerulonephritis was induced by two injections of the anti-Thy 1.1 antibody (OX-7) in rats, the therapeutic efficacy of rat bone marrow stem cell (rBMSC) sheet transplantation was evaluated. The rBMSC-sheets were prepared using the temperature-responsive cell-culture surfaces and transplanted as patches onto the surface of two kidneys of each rat at 24 h after the first injection of OX-7. At 4 weeks, retention of the transplanted MSC-sheets was confirmed, and the animals with MSC-sheets showed significant reductions in proteinuria, glomerular staining for extracellular matrix protein, and renal production of TGFß1, PAI-1, collagen I, and fibronectin. The treatment also ameliorated podocyte and renal tubular injury, as evidenced by a reversal in the reductions of WT-1, podocin, and nephrin and by renal overexpression of KIM-1 and NGAL. Furthermore, the treatment enhanced gene expression of regenerative factors, and IL-10, Bcl-2, and HO-1 mRNA levels, but reduced TSP-1 levels, NF-kB, and NAPDH oxidase production in the kidney. These results strongly support our hypothesis that MSC-sheets facilitated MSC transplantation and function, and effectively retarded progressive renal fibrosis via paracrine actions on anti-cellular inflammation, oxidative stress, and apoptosis and promoted regeneration.

Engineering human stem cell-derived islets to evade immune rejection and promote localized immune tolerance.

Immunological protection of transplanted stem cell-derived islet (SC-islet) cells is yet to be achieved without chronic immunosuppression or encapsulation. Existing genetic engineering approaches to produce immune-evasive SC-islet cells have so far shown variable results. Here, we show that targeting human leukocyte antigens (HLAs) and PD-L1 alone does not sufficiently protect SC-islet cells from xenograft (xeno)- or allograft (allo)-rejection. As an addition to these approaches, we genetically engineer SC-islet cells to secrete the cytokines interleukin-10 (IL-10), transforming growth factor ß (TGF-ß), and modified IL-2 such that they promote a tolerogenic local microenvironment by recruiting regulatory T cells (Tregs) to the islet grafts. Cytokine-secreting human SC-ß cells resist xeno-rejection and correct diabetes for up to 8 weeks post-transplantation in non-obese diabetic (NOD) mice. Thus, genetically engineering human embryonic SCs (hESCs) to induce a tolerogenic local microenvironment represents a promising approach to provide SC-islet cells as a cell replacement therapy for diabetes without the requirement for encapsulation or immunosuppression.

Enhanced protein translocation to mammalian cells by expression of EtgA transglycosylase in a synthetic injector E. coli strain.

BACKGROUND: Bacterial type III secretion systems (T3SSs) assemble a multiprotein complex termed the injectisome, which acts as a molecular syringe for translocation of specific effector proteins into the cytoplasm of host cells. The use of injectisomes for delivery of therapeutic proteins into mammalian cells is attractive for biomedical applications. With that aim, we previously generated a non-pathogenic Escherichia coli strain, called Synthetic Injector E. coli (SIEC), which assembles functional injectisomes from enteropathogenic E. coli (EPEC). The assembly of injectisomes in EPEC is assisted by the lytic transglycosylase EtgA, which degrades the peptidoglycan layer. As SIEC lacks EtgA, we investigated whether expression of this transglycosylase enhances the protein translocation capacity of the engineered bacterium. RESULTS: The etgA gene from EPEC was integrated into the SIEC chromosome under the control of the inducible tac promoter, generating the strain SIEC-eEtgA. The controlled expression of EtgA had no effect on the growth or viability of bacteria. Upon induction, injectisome assembly was ~ 30% greater in SIEC-eEtgA than in the parental strain, as determined by the level of T3SS translocon proteins, the hemolytic activity of the bacterial strain, and the impairment in flagellar motility. The functionality of SIEC-eEtgA injectisomes was evaluated in a derivative strain carrying a synthetic operon (eLEE5), which was capable of delivering Tir effector protein into the cytoplasm of HeLa cells triggering F-actin polymerization beneath the attached bacterium. Lastly, using ß-lactamase as a reporter of T3SS-protein injection, we determined that the protein translocation capacity was ~ 65% higher in the SIEC-EtgA strain than in the parental SIEC strain. CONCLUSIONS: We demonstrate that EtgA enhances the assembly of functional injectisomes in a synthetic injector E. coli strain, enabling the translocation of greater amounts of proteins into the cytoplasm of mammalian cells. Accordingly, EtgA expression may boost the protein translocation of SIEC strains programmed as living biotherapeutics.

