Bioconjugate Chemistry

The contemporary shift toward multispecific antibodies, antibody-drug conjugates (ADCs), and bespoke glycoengineered therapeutics have exposed the limitations of standard genomic engineering tools. This paper presents a novel iterative engineering paradigm utilizing the Leap-In Transposase(R) platform. By leveraging a suite of three mutually orthogonal transposase-transposon systems, we demonstrate the sequential modification of the Chinese Hamster Ovary (CHO) genome to achieve three distinct functional outcomes: (i) First, the creation of a glutamine synthetase (GS)-deficient host (CHO-K1-GS) via targeted knockdown, (ii) Second, the integration of multiple copies of a model therapeutic IgG1 for expression, and (iii) Third, the subsequent knockdown of the fucosylation pathway to modulate the glycan profile of the expressed IgG1. Genetic stability (copy number & sequence) of each integration event was confirmed using Targeted Locus Amplification (TLA) and Next-Generation Sequencing (NGS). Functional stability (expression levels, metabolic phenotype, and glycan phenotypes) was confirmed using standard cell culture and analytical techniques. Crucially, the truly orthogonal nature of the transposase-transposon pairs prevents cross-mobilization and ensures the structural and functional integrity of previously integrated cargo. This study establishes a "What You See Is What You Get" (WYSIWYG) methodology that provides a robust, scalable, and predictable framework for developing next-generation complex biopharmaceutical manufacturing cell lines.

Fluorescent proteins (FPs) are essential tools for biological imaging but are limited by photobleaching, a light-induced loss of fluorescence intensity that reduces spatial and temporal resolution. Despite extensive use, the molecular mechanisms underlying FP photobleaching remain poorly understood due to the diversity of FPs and the complexity of their photochemistry. Existing approaches either monitor fluorescence decay in live cells, reflecting imaging conditions but lacking molecular detail, or rely on in vitro spectroscopy of purified proteins, providing mechanistic insight but often limited to individual FPs. We introduce a quantitative workflow bridging these approaches by combining live-cell measurements with in vitro spectroscopy. In vitro measurements are performed on a dedicated setup that simultaneously monitors absorption, emission, and fluorescence decay during photobleaching. Applied to six FPs spanning different chromophores, emission ranges and sequences, this approach reveals that photobleaching strongly depends on FP. It involves multiple chemical pathways, including oxidation, dimerization, and backbone cleavage. Spectroscopic analysis uncovers a heterogeneous ensemble of photoproducts with distinct photophysical properties that can remain optically active during irradiation, including shortened fluorescence lifetimes or altered absorption spectra. These findings demonstrate that FP photobleaching cannot be described as a simple ON-OFF process but involves complex transformations affecting both fluorescence intensity and lifetime. Such transformations can introduce significant biases in quantitative imaging, particularly in advanced techniques such as FLIM and FRET. Finally, we introduce quantitative indicators enabling robust comparison of FP photostability across experimental conditions. This framework provides a comprehensive approach for understanding and quantifying photobleaching and its implications for fluorescence imaging.

Cholesterol is a key component of cellular membranes, regulating membrane organization, fluidity, and signaling. However, cholesterol analysis remains technically challenging, as no single method currently allows both accurate quantification and spatially resolved visualization. Biochemical assays provide accurate quantification but lack spatial resolution, whereas imaging strategies can perturb membrane organization or cholesterol accessibility. Here, we describe optimized protocols using fluorescent D4 probes derived from the cholesterol-binding domain of perfringolysin O (D4-mCherry and D4-GFP) to detect, visualize, and quantify cholesterol in biological samples. We detail procedures for probe production, purification, and application, and establish conditions that ensure robust and reproducible labeling of membrane-accessible cholesterol. By combining fluorescence-based imaging with quantitative analysis, this approach enables the assessment of cholesterol distribution while preserving its native membrane environment. The proposed methodology provides a versatile and reliable framework for studying cholesterol in a wide range of experimental systems.

