Nature Biomedical Engineering2021ResearchNon-viral Gene Delivery
Ian C. Miller, Ali Zamat, Lee-Kai Sun, Hathaichanok Phuengkham, Adrian M. Harris, Lena Gamboa, Jason Yang, John P. Murad, Saul J. Priceman, Gabriel A. Kwong
CAR T cell therapy for solid malignancies typically results in poor responses. Systemic administration of immunomodulatory biologics (cytokines, BiTEs) can augment T cell activity but off-target toxicity narrows the therapeutic window. A method to spatially and temporally control transgene expression by engineered T cells at tumor sites—without systemic exposure—could improve safety and efficacy. ### Thermal Switch Engineering | Parameter | Result | |---------------|------------| | Best HSE repeat number | 7 repeats (5H-7H best; 2H-4H lower response) | | Best core promoter | YB (synthetic) — ~60-fold induction.
Read the article →Seminars in Immunology2021ReviewNon-viral Gene Delivery
Est-Witte Se, Livingston Nk, Omotoso Mo, Green Jj, Schneck Jp.
T cell therapies (TIL, TCR, CAR T) show clinical efficacy but are limited by high cost, lengthy ex vivo manufacturing, patient variability, and poor persistence. Nanoparticles offer a modular, universal platform to improve T cell therapy at every stage—from antigen presentation and T cell activation to T cell maintenance—while reducing cost and broadening patient accessibility. Nano-aAPC E+E expanded Kb-TRP2 cognate cells from ~0.03% to 17.6% by day 7; nano-aAPCs expanded MART-1-specific CD8⁺ T cells more effectively than autologous DCs and CD3/CD28 Dynabeads, with higher stem cell memory.
Read the article →Nature Communications2020ResearchNon-viral Gene Delivery
N.N. Parayath, S.B. Stephan, A.L. Koehne, P.S. Nelson, M.T. Stephan
CAR T and TCR T cell therapies are powerful but face prohibitive costs and manufacturing complexity due to the need for leukapheresis, ex vivo transduction, expansion, and quality control—all under GMP conditions. An "off-the-shelf" injectable nanocarrier that can transiently reprogram circulating T cells in vivo would bypass these barriers, enabling scalable, cost-effective, and repeatable dosing for a wide range of diseases including cancer. ### NP Characterization & Ex Vivo Transfection | Parameter | Result | |---------------|------------| | NP size (PBAE/mRNA polyplex) | ~100 nm | | mRNA encapsulation efficiency | >90% | | T cell transfection efficiency |.
Read the article →Nature Communications2020ResearchNon-viral Gene Delivery
John C. Charpentier, Di Chen, Philip E. Lapinski, Jackson Turner, Irina Grigorova, Joel A. Swanson, Philip D. King
Macropinocytosis has been shown to regulate the growth of Ras-transformed tumor cells under amino acid (AA)-limiting conditions by providing extracellular proteins that are degraded into AA to fuel cell growth and activate mTORC1. However, whether macropinocytosis regulates the growth of non-transformed mammalian cells, such as primary T lymphocytes, was unknown. Understanding whether macropinocytosis is required for T cell growth has. ### T Cell Macropinocytosis (Probe Uptake) | Cell Type | Probe | Stimulation | Fold Increase (Stimulated vs. Unstimulated) |.
Read the article →Cancer Immunology Research (AACR)2020ResearchNon-viral Gene Delivery
Dylan J. Drakes, Sarwish Rafiq, Terence J. Purdon, Andrea V. Lopez, Smita S. Chandran, Christopher A. Klebanoff, Renier J. Brentjens
T-cell receptor (TCR)-modified T-cell gene therapy can target a variety of extracellular and intracellular tumor-associated antigens, yet has had little clinical success. A potential explanation for limited antitumor efficacy is a lack of T-cell activation in vivo, particularly when tumor cells downregulate costimulatory molecules. A method to provide a durable proinflammatory signal to TCR-modified T cells could enhance persistence, effector. ### In Vitro T Cell Activation & Cytotoxicity | Parameter | Control | mIL12 pmel-1 | mIL18 pmel-1 | |---------------|-------------|------------------|------------------| | IFNγ secretion | Baseline | Significantly.