Electrogenetics: Bridging synthetic biology and electronics to remotely control the behavior of mammalian designer cells.

Electrogenetics, the combination of electronics and genetics, is an emerging field of mammalian synthetic biology in which electrostimulation is used to remotely program user-designed genetic elements within designer cells to generate desired outputs. Here, we describe recent advances in electro-induced therapeutic gene expression and therapeutic protein secretion in engineered mammalian cells. We also review available tools and strategies to engineer electro-sensitive therapeutic designer cells that are able to sense electrical pulses and produce appropriate clinically relevant outputs in response. We highlight current limitations facing mammalian electrogenetics and suggest potential future directions for research.

Versioning biological cells for trustworthy cell engineering.

"Full-stack" biotechnology platforms for cell line (re)programming are on the horizon, thanks mostly to (a) advances in gene synthesis and editing techniques as well as (b) the growing integration of life science research with informatics, the internet of things and automation. These emerging platforms will accelerate the production and consumption of biological products. Hence, traceability, transparency, and-ultimately-trustworthiness is required from cradle to grave for engineered cell lines and their engineering processes. Here we report a cloud-based version control system for biotechnology that (a) keeps track and organizes the digital data produced during cell engineering and (b) molecularly links that data to the associated living samples. Barcoding protocols, based on standard genetic engineering methods, to molecularly link to the cloud-based version control system six species, including gram-negative and gram-positive bacteria as well as eukaryote cells, are shown. We argue that version control for cell engineering marks a significant step toward more open, reproducible, easier to trace and share, and more trustworthy engineering biology.

Cell behaviors within a confined adhesive area fabricated using novel micropatterning methods.

In the field of cell and tissue engineering, there is an increasing demand for techniques to spatially control the adhesion of cells to substrates of desired sizes and shapes. Here, we describe two novel methods for fabricating a substrate for adhesion of cells to a defined area. In the first method, the surface of the coverslip or plastic dish was coated with Lipidure, a non-adhesive coating material, and air plasma was applied through a mask with holes, to confer adhesiveness to the surface. In the second method, after the surface of the coverslip was coated with gold by sputtering and then with Lipidure; the Lipidure coat was locally removed using a novel scanning laser ablation method. These methods efficiently confined cells within the adhesive area and enabled us to follow individual cells for a longer duration, compared to the currently available commercial substrates. By following single cells within the confined area, we were able to observe several new aspects of cell behavior in terms of cell division, cell-cell collisions, and cell collision with the boundary between adhesive and non-adhesive areas.

Combined gene and environmental engineering offers a synergetic strategy to enhance r-protein production in Chinese hamster ovary cells.

Environmental growth-inhibition conditions (GICs) have been used extensively for increasing cell-specific productivity (qP ) of Chinese hamster ovary (CHO) cells, with the most common being temperature downshift and sodium butyrate (NaBu) treatment. B lymphocyte-induced maturation protein-1 (BLIMP1) overexpression in CHO cells can also inhibit cell growth and increase product titers and qP . Given the similar responses, this study evaluated the individual and combined effects of BLIMP1 expression, low temperature, and NaBu treatment on culture performance, cell metabolism, and recombinant protein production of CHO cells. As expected, all three interventions decreased cell growth, arrested cells in G1/G0 cell cycle phase, and increased qP . However, CHO cells presented different responses when considering cell viability, recombinant gene expression, and cell metabolism that indicated differences in the molecular loci by which BLIMP1 and GICs generated higher productivities. Combinations of BLIMP1 expression and GICs acted synergistically to inhibit cell growth and maximize r-protein production, with the BLIMP1/NaBu condition leading to the most significant improvements in product titers and qP . This latter condition also proved to substantially increase product yields (up to 9.8 g immunoglobulin G1 [IgG1]/L and 2.2 g erythropoietin-Fc [EPO-Fc]/L) and qP (up to 179 pg/cell/day [pcd] for IgG1 and 30 pcd for EPO-Fc) in high-density perfusion cultures. These findings offered mechanistic insights about the productivity-enhancing effects of BLIMP1 and GICs, as well as their complementarity for generating highly productive processes.