Instability of the inverted terminal repeats (ITRs) in AAV transfer plasmids has long hindered consistent and efficient production of therapeutic AAV vectors. The palindromic, GC-rich ITR sequence readily forms secondary structures, making them highly mutable in transfer plasmids. Indeed, a recent survey observed mutated ITRs in [~]40% of AAV transfer plasmids from labs around the world. Conventional strategies to mitigate this issue - such as using specialized E. coli strains, suboptimal culture conditions, or modified ITR sequences - have limited effect and often compromise plasmid and AAV yield. Here, by combinatorial optimization of the plasmid backbone structure and ITR flanking sequences, we established MuteFree, an AAV transfer plasmid system that eliminated ITR mutations for both single-stranded AAV (ssAAV) and self-complementary AAV (scAAV). Specifically, MuteFree reduced ITR mutation rates from a range of 32-100% in various transfer plasmids tested to 0% after serial passage of host E. coli for >160 population doublings. Moreover, in three GMP-grade AAV plasmid manufacturing projects initially cancelled due to severe and incurable ITR mutations, replacing conventional backbone with MuteFree completely solved the problem, reducing mutation occurrence to zero under standard GMP manufacturing conditions. Notably, MuteFree supports the packaging of potent AAV virus. The MuteFree system thus presents a robust solution to ITR instability, enabling high-fidelity and high-yield AAV production of AAV-based gene therapy vectors that is fully compatible with existing GMP manufacturing workflows.

The clinical translation of RNA interference (RNAi) therapeutics remains limited by inefficient delivery and cancer-target accumulation. Here, we report the development of a new cationic liposome (CLP) nanocarrier engineered for delivery and controlled-release of small interfering RNA (siRNA) targeting the epidermal growth factor receptor (EGFR) in human colorectal cancer. CLPs were synthesized from ethylphosphocholine-based lipids and PEGylated components, with folic acid (FA) tissue-specific ligand and fluorophore labelling. These nanocarriers exhibited robust physicochemical stability across a broad pH and temperature range, efficient siRNA complexation, and nuclease-protection of siRNA. Functional studies revealed that CLP-siEGFR achieved effective cytosolic siRNA cargo release and EGFR silencing in vitro, proving to be more effective than conventional lipid-based transfection systems. In human xenograft models, intravenously administered CLP-siEGFR showed enhanced tumor localization, prolonged siRNA retention, and significant tumor growth suppression, accompanied by marked downregulation of EGFR. Importantly, systemic dosing was well-tolerated, with no evidence of hepatotoxicity, nephrotoxicity, or hematological abnormalities. These results position CLP nanocarriers as an effective platform for targeted RNAi therapeutics, offering translational potential for precision oncology applications.

Despite >50 years of methods development, specific antibodies are still generated at low throughput and remain in high demand across biotechnology. Most biologics and immunoprobes are monoclonal antibodies, developed using a combination of inoculating animals with a target antigen, engineered candidate libraries, and multiple rounds of selection using phage or yeast display. Here we introduce a synthetic biology scheme to eliminate the need for nearly all of these steps, by combining Surface display on E. coli and Phage display with the microvirus {Phi}X174, Assisting Continuous Evolution (SurPhACE). Instead of building libraries for screening, SurPhACE runs a closed evolutionary program. A typical experiment can have 1011 mutant candidates under active selection, with complete turnover of the mutant population every 30min, or >5x1012 unique mutants per day, using less than 100mL of bacterial culture media. We demonstrate SurPhACE for optimizing a nanobody to a related epitope, and develop novel nanobodies for an arbitrary target using a minimal starting library to establish a proof of concept and identify best practices for this scalable method for generating protein binders.

Efficient production of human proteins for the development of tool compounds and biologics depends on a detailed understanding of the protein expression machinery in mammalian cells. Codon optimization is widely believed to enhance protein yield, yet its impact in homologous mammalian systems remains poorly defined. Here, we systematically compare five codon usage strategies reflecting common assumptions about rare codons, RNA stability, and synthesis efficiency. We developed pTipi, an efficient open-source mammalian expression vector, and evaluated its performance in antibody production. We generated plasmids for common epitope tag antibodies such as V5, anti-biotin and anti-His for distribution by Addgene. To compare codon usage schemes, we performed a bake-off of 18 human and murine Wnt pathway glycoproteins in mammalian cells. Small-scale expression screens revealed that codon optimization did not provide a general advantage over native coding sequences, while strategies prioritizing RNA stability consistently reduced expression. Interestingly, a skewed codon scheme using the most abundant codons produced yields comparable to native sequences and occasionally enhanced protein output. To enable flexible evaluation of codon strategies, we implemented a Golden Gate-compatible pTipi platform for efficient synthetic gene incorporation. We conclude that native codons are sufficient for robust homologous mammalian expression of glycoproteins, while selective codon skewing can be beneficial for some targets.

