Virus-free CRISPR knock-in of a chimeric antigen receptor into KLRC1 generates potent GD2-specific natural killer cells.

Natural killer (NK) cells are an appealing off-the-shelf, allogeneic cellular therapy due to their cytotoxic profile. However, their activity against solid tumors remains suboptimal in part due to the upregulation of NK-inhibitory ligands, such as HLA-E, within the tumor microenvironment. Here, we utilize CRISPR-Cas9 to disrupt the KLRC1 gene (encoding the HLA-E-binding NKG2A receptor) and perform non-viral insertion of a GD2-targeting chimeric antigen receptor (CAR) within NK cells isolated from human peripheral blood. Genome editing with CRISPR/Cas9 ribonucleoprotein complexes yields efficient genomic disruption of the KLRC1 gene with 98% knockout efficiency and specific knock-in of the GD2 CAR transgene as high as 23%, with minimal off-target activity as shown by CHANGE-Seