Genome editing of allogeneic T cells can provide “off-the-shelf” alternatives to autologous chimeric antigen receptor (CAR) T cell therapies. Disruption of T cell receptor α chain (TRAC) to prevent graft-versus-host disease (GVHD) and removal of CD52 (cluster of differentiation 52) for a survival advantage in the presence of alemtuzumab have previously been investigated using transcription activator–like effector nuclease (TALEN)-mediated knockout. Here, we deployed next-generation CRISPR-Cas9 editing and linked CAR expression to multiplexed DNA editing of TRAC and CD52 through incorporation of self-duplicating CRISPR guide RNA expression cassettes within the 3’ long terminal repeat of a CAR19 lentiviral vector. Three cell banks of TT52CAR19 T cells were generated and cryopreserved. A phase 1, open-label, non-randomized clinical trial was conducted and treated six children with relapsed/refractory CD19-positive B cell acute lymphoblastic leukemia (B-ALL) (NCT04557436). Lymphodepletion included fludarabine, cyclophosphamide, and alemtuzumab and was followed by a single infusion of 0.8 × 10 ⁶ to 2.0 × 10 ⁶ CAR19 T cells per kilogram with no immediate toxicities. Four of six patients infused with TT52CAR19 T cells exhibited cell expansion, achieved flow cytometric remission, and then proceeded to receive allogeneic stem cell transplantation. Two patients required biological intervention for grade II cytokine release syndrome, one patient developed transient grade IV neurotoxicity, and one patient developed skin GVHD, which resolved after transplant conditioning. Other complications were within expectations, and primary safety objectives were met. This study provides a demonstration of the feasibility, safety, and therapeutic potential of CRISPR-engineered immunotherapy.

We developed an allogeneic matched donor CD19 chimeric antigen receptor (CAR) product (CAR–donor lymphocyte infusion [DLI]) for adult patients with B-cell acute lymphoblastic leukemia (B-ALL) after failure of allogeneic stem-cell transplantation (allo-SCT). We evaluate the risks and benefits of pre–CAR-DLI lymphodepleting chemotherapy (LD) and the efficacy of repeat CAR-DLI dosing per conventional DLI protocols. Primary outcomes were toxicity and feasibility of CAR-DLI manufacture; secondary outcomes included CAR-DLI engraftment, expansion, and persistence. A total of 17 allo-SCT donors received leukapheresis and 14 patients with B-ALL (median age, 43 years) received infusion. Median disease burden at registration was 50.5% bone marrow blasts (range, measurable residual disease [MRD] to 100%). Patients 1 to 7 received CAR-DLI alone (CAR-DLI-alone); patients 8 through 14 received CAR-DLI and LD with fludarabine/cyclophosphamide (CAR-DLI+LD). CAR-DLI+LD vs CAR-DLI-alone was associated with superior peak CAR-DLI engraftment (93 134 vs 8010 copies per μg genomic DNA [gDNA]), expansion (858 101 vs 39 038 copies per μg gDNA per 28 days) and persistence (median, 197 vs 32 days). CAR-DLI+LD was not associated with more immunotoxicity than CAR-DLI-alone, and graft-versus-host disease (GVHD; grade 1, skin) affected only 2 of 14 patients (14%). CAR-DLI+LD vs CAR-DLI-alone conferred superior event-free-survival and overall survival at 12 months (57% vs 29%; 83% vs 29%). Repeat CAR-DLI dosing was administered to 8 of 14 (57%) patients with morphological/MRD+ relapse, but with minimal engraftment/expansion or toxicity/efficacy. CAR-DLI+LD has a tolerable safety profile without significant GVHD and is associated with significantly better outcomes than CAR-DLI-alone. Repeat CAR-DLI dosing beyond dose 1 was not found to be effective in this analysis. This trial was registered at www.clinicaltrials.gov as #NCT02893189.

The present study emphasizes the stabilization of the biologically active anatase form of titanium dioxide (TiO ₂ ) nanoparticles (NP). TiO ₂ NP require stringent conditions for chemical synthesis and are usually a mixture of biologically inactive bulk rutile and the active bulk anatase forms. We utilized the culture pellet of the Exiguobacterium aestuarii SBG4 MH185868 to synthesize and stabilize the anatase form of TiO ₂ NP. The NP showed λ _max at ∼350 nm and scanning electron microscope micrographs indicated their oval and spherical shape. Steric stabilized anatase TiO ₂ NP exhibited substantial cytotoxicity of up to 80% reduction in cell viability at 100 µg against cervical cancer derived HeLa and SiHa cell lines, whereas the rutile form showed least cytotoxicity. Clonogenic inhibition assay of HeLa cells showed dose-dependent decline with a 75% reduction in colony formation at 100 µg TiO ₂ NP and cell migration assay revealed significant inhibition in recovery of the wound/scratch in presence of anatase form of TiO ₂ NP (10–33% at 24 h and 42–79% at 48 h). Co-incubation of HeLa cells with anatase TiO ₂ NP in chorioallantoic membrane of embryonated chick eggs prevented the formation of new capillaries (20 ± 5% compared with control groups), indicating appreciable anti-angiogenic activity of the NP. Further, TiO ₂ NP tagged with doxorubicin and paclitaxel exhibited enhanced cytotoxicity against cancer cells at very low concentrations of 9 and 120 nM itself, indicating their anti-tumor potential. In conclusion, biomimetic anatase TiO ₂ NP have significant anti-proliferative and anti-angiogenic activity and can have potential application in tagging with generic anti-cancer drugs for enhanced cytotoxicity against cancer cells.