Engineering macrophages with chimeric antigen receptors is emerging as a promising cancer therapeutic. Chimeric antigen receptor-expressing macrophages (CAR-Ms) engineered to recognize tumor-specific antigens have been shown to inhibit tumor growth and activate adaptive immune responses, leading to robust tumor control in animal studies. Based on this work, clinical trials have been initiated. While the trials have shown promise, challenges remain. The dynamic interactions between CAR-Ms and cancer cells and the exact mechanisms driving anti-tumor effects remain poorly defined. Defining the dynamic interactions between CAR-Ms and cancer cells will provide critical insights for optimizing future CAR-M design and improving therapeutic efficacy. We sought to directly visualize CAR-M interactions with glioblastoma cells at high-resolution and in real-time using CAR-Ms engineered to recognize Neural-Glial Antigen 2 (NG2), an antigen expressed on glioblastoma cells. Using patient-derived glioblastoma cells, we formed glioblastoma spheroids and embedded them in a 3D matrix together with CAR-Ms. Using time-lapse microscopy, as expected, we found that NG2-targeting CAR-Ms engulfed glioblastoma cells. However, excitingly, we found that NG2-targeting CAR-Ms blocked >85% of glioblastoma cell invasion in 3D. This inhibition of glioblastoma invasion was not due to a significant change in CAR-M polarization states. Together, these data suggest that NG2-targeting CAR-Ms both engulf glioblastoma cells and block glioblastoma invasive behavior.

A programmable 4-input cascade DNA logic gate utilizing toehold mediated strand displacement (TMSD) was implemented on a 3D printed hybrid paper-polymer vertical flow device (3D HPVF) for on/off sensitive and specific fluorescence detection of platelet derived growth factor BB (PDGF BB). Polypropylene was 3D printed directly on paper and thermally cured to create micro paper analytical devices ({micro}PADs). The 3D HPVF comprised of three layers of {micro}PADs enclosed in a casing that clamped each {micro}PAD securely to ensure seamless and efficient wicking between layers. In the presence of PDGF BB, a partially complementary strand to a PDGF B aptamer (PDGF B Apt), cApt, was liberated from a PDGF B Apt/cApt duplex in solution. The solution was then deposited on the 3D HPVF with a dimeric g-quadruplex hairpin. The 4-nucleotide toehold region on the cApt started the hybridization reaction with the dimeric g-quadruplex hairpin (dGH) opening it up allowing formation of a dimeric g-quadruplex structure that binds with thioflavin T (ThT) with enhanced fluorescence intensity at room temperature. The 3D HPVF exhibits a pico molar range of detection from 10pM to 100pM with a 10pM limit of detection (LOD) for PDGF BB concentrations relevant for pregnant women predisposed to early-onset preeclampsia with clear differentiation when compared to similarly competing analytes PDGF AA and AB.

Precise control over viral tropism remains a major challenge in the development of gene delivery technologies. We present MATCH (Modulation of AAV Tropism through Conjugation to Homing proteins), a modular biochemical method that enables programmable, receptor-guided retargeting of adeno-associated viruses (AAVs) through site-specific covalent protein conjugation. By incorporating a SpyTag peptide motif into selected AAV capsid loops, MATCH allows one-step, stoichiometrically defined attachment of recombinant SpyCatcher-linked targeting proteins to the viral surface. Using mosaic AAV-DJ and AAV9 capsids with controlled SpyTag incorporation, we achieve efficient assembly and tunable ligand display. MATCH-AAVs conjugated to an anti-CD3 single-chain antibody efficiently activate and transduce resting human T cells within mixed PBMC populations in vitro, achieving transduction levels of up to [~]58% of total PBMCs. Conjugation to transferrin receptor (TfR1)-binding proteins yielded enhanced brain transduction in vivo, with murine TfR1-targeted MATCH-AAV9 exhibiting up to an 84-fold increase in brain expression relative to wild-type AAV9. Human TfR1-targeted vectors similarly enabled robust, receptor-dependent transduction both in vitro and in humanized mouse models. Both TfR-targeted vectors enabled widespread transduction of the parenchyma, consistent with TfR1-mediated crossing of the blood-brain barrier. Finally, we establish a streamlined one-pot "Mix-and-MATCH" production strategy in which capsid and targeting ligands are co-expressed during vector generation, yielding functional, targeted AAVs at titers comparable to conventional production. This simple and generalizable synthetic-biology approach provides a versatile toolkit for rational AAV tropism engineering, offering a scalable route to custom vector design for research and therapeutic applications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/715691v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@170a7c1org.highwire.dtl.DTLVardef@11599acorg.highwire.dtl.DTLVardef@11c0982org.highwire.dtl.DTLVardef@1b43973_HPS_FORMAT_FIGEXP M_FIG C_FIG

