Cellular Engineering Research

Direct in vivo CAR T cell engineering - Adoptive cell therapy using chimeric antigen receptor (CAR) T cells is effective against B cell malignancies; however, the complex manufacturing process and financial realities constrain the scalability of the approach. - The in vivo generation of CAR T cells, and possibly other immune cells, using off-the-shelf products therefore has numerous logistical and functional advantages. - In preclinical models, in vivo gene delivery using nanoparticles or viral vectors has yielded CAR T cells with therapeutic equivalency to ex vivo generated CAR T cells. 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 vivostrategies and discuss emerging data from preclinical models to highlight the potency of the in vivoapproach, the applicability for new disease indications, and the remaining challenges associated with clinical readiness, including delivery specificity, long term efficacy, and safety. https://lnkd.in/ewfs7vez - Ongoing research efforts are attempting to determine how to best target and leverage effector cells of interest (T cells, macrophages etc.), understand how direct in vivo CAR generation interfaces with other immune cells, and optimize design elements of the viral vectors or nanoparticle and nucleic acid formulations.

🧬 CAR T cells demonstrate the power of engineered cells as therapeutics. But they fail for most patients. Can we make them better by gene editing? Our paper in Nature presents a CRISPR platform for optimizing immunotherapies & discovering boosters of CAR T cell function. ⚙️ We developed CELLFIE (“cell engineering for immunotherapy enhancement”), a CRISPR platform to make & test gene-edited CAR T cells at scale. CELLFIE supports in vitro & in vivo screens with various clinically relevant readouts, plus combinatorial & base-editing screens. 🩸 Using CELLFIE, we conducted 58 genome-wide CRISPR screens, with readouts for CAR T cell proliferation, target cell recognition, activation, apoptosis & fratricide, and exhaustion. The screens identified known genes (PD-1, CTLA4, TIM3, TIGIT etc.) and promising new hits. 🐭 But not everything that makes CAR T cells proliferate or kill better in vitro translates into more effective therapies. For scalable validation in mice, we conducted pooled in vivo CRISPR screening and observed strong positive effects of RHOG, PRDM1, and FAS knockouts. 🐁 We performed extensive in vivo validations and found that RHOG knockout CAR T cells achieve strong reductions in cancer cell numbers and prolonged survival in an aggressive mouse model of human leukemia, with consistent results across different CARs and T cell donors. 🔍 RHOG is a small GTPase involved in cell signaling. How does it influence CAR T cells ? We found that RHOG knockout increases the proliferative capacity of CAR T cells and helps them retain a highly functional state with reduced exhaustion and enhanced memory phenotype. 💪 We also observed prolonged survival for FAS knockout CAR T cells, likely because these cells are less effective at killing each other (“fratricide”). Combining RHOG & FAS knockout, we obtained more & better CAR T cells, which further improved survival in leukemic mice. 🔬 From a technical perspective, we are excited how our new in vivo CROP-seq method improves gRNA detection (reading from an mRNA transcript as in https://lnkd.in/eaKPi335) and reduces experimental noise (by using UMIs), which enables larger screens with fewer mice. 🔥 What’s next? Our discovery of strong combined effects for RHOG & FAS knockout underlines the potential of synergistic gene edits for boosting CAR T cell function. We thus integrated combinatorial screening into CELLFIE, using the Blainey lab’s CROPseq-multi method. ⚕️ Our CELLFIE platform supports clinical translation of CRISPR-boosted CAR T cells. For example, to avoid the DNA double-strand breaks introduced by CRISPR knockout, we performed a tiling base-editing screen across RHOG and identified promising gRNA for clinical testing. 📑 Check out our paper titled “Systematic discovery of CRISPR-boosted CAR T cell immunotherapies” at Nature (open access): https://lnkd.in/eVTKrTjY. Feedback & suggestions are very welcome.

Thrilled to share our latest research published in ACS Nano on an enzyme-free method for detaching adherent cells from culture surfaces. Traditional enzymatic and mechanical detachment methods can damage cells, require multiple steps, and generate significant amount medical waste (~300 million liters annually). Our platform uses alternating electrochemical redox cycling on a conductive polymer nanocomposite to release cells in minutes while preserving >90 % viability,eliminating the need for proteolytic enzymes and reducing waste. By applying a low-frequency alternating voltage, we dynamically disrupt cell adhesion and create a biocompatible, electrically tunable interface that supports gentle, rapid detachment. Tested with human cancer cells, this method achieved detachment efficiencies up to 95 % while maintaining high viability — and is well suited for automation and scalable biomanufacturing. Beyond improving routine cell culture, this technology opens doors to automated, contamination-conscious workflows for cell therapies, tissue engineering, regenerative medicine, and high-throughput applications like drug screening. The approach also offers exciting opportunities to integrate electrochemical control into bioelectronic systems for next-generation platforms. https://lnkd.in/e7sW947N https://lnkd.in/egDC-CNJ