Regenerative strategies for the consequences of myocardial infarction: Chronological indication and upcoming visions.

Heart muscle injury and an elevated troponin level signify myocardial infarction (MI), which may result in defective and uncoordinated segments, reduced cardiac output, and ultimately, death. Physicians apply thrombolytic therapy, coronary artery bypass graft (CABG) surgery, or percutaneous coronary intervention (PCI) to recanalize and restore blood flow to the coronary arteries, albeit they were not convincingly able to solve the heart problems. Thus, researchers aim to introduce novel substitutional therapies for regenerating and functionalizing damaged cardiac tissue based on engineering concepts. Cell-based engineering approaches, utilizing biomaterials, gene, drug, growth factor delivery systems, and tissue engineering are the most leading studies in the field of heart regeneration. Also, understanding the primary cause of MI and thus selecting the most efficient treatment method can be enhanced by preparing microdevices so-called heart-on-a-chip. In this regard, microfluidic approaches can be used as diagnostic platforms or drug screening in cardiac disease treatment. Additionally, bioprinting technique with whole organ 3D printing of human heart with major vessels, cardiomyocytes and endothelial cells can be an ideal goal for cardiac tissue engineering and remarkable achievement in near future. Consequently, this review discusses the different aspects, advancements, and challenges of the mentioned methods with presenting the advantages and disadvantages, chronological indications, and application prospects of various novel therapeutic approaches.

Current Perspectives on "Off-The-Shelf" Allogeneic NK and CAR-NK Cell Therapies.

Natural killer cells (NK cells) are the first line of the innate immune defense system, primarily located in peripheral circulation and lymphoid tissues. They kill virally infected and malignant cells through a balancing play of inhibitory and stimulatory receptors. In pre-clinical investigational studies, NK cells show promising anti-tumor effects and are used in adoptive transfer of activated and expanded cells, ex-vivo. NK cells express co-stimulatory molecules that are attractive targets for the immunotherapy of cancers. Recent clinical trials are investigating the use of CAR-NK for different cancers to determine the efficiency. Herein, we review NK cell therapy approaches (NK cell preparation from tissue sources, ways of expansion ex-vivo for "off-the-shelf" allogeneic cell-doses for therapies, and how different vector delivery systems are used to engineer NK cells with CARs) for cancer immunotherapy.

Engineering mammalian living materials towards clinically relevant therapeutics.

Engineered living materials represent a new generation of human-made biotherapeutics that are highly attractive for a myriad of medical applications. In essence, such cell-rich platforms provide encodable bioactivities with extended lifetimes and environmental multi-adaptability currently unattainable in conventional biomaterial platforms. Emerging cell bioengineering tools are herein discussed from the perspective of materializing living cells as cooperative building blocks that drive the assembly of multiscale living materials. Owing to their living character, pristine cellular units can also be imparted with additional therapeutically-relevant biofunctionalities. On this focus, the most recent advances on the engineering of mammalian living materials and their biomedical applications are herein outlined, alongside with a critical perspective on major roadblocks hindering their realistic clinical translation. All in all, transposing the concept of leveraging living materials as autologous tissue-building entities and/or self-regulated biotherapeutics opens new realms for improving precision and personalized medicine strategies in the foreseeable future.

Engineering living therapeutics with synthetic biology.

The steadfast advance of the synthetic biology field has enabled scientists to use genetically engineered cells, instead of small molecules or biologics, as the basis for the development of novel therapeutics. Cells endowed with synthetic gene circuits can control the localization, timing and dosage of therapeutic activities in response to specific disease biomarkers and thus represent a powerful new weapon in the fight against disease. Here, we conceptualize how synthetic biology approaches can be applied to programme living cells with therapeutic functions and discuss the advantages that they offer over conventional therapies in terms of flexibility, specificity and predictability, as well as challenges for their development. We present notable advances in the creation of engineered cells that harbour synthetic gene circuits capable of biological sensing and computation of signals derived from intracellular or extracellular biomarkers. We categorize and describe these developments based on the cell scaffold (human or microbial) and the site at which the engineered cell exerts its therapeutic function within its human host. The design of cell-based therapeutics with synthetic biology is a rapidly growing strategy in medicine that holds great promise for the development of effective treatments for a wide variety of human diseases.