Novel strategies for treating bacterial infections are needed to combat the growing threat of antibiotic resistance. Here we sought to engineer and produce phage-like particles to either harness the microbiome to secrete therapeutics or to hijack pathogenic bacteria for treatment and prevention of disease. For this, we used the P2/P4 system to design, produce and test P4 phage-mediated single- and dual-action antimicrobial prototypes. Upon successful completion of the in vitro proof of concept experiments, we focused on optimizing early-stage bioprocessing for in vivo studies, leading to 1011 plaque forming units (PFU) per mL and 0.25 endotoxin units (EU) per 109 PFU. We also challenged the P4 viral vector packaging limit by deleting the sid gene to package the payload into P2-sized capsids ([~]25.8 kb cargo capacity). Importantly, repressing the therapeutic payload during the production of particles improved viral titers about 2 logs, maintained viral payload sequence integrity and improved post-transduction functional activity. Altogether, this study demonstrates the potential of novel phage-based antimicrobials to go beyond elimination of bacteria. The in vitro optimized P2/P4 system constitutes a promising platform technology for in vivo evaluations of targeted antimicrobial candidates paving the way for future antimicrobial research in animal models of infection.

Medulloblastoma (MB) is an aggressive central nervous system (CNS) malignancy that primarily affects children and frequently exhibits metastasis to the leptomeninges of the brain and spinal cord. We developed a {beta}-Cyclodextrin-poly({beta}-Amino Ester) nanoparticle system to deliver the histone deactylase inhibitor (HDACi) Panobinostat to MB by the intrathecal route. Various imaging methods were utilized to study nanoparticle and payload fate following infusion into the cerebrospinal fluid (CSF) of mice via cisterna magna or lumbar access points. Nanoparticles dramatically improved penetration of hydrophobic small molecules into distal regions of the spinal cord. Panobinostat-loaded nanoparticles were effective at treating patient-derived MB, activating pharmacodynamic targets, slowing growth of the primary tumor, decreasing incidence of metastasis at the time of death, and ultimately prolonging survival. These studies provide insight into the mechanisms mediating transport of colloids and therapeutic molecules in the subarachnoid space and highlight new approaches for treating metastatic disease in the CNS.

Pseudomonas aeruginosa is an adaptable organism, frequently found in chronic infections, and for which antimicrobial resistance is a growing concern. Therefore, there is an urgent need for alternative therapeutic strategies. Cationic antimicrobial peptides (AMPs) offer potent bactericidal activity but suffer from limited selectivity and potential host toxicity. To enhance species-specific targeting, we designed two prodrug variants of the AMP D-Bac8CLeu2,5 - EEEE-D-Bac8CLeu2,5 and ELEG-D-Bac8CLeu2,5 -- engineered for activation by the P. aeruginosa extracellular aminopeptidase PaAP. While both prodrug motifs effectively neutralized the positive charge of D-Bac8CLeu2,5 and prevented DNA-peptide complex formation, EEEE-D-Bac8CLeu2,5 showed negligible antimicrobial activity due to slow and incomplete activation. In contrast, ELEG-D-Bac8CLeu2,5 underwent rapid PaAP-mediated activation, restoring bactericidal activity in planktonic cultures and biofilms. PaAP contributed significantly to complete prodrug activation, particularly within biofilms, where the accumulation of partially activated intermediates correlated with biphasic killing kinetics. The prodrug showed reduced activity against other ESKAPEE pathogens, demonstrating selective activation by P. aeruginosa. Experiments selecting resistant bacteria revealed distinct mutations in lipopolysaccharide biosynthesis pathways for D-Bac8CLeu2,5 and the prodrug, with limited cross-resistance. These findings establish aminopeptidase-activated AMP prodrugs as a promising approach for species-selective antimicrobial therapy and highlight the feasibility of exploiting bacterial enzymes for controlled antimicrobial peptide activation. Table of contents graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/715093v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@4a5505org.highwire.dtl.DTLVardef@13e578org.highwire.dtl.DTLVardef@3e3080org.highwire.dtl.DTLVardef@e24266_HPS_FORMAT_FIGEXP M_FIG C_FIG