Modification Strategies for Engineering NK Cell Surface Surface engineering of natural killer (NK) cells has become a key strategy to enhance their efficacy in immunotherapy. By modifying surface receptors and functional molecules, researchers aim to improve NK cell targeting, persistence, and cytotoxicity, leading to more effective cancer and infectious disease treatments. One of the most advanced surface engineering technologies is the introduction of chimeric antigen receptors (CARs) into NK cells. CAR-NK cells are engineered to express synthetic receptors that recognize specific tumor antigens, thereby enhancing their ability to target cancer cells with high precision. Unlike CAR-T cells, CAR-NK cells have a lower risk of graft-versus-host disease (GVHD) and lower cytokine release syndrome (CRS), making them safer for clinical use. Modifying NK cells to overexpress natural activating receptors such as NKG2D or CD16 can enhance their cytotoxic potential. These receptors can enhance tumor cell recognition of stress ligands or enhance antibody-dependent cellular cytotoxicity (ADCC) when paired with therapeutic antibodies. Nanotechnology has enabled the functionalization of NK cell surfaces with nanomaterials. These nanostructures can be equipped with ligands, drugs, or cytokines to enhance tumor targeting, activation, and persistence in the tumor microenvironment. Binding immune checkpoint inhibitors directly to the NK cell surface (such as PD-1 or TIGIT blockers) helps overcome inhibitory signals from the tumor microenvironment and restore NK cell activity. Overall, NK cell surface engineering is transforming immunotherapy, addressing challenges such as tumor immune evasion and poor NK cell persistence. These innovations pave the way for highly effective, targeted cancer treatments, bringing new hope to patients with difficult-to-treat diseases. Reference [1] Hao Zhang et al., Bioactive Materials 2024 (https://lnkd.in/e4aZmbWr) #Immunotherapy #NKCells #CellEngineering #CancerResearch #CAR_NKCells #Nanomaterials #TumorMicroenvironment #BiomedicalInnovation #PrecisionMedicine #CancerImmunology #OncologyBreakthroughs #GeneEditing #NKCellTherapy #CancerTreatment

𝐖𝐡𝐚𝐭 𝐢𝐟 𝐲𝐨𝐮 𝐜𝐨𝐮𝐥𝐝 𝐟𝐢𝐧𝐞-𝐭𝐮𝐧𝐞 𝐓 𝐜𝐞𝐥𝐥𝐬 𝐥𝐢𝐤𝐞 𝐚 𝐬𝐲𝐦𝐩𝐡𝐨𝐧𝐲, 𝐧𝐨𝐭 𝐚 𝐬𝐨𝐥𝐨? 𝐒𝐜𝐢𝐞𝐧𝐭𝐢𝐬𝐭𝐬 𝐡𝐚𝐯𝐞 𝐝𝐞𝐯𝐞𝐥𝐨𝐩𝐞𝐝 𝐚 𝐧𝐞𝐰 𝐰𝐚𝐲 𝐭𝐨 𝐜𝐫𝐞𝐚𝐭𝐞 𝐡𝐢𝐠𝐡𝐥𝐲 𝐜𝐮𝐬𝐭𝐨𝐦𝐢𝐬𝐞𝐝 𝐓 𝐜𝐞𝐥𝐥𝐬 that can target disease more precisely. Instead of ending up with a messy mix of partly edited cells, this method helps researchers keep only the ones with all the right edits. No extra labels or tags needed! 𝐓𝐡𝐞 𝐬𝐞𝐜𝐫𝐞𝐭 𝐥𝐢𝐞𝐬 𝐢𝐧 𝐚 𝐜𝐥𝐞𝐯𝐞𝐫 𝐭𝐨𝐨𝐥 𝐜𝐚𝐥𝐥𝐞𝐝 𝐒𝐄𝐄𝐃-𝐒𝐞𝐥𝐞𝐜𝐭𝐢𝐨𝐧. It uses synthetic "disruptors" to link the integration of a new gene with the removal of a native protein. That means you can tell which cells have been fully edited just by checking what’s missing on their surface. 𝐓𝐡𝐞 𝐭𝐞𝐚𝐦 𝐭𝐞𝐬𝐭𝐞𝐝 𝐭𝐡𝐢𝐬 𝐚𝐩𝐩𝐫𝐨𝐚𝐜𝐡 𝐨𝐧 𝐭𝐡𝐫𝐞𝐞 𝐜𝐫𝐢𝐭𝐢𝐜𝐚𝐥 𝐭𝐚𝐫𝐠𝐞𝐭𝐬 𝐫𝐞𝐥𝐚𝐭𝐞𝐝 𝐭𝐨 𝐓 𝐜𝐞𝐥𝐥 𝐟𝐮𝐧𝐜𝐭𝐢𝐨𝐧: 🟠 Specificity (what the T cell recognises) 🟠 Co-receptors (how it responds) 🟠 MHC expression (how it communicates with other immune cells) 𝐓𝐡𝐞 𝐫𝐞𝐬𝐮𝐥𝐭𝐬? ✅ 98% purity for individual edits ✅ 90% purity even when doing six changes at once (three knock-ins and three knockouts) ✅ Fully compatible with clinical-grade manufacturing workflows 𝐓𝐡𝐢𝐬 𝐦𝐢𝐠𝐡𝐭 𝐬𝐞𝐞𝐦 𝐥𝐢𝐤𝐞 𝐚 𝐭𝐞𝐜𝐡𝐧𝐢𝐜𝐚𝐥 𝐭𝐰𝐞𝐚𝐤, but it opens the door to faster, cleaner, and more precise production of next-generation cell therapies. Imagine the impact for cancer, autoimmunity, and even infectious disease treatments. 💡 𝐖𝐡𝐚𝐭 𝐬𝐭𝐚𝐧𝐝𝐬 𝐨𝐮𝐭 𝐭𝐨 𝐦𝐞 is how this tackles one of the most frustrating bottlenecks in cell therapy manufacturing, which is purity without complexity. Exactly what the field needs to move from promise to product. 👣 Follow me for more insights on cell engineering, biotech breakthroughs, and the science behind tomorrow’s therapies. https://lnkd.in/eqymad4p