Read the article →Proceedings of the National Academy of Sciences (PNAS)2020ResearchNon-viral Gene Delivery
Bradley Vis, Rachel E. Hewitt, Tom P. Monie, Camilla Fairbairn, Suzanne D. Turner, Stephen D. Kinrade, Jonathan J. Powell
Ultrasmall silica nanoparticles (USSN, <10 nm diameter) were recently shown to stimulate T lymphocytes directly at relatively low exposure doses, but the underlying mechanisms and associated cell signaling were unknown. Understanding whether USSN directly engage the T cell receptor (TCR) complex or act through other pathways is critical for both safety assessment and therapeutic translation of these inexpensive, rapidly dissolving nanoparticles. ### USSN Activation of T Cells (CD69 Expression, 24 h) | Cell Type | Control (%) | USSN (800 μM) (%) | Fold Increase | |---------------|-----------------|-----------------------|------------------| | CD4⁺ T cells (n=23).
Read the article →Nature Biotechnology2020ResearchNon-viral Gene Delivery
David N. Nguyen, Theodore L. Roth, P. Jonathan Li, Peixin Amy Chen, Ryan Apathy, Murad R. Mamedov, Linda T. Vo, Victoria R. Tobin, Daniel Goodman, Eric Shifrut, Jeffrey A. Bluestone, Jennifer M. Puck, Francis C. Szoka, Alexander Marson
CRISPR-Cas9 genome editing in clinically relevant primary cells is limited by inefficient homology-directed repair (HDR) and cytotoxicity of exogenous DNA. Improvements are needed to increase editing efficiency, cell viability, and generalizability across cell types for adoptive cell therapies and research. tCTS shuttle: Facing orientation of tCTS on both 5′ and 3′ homology arms improved knock-in at TRAC. Representative flow cytometry: dsDNA vs tCTS — 45.7% vs 68.7%, 16.9% vs 54.1%, 18.7% vs 47.3%, 24.1% vs 51.3% across.
Read the article →Frontiers in Bioengineering and Biotechnology2020ResearchDrug Delivery
Serkan Yaman, Harish Ramachandramoorthy, Gizem Oter, Daria Zhukova, Tam Nguyen, Manoj K. Sabnani, Jon A. Weidanz, Kytai T. Nguyen
Melanoma is an aggressive skin cancer with limited treatment efficacy due to non-specific drug targeting, severe side effects, and multidrug resistance. Current cell-based immunotherapies are costly, complex, and carry long-term autoimmune risks. There is a need for a targeted, biocompatible drug delivery system that selectively recognizes melanoma cells and provides sustained drug release. Physicochemical properties: T-MNPs (1:2) were 193 ± 56 nm, PDI 0.265, zeta −36 mV; stable in saline for 48 h. Drug loading 61%; sustained trametinib release over 28 days, slowest at highest membrane ratio (1:2). - TCR.
Read the article →Nature2019ResearchNon-viral Gene Delivery
Haig Aghajanian, Toru Kimura, Joel G. Rurik, Aidan S. Hancock, Michael S. Leibowitz, Li Li, John Scholler, James Monslow, Albert Lo, Wei Han, Tao Wang, Kenneth Bedi, Michael P. Morley, Ricardo A. Linares Saldana, Nikhita A. Bolar, Kendra Mcdaid, Charles-Antoine Assenmacher, Cheryl L. Smith, Dagmar W
Fibrosis is observed in nearly every form of myocardial disease and contributes to heart failure, yet clinical interventions targeting fibrosis remain limited. No therapies directly target excessive cardiac fibrosis, and very few interventions improve cardiac function in patients with impaired compliance. Genetic ablation of cardiac fibroblasts after injury reduces fibrosis and improves function in mice, but a clinically translatable method to. ### OVA Proof-of-Concept (Conditional Cardiac Fibroblast Targeting) | Parameter | OVA;Postnᴹᶜᴹ + Tam + OT-I | Controls | Significance |.
Read the article →Nature Communications2018ResearchNon-viral Gene Delivery
Ewoud B. Compeer, Felix Kraus, Manuela Ecker, Gregory Redpath, Mayan Amiezer, Nils Rother, Philip R. Nicovich, Natasha Kapoor-Kaushik, Qiji Deng, Guerric P.B. Samson, Zhengmin Yang, Jieqiong Lou, Michael Carnell, Haig Vartoukan, Katharina Gaus, Jérémie Rossy
Polarized endocytic recycling of the T cell receptor (TCR) to the immunological synapse is essential for T cell activation, yet the cellular mechanisms that coordinate internalization of surface receptors with sustained delivery back to the plasma membrane remain incompletely understood. The role of flotillins in defining a clathrin-independent endocytic route and supporting receptor recycling is unclear, particularly in the context of TCR. ### TCR Internalization Dynamics | Finding | Detail | |-------------|------------| | TCR internalization | Constitutive in resting cells; rapidly increased upon activation (most molecules internalized within 20 s) | |.