Chimeric antigen receptor T (CAR-T) cell therapy is transforming the treatment landscape of hematological malignancies. However, manufacturing with integrating viral vectors is costly, slow, and carries risks including insertional mutagenesis, pro-longed B cell aplasia, and other long-term toxicities. Expression of CAR with mRNA can reduce cost, manufacturing timelines, and improve safety. However, the short-lived expression necessitates frequent repeat dosing. Here, we describe a modified self-amplifying RNA (saRNA) platform for engineering CAR T cells with prolonged CAR expression and enhanced durability of tumor control relative to mRNA CAR T cells. In an acute lymphoblastic leukemia (ALL) xenograft model, saRNA CAR T cells achieve superior tumor suppression and prolong survival. Further, a single-strand modified saRNA supports the co-expression of multiple proteins, enabling the construction of advanced CAR systems, such as OR- and AND-gated logic CAR T cells. Together, these results highlight saRNA as a powerful and versatile platform for CAR T cell engi-neering with favorable safety, efficacy, and accessibility.

KRAS mutations drive multiple cancers and represent an important therapeutic target, together with other oncogenic regulators such as MYC, KIT, and BCL2 that are critically involved in pancreatic cancer. Here we describe a novel therapeutic strategy based on stable nucleolipid-modified G-quadruplexes (NLG4). Cell viability assays demonstrate that NLG4 strongly inhibit pancreatic cancer cell proliferation, whereas non-lipidic G-quadruplex sequences display minimal activity under comparable conditions. Owing to their distinctive physicochemical properties, including stabilization of parallel G-quadruplex structures and self-assembly into micellar aggregates, NLG4 efficiently internalize into cells and interact with key G-quadruplex unfolding factors such as UP1. This interaction leads to a marked downregulation of KRAS, c-MYC, c-KIT, and BCL2 expression. Suppression of these oncogenes profoundly affects pancreatic cancer cell fate, as evidenced by reduced expression of proliferation (Ki67) and anti-apoptotic (BCL2) markers. In addition, NLG4 treatment decreases inflammatory signaling mediated by NF-{kappa}B and inhibits major pro-proliferative kinase pathways, including ERK, AKT, and phosphorylated AKT. The therapeutic relevance of this decoy strategy is further supported by the observed potentiation of gemcitabine antitumor activity. Overall, these findings highlight NLG4 as a promising anticancer approach that simultaneously targets multiple oncogenic pathways through G-quadruplex-based decoy mechanisms, with translational potential for future pancreatic cancer treatment.

Systemic delivery of adeno-associated virus (AAV) for gene therapy of central nervous system (CNS) disorders is limited by inefficient blood-brain barrier (BBB) penetration and dose-limiting toxicity in peripheral organs, notably the liver and dorsal root ganglia (DRG)1-5. Here, we report the development of novel AAV variants via a proprietary capsid engineering platform (REACH). In non-human primates (NHPs), intravenous administration of lead variants resulted in transgene expression levels in the brain that were 600-2000 fold higher than AAV9 at the RNA level, concomitant with a 10-50 fold reduction in liver tropism and minimal off-target exposure in the heart and DRG. These engineered capsids achieve unprecedented, pan-CNS transduction with a markedly improved safety profile, representing a transformative platform for treating a broad spectrum of neurological diseases.

Pancreatic ductal adenocarcinoma (PDAC) remains difficult to treat with nucleic acid therapeutics because efficient intratumoral delivery is limited and off-target liver accumulation is common. Here, we developed a structure-activity map for intraperitoneally administered mRNA lipid nanoparticles (mRNA-LNPs) to identify formulation features that improve delivery to pancreatic tumors while reducing liver expression. A full-factorial library of 48 mRNA-LNP formulations was generated by varying ionizable lipid, sterol, phospholipid, and PEG-lipid components. Formulations were characterized for size, polydispersity, zeta potential, and encapsulation, then evaluated in an orthotopic KPC8060 pancreatic tumor model after intraperitoneal administration of firefly luciferase mRNA-loaded LNPs. Biodistribution was assessed by Rhodamine B fluorescence and functional delivery by luciferase expression 12 h after dosing. Lipid composition strongly influenced both physicochemical properties and in vivo performance. G0-C14-based formulations produced the smallest and most homogeneous particles, whereas FTT5-containing formulations were generally larger. Across the 48-formulation library, mRNA expression and nanoparticle biodistribution varied significantly among tumor, pancreas, liver, and spleen. Statistical, decision-tree, and predictive modeling analyses identified composition rules associated with organ-selective delivery. High tumor expression was associated primarily with G0-C14 combined with DSPC and {beta}-sitosterol, whereas liver expression was favored by C12-200 or DLin-MC3-DMA with DOPE and DSPE-PEG. Notably, a G0-C14/DSPC/DSPE-PEG formulation emerged as a lead candidate, producing a greater than 6-fold increase in tumor luciferase signal relative to the library median while reducing liver exposure by approximately 60%. Histopathology showed no treatment-related liver or lung toxicity. These findings define actionable formulation rules for tuning intraperitoneal mRNA-LNP delivery in PDAC and support further development of tumor-selective mRNA therapeutics for pancreatic cancer.