🔥 New Breakthrough in Stem Cell Engineering: Microglia in Just 4 Days 🧠 I’m thrilled to share exciting news from a team at Wyss Institute at Harvard University & HMS (led by George Church) published in Nature Communications on June 10, 2025. 🔬Team Acheivement: Leveraged a high-throughput, single-cell transcription factor (TF) screening approach to systematically test combinations that drive human iPSCs toward microglia identity. Over two iterative screening rounds, they identified a powerful cocktail of six TFs - SPI1, CEBPA, FLI1, MEF2C, CEBPB & IRF8 that rapidly reprogram iPSCs into microglia-like cells (TFiMGLs) in just 4 days, skipping the typical 35-day conventional protocol. 🧩 Why it matters? These lab-derived cells display key molecular and functional characteristics of native microglia (e.g. response to ADP and interferon-γ stimulation). Unlike conventional methods relying on lengthy small‑molecule cocktails and co‑culture steps, this TF-based strategy is robust, reproducible, and quicker. The platform also produced a causal gene regulatory network, offering insight into how TFs drive cell fate is a powerful blueprint for future cell engineering . 🌍 Broader implications: Disease modeling: Enables rapid production of human microglia for neurodegenerative or neuroinflammation research across Alzheimer’s, Parkinson’s, ALS, MS, and more . Cell therapy & drug discovery: Offers a scalable, tunable approach to generate other complex cell types that have been historically difficult to produce. Synthetic biology & personalized medicine: Iterative TF screening + single‑cell analysis = a plug‑and‑play platform for targeted cellular engineering. 💡 Key Takeaway: This iterative TF‑based reprogramming platform is a game‑changer - getting us microglia fast and setting the stage for broader cell‑fate breakthroughs. Truly a transformative stride in regenerative & synthetic biology. Would love to hear your thoughts: how do you envision this method reshaping your research, therapeutic strategy, or drug pipeline? #StemCellEngineering #iPSC #TranscriptionFactors #Microglia #Neurobiology #SyntheticBiology #RegenerativeMedicine 📢more in comments

A breakthrough in CAR-immune cell engineering. Ludwig Cancer Research scientists have devised new types of CAR-T cells that can be switched on to varying degrees of intensity and switched off on demand with existing drugs. ⬇️ ❗️ The trouble is that many solid tumor antigens are also found on healthy cells, raising the risks of so-called "off-tumor, on-target" effects. These can provoke destructive immune responses that are difficult to control and potentially lethal to the patient. Conversely—and perhaps more often—the immunosuppressive conditions of the solid tumor microenvironment can push anti-tumor T cells, including those equipped with CARs, into a state of dysfunction known as "exhaustion." 💡 Having the ability to remotely switch CAR-T cells on to varying degrees using different doses of an activating drug—and then off on demand, as needed—would improve the safety of this therapy. Further, the remote control of CAR-T cell activity could also mitigate T cell exhaustion, improving the durability of patient responses to the therapy. 💡💡➡️ To enable control of CAR activity, scientists separated the antigen-sensing moiety (the antibody fragment) and the activation domain (CD3-ζ) into two separate chains, the "receptor chain" and the "signaling chain." Tapping the expertise of Bruno Correia, they also included an extra module that can dimerize the two chains upon application of a cancer drug called "venetoclax." When it binds those external modules, the venetoclax molecule acts like a bridge, bringing the two chains together to create an active CAR complex. The intensity of the subsequent CAR-T cell response depends on how much of the drug is used. The researchers named this CAR construct the "inducible-ON" (iON) CAR. 💡💡➡️ To be truly safe, however, the CAR-T cells also need to be switched off promptly if they pose a danger to patients. To that end, the researchers added an additional druggable component onto the CD3- ζ signaling chain that is responsive to another approved cancer drug named "lenalidomide." This binding, however, marks the receptor for degradation by the cell's waste-disposal machinery. The researchers show that the all-in-one iON/OFF CAR (iONØ-CAR) T cells can be switched on by venetoclax and quickly deactivated—within 4-6 hours—by lenalidomide. ✅ Researchers plan to characterize the performance of their iON and iONØ-CARs against various tumor models further. They will also investigate whether remotely controlling the cells can reduce the toxicity associated with overactive CAR-T responses. Additionally, they will assess if periodic breaks can enhance the long-term control of tumors. https://lnkd.in/e-pjb-ax #cancer #cancerresearch #immunology #immunooncology #immunotherapy #celltherapy #cartcelltherapy #tumormicroenvironment #cellenginieering #chemotherapy #solidtumor

Following our recent breakthrough in developing mouse mini-intestines for ex vivo tumor development (https://lnkd.in/eAc6YzAr) and building on our ability to generate in vitro models of healthy human colon (https://lnkd.in/ep7Xni-3), we asked ourselves: can this technology be applied to cells from colorectal cancer patients? We're thrilled to announce that our latest publication provides the answer: https://rdcu.be/dMuAr We've created long-lived human 'mini-colons' that stably integrate patient cancer cells and their native tumor microenvironment. This innovative format is optimized for real-time, high-resolution evaluation of cellular dynamics, offering exciting experimental possibilities. Our research highlights include: 1) Multi-faceted evaluation of drug efficacy, toxicity, and resistance in anti-cancer therapies. 2) Discovery of a cancer-associated fibroblast (CAF)-triggered mechanism driving colorectal cancer invasion. 3) Identification of immunomodulatory interactions among different components of the tumor microenvironment. This work has been led by Luis Francisco Lorenzo Martín, with invaluable support from Nicolas Broguiere, Jakob Langer, Lucie Tillard, Mike Nikolaev, George Coukos, and Krisztian Homicsko. Thank you all!! #Organoid #Tumoroid #Bioengineering #CancerResearch #TeamScience

Announcing our latest publication from the #Heilshorn_Biomaterial_Lab! In our new collaborative work, led by brilliant Betty Cai and supervised by Sarah Heilshorn and Sungchul Shin, we developed an integrated fabrication and #endothelialization strategy that directly generates branched, endothelial cell-lined networks using a #diffusion_based, embedded 3D #bioprinting process for the first time. This #innovation not only addresses long-standing challenges in #vascular biofabrication, such as cell uniformity, seeding efficiency, and multi-cell type #patterning but also paves the way for engineering more complex, multi-cellular vasculature. Learn more about how we patterned both #arterial and #venous endothelial cells within a single network to enhance geometric complexity and #phenotypic heterogeneity by reading the full article via the link below: https://lnkd.in/gdcv-hW3 Betty Cai, David Kilian, Julien Roth, Alexis Seymour, Lucia Brunel, Daniel Ramos, @Ricardo J Rios, @Isabella M Szabo, Sean Chryz Iranzo, @Andy Perez, Ram Rao MD PhD, Sungchul Shin, Sarah Heilshorn Stanford University, DTU Health Tech, University of Washington, Seoul National University #Biofabrication #3DBioprinting #TissueEngineering #Bioprinting #VascularEngineering #Endothelialization #Biomaterials #RegenerativeMedicine #BiomedicalEngineering #Innovation #ScientificResearch #CellBiology #VascularNetworks #AdvancedManufacturing #MedicalInnovation #DiffusionBased #EmbeddedBioprinting #MultiCellularSystems #MaterialsEngineering #FutureOfMedicine #Arterial #Venous #ScienceInnovation #HealthcareInnovation #BiomedicalResearch #ScientificPublication