Read the article →Nature Communications2017ResearchNon-viral Gene Delivery
H.F. Moffett, M.E. Coon, S. Radtke, S.B. Stephan, L. Mcknight, A. Lambert, B.L. Stoddard, H.P. Kiem, M.T. Stephan
Therapies based on genetically engineered immune cells (CAR T cells, HSCs) are transforming medicine, but the viral and electroporation methods used to create these cytoreagents are complex, expensive, and time-consuming. There is a growing need for transient "hit-and-run" genetic programming (transcription factors, genome-editing nucleases) where permanent transgene integration is undesirable or dangerous. A simple, non-viral method to. ### NP Characterization & Transfection Efficiency | Parameter | Result | |---------------|------------| | NP size | 109.6 ± 26.6 nm | | Zeta potential | 1.1 ± 5.3 mV (near-neutral) | | T cell transfection efficiency |.
Read the article →Molecular Therapy2016ResearchNon-viral Gene Delivery
Ans De Beuckelaer, Charlotte Pollard, Sandra Van Lint, Kenny Roose, Lien Van Hoecke, Thomas Naessens, Vimal Kumar Udhayakumar, Muriel Smet, Niek Sanders, Stefan Lienenklaus, Xavier Saelens, Siegfried Weiss, Guido Vanham, Johan Grooten & Stefaan De Koker
mRNA vaccines are promising for inducing cytolytic CD8⁺ T cell responses, and lipoplex carriers improve mRNA delivery to dendritic cells. However, the innate factors that regulate T cell induction by mRNA lipoplex vaccines remain unresolved. In particular, type I interferons (IFNs) are induced by mRNA but can either promote or inhibit T cell immunity depending on context. mRNA lipoplexes induced potent type I IFN responses after subcutaneous, intradermal, and intranodal injection. Naked mRNA also induced IFN-β, whereas liposomes alone did not, indicating IFN induction is inherent to mRNA.
Read the article →Proceedings of the National Academy of Sciences (PNAS)2015ResearchNon-viral Gene Delivery
Tim Willinger, Matthew Staron, Shawn M. Ferguson, Pietro De Camilli, Richard A. Flavell
Prolonged T-cell receptor (TCR) signaling is required for T lymphocyte proliferation, yet TCR ligation paradoxically causes internalization and down-modulation of the TCR from the cell surface. It is unknown how TCR signaling is sustained for many hours despite lower surface expression. Understanding whether TCR signaling continues from intracellular compartments after internalization has significant implications for T cell biology and. ### TCR Internalization & Signal Strength | Parameter | Dnm2 HET | Dnm2 KO | Significance | |---------------|--------------|-------------|------------------| | TCR down-modulation (8 h post-stimulation) | ~50% reduction.
Read the article →The Journal of Immunology2013ResearchNon-viral Gene Delivery
Ying-Chun Lo, Michael A. Edidin, Jonathan D. Powell
Soluble anti-CD3 antibodies are potent T cell mitogens but activate all T cells regardless of TCR specificity, limiting their therapeutic utility for boosting antigen-specific responses. TCRs cluster into microclusters on the surface of antigen-experienced T cells but remain dispersed on naive T cells. This difference in receptor organization could be exploited to selectively activate only previously activated T cells using anti-CD3 antibodies. ### TCR Clustering (Confocal Microscopy) | T Cell State | Microclusters/Cell | Average Cluster Area (μm²) | Mean Intensity |.
Read the article →Journal of Biological Chemistry1987ResearchNon-viral Gene Delivery
Yasuhiro Minami, Lawrence E. Samelson, Richard D. Klausner
The T cell antigen receptor (TCR) plays a critical role in T cell activation, but the dynamics of TCR internalization and its regulation were poorly understood. It was known that phorbol esters (activators of protein kinase C) lead to loss of surface TCRs, but the mechanisms—whether internalization was signal-dependent and whether receptors continuously cycle—remained unclear. Understanding TCR trafficking has implications for receptor signaling. ### Basal TCR Internalization | Condition | % Internalized (30 min at 37°C) | Significance | |---------------|-------------------------------------|------------------| | ¹²⁵I-A2B4-2 (whole IgG) | 20-30% | No increase.
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