Transforming growth factor-beta (TGF-{beta}), a potent promoter of extracellular matrix deposition and suppressor of infiltrating immunity, has arisen as an attractive target for improving outcomes in tissue fibrosis and cancer immune therapy. Despite the promise of TGF-{beta} inhibitors for attenuating the progression of fibrotic disorders or as adjuncts for cancer immunotherapy, current systemically administered inhibitors that target the ligand or receptors have significant on-target liabilities, including cardiotoxicity and development of pre-malignant cutaneous squamous lesions. Recently, an engineered mini monomer of TGF-{beta} (mmTGF-{beta}), which potently and specifically inhibits TGF-{beta} activity, was shown to strongly synergize with checkpoint inhibitors to suppress cancer progression in an aggressive model of melanoma when genetically delivered using an engineered form of vaccinia virus that preferentially infects cancer cells. Despite these promising results, however, a significant fraction of the mmTGF-{beta} was found to misfold, likely due to mispairing of the cysteines that comprise its cystine knot. Here, we demonstrate that inclusion of a modified form of the TGF-{beta} pro-domain that lacks its dimerization motif, the bowtie knot, dramatically improves both the folding and inhibitory activity upon secretion by mammalian cells, thus overcoming one of the major limitations of genetically delivering mmTGF-{beta}. Furthermore, we show that fusion of mmTGF-{beta} to a CD44 binding domain enhances the inhibitory potential of mmTGF-{beta} on immune cells, and on other cell types which express CD44, by more than 30-fold compared to cells negative for CD44. Together, these modifications provide a framework for further enhancing the efficacy and safety of mmTGF-{beta} for cancer immune therapy, and possibly also tissue fibrosis, when delivered genetically using vaccinia, or other related approaches.

Genetic engineering experiments and therapies are constrained by the size of DNA integrations into human cells genomes. Existing AAV, lentiviral, and non-viral methods rapidly decrease in integration efficiency beyond [~]5kb of sequence. Through systematic evaluation of non-viral DNA template formats, we identified circular ssDNA and dsDNA as capable of mediating >5kb integrations. Large circular DNA delivery efficiency and its impacts on cell viability and payload expression could be significantly improved with small DNA "helper" plasmids, mRNA-encoded nucleases, and sequence design optimizations. Collectively, these modifications enabled ultra-large--up to 10 kb DNA--integrations at >20% efficiency in primary human T cells at the TRAC locus and at >60% efficiency in human iPSCs at the AAVS1 locus. Finally, we demonstrate that GMP clinical-manufactured T cells with ultra-large integrations are functional in vitro and in vivo. Overall, we identified optimal template architectures, delivery modes, and sequence design rules for ultra-large DNA integrations in both research and clinical settings to accelerate basic genetic research and next-generation cellular therapies.

Optogenetics enables researchers to control protein localization, interactions, and activity using photosensitive domains. The key desired properties for optogenetic tools include broad applicability, tight light-regulated control with high dynamic range, and tunability. Previously, we described an engineered light-sensitive switch, LightR, composed of two VVD domains connected by a flexible linker, enabling light-dependent allosteric control of protein activity through site-specific insertion. Here, we introduce enhanced LightR variants with improved dynamic range and faster activation kinetics. Through targeted modifications to the VVD domains and linker region, we optimized a LightR-regulated Src kinase (LightR-Src) activity and generated two LightR-Src variants: one supporting sustained Src activation comparable to constitutively active Src, and another enabling rapid, reversible control, ideal for modeling transient signaling events suitable to mimic Src signaling in living cells. These modifications expand the versatility of LightR-based tools, facilitating their use in diverse optogenetic applications requiring high dynamic range of regulation and fast control of targeted proteins.