Chimeric Antigen Receptor T (CAR T) cell therapy targeting the CD19 antigen represents a significant advancement in the treatment of various subtypes of CD19-expressing hematologic malignancies. Nevertheless, the current commercial CAR T manufacturing process is intricate, time-consuming, and costly, involving a supply chain that initiates at the treating center with mononuclear cell apheresis, followed by cryopreservation and shipping to and from a centralized third-party manufacturing facility. This process can introduce treatment delays due to logistical requirements. We hypothesized that point-of-care (POC) manufacturing of CAR T cells could reduce vein to vein time which is one of the major challenges with commercial CAR T processes. This study presents the outcomes of large-scale, clinical-grade, in-house POC CAR T manufacturing, including assessments of transduction efficiency, vector copy number, phenotype, and effector function of CAR T cells. Utilizing the CliniMACS Prodigy system, we processed mononuclear cells from fresh leukapheresis harvests of healthy donors. The cells underwent automated processing and activation with TransAct, a polymeric nanomatrix activation reagent containing CD3/CD28-specific antibodies. Following activation, the cells were transduced and expanded in the Centricult-unit under stabilized conditions, with automated feeding and media exchange. For transduction, we employed the Miltenyi Lentigen third-generation lentiviral vector, which delivers a second-generation CD19 CAR construct with a 4-1BB costimulatory domain and TNFRSF19 transmembrane domain. Our POC manufacturing data demonstrated the successful generation of large-scale CAR T cells, sufficient to prepare multiple doses within a shortened production period of 9 days, followed by cryopreservation for quality control assessment and batch certification for patient infusion. The transduction efficiencies, phenotype, and function of the Lentigen CD19 CAR T cells were comparable to previously published procedures, with overall T-cell yields sufficient for therapeutic application. CAR expression was evaluated by flow cytometry and vector copy number. In-process testing ensured the quality and suitability of CAR T cells in terms of sterility and cell number for the desired dose preparations. The final product was also subjected for the assessment of any replicative competent lentivirus. CAR-mediated target cell killing was assessed via co-culture assays, and cytokine release was measured by ELISA. To evaluate the shelf life of the cryopreserved product, stability and viability were checked at approximately 6 and 12 months. Our results indicate that in-house POC manufacturing at King Faisal Specialist Hospital & Research Centre (KFSH&RC) enables regulatory-compliant production of CAR T cells, eliminating the need for third-party shipping logistics.

Ionizable lipid nanoparticles (LNPs) are widely used for delivery of CRISPR/Cas9 payloads to hepatocytes, but conventional hepatic uptake is strongly influenced by adsorption of apolipoprotein E and subsequent low-density lipoprotein receptor (LDLR)-mediated internalization. This dependence may limit specificity and reduce efficacy in LDLR-deficient settings. Here, we designed an aptamer-functionalized LNP platform to enable hepatocyte-selective genome editing through an LDLR-independent route and validated its performance using a genetically defined LDLR-knockout HepG2 model. Ionizable LNPs co-encapsulating Cas9 mRNA and an LDLR-targeting guide RNA were surface-decorated with the hepatocellular carcinoma-targeting TLS11a aptamer using thiol-maleimide chemistry. Comprehensive physicochemical analysis using cryo-electron microscopy, dynamic light scattering, pKa titration, UV and circular dichroism spectroscopy, X-ray photoelectron spectroscopy, and molecular beacon assays confirmed uniform nanoparticles of approximately 105 nm, preserved mRNA integrity, retained endosomal charge-switching behavior with a pKa of approximately 6.3 to 6.5, and maintained correctly folded surface-displayed TLS11a. TLS11a decoration increased Cas9 mRNA delivery to HepG2 cells from 39% to 79% Cy5-positive cells, while reducing uptake in receptor-low control cells, supporting aptamer-associated and cell-preferential delivery. In parallel, CRISPR/Cas9-mediated deletion of LDLR exon 2 generated a validated LDLR-deficient HepG2 line, confirmed at genomic, transcript, and protein levels. LDLR loss reduced LDL binding and uptake by approximately 85%, while transferrin uptake was preserved, indicating selective impairment of LDLR-dependent endocytosis. Cholesterol depletion activated the SCAP-SREBP-2 pathway and induced cholesterol biosynthesis genes. Together, these findings establish a modular aptamer-guided LNP system for targeted genome-editing delivery and a validated LDLR-null hepatocyte model for studying LDLR-dependent biology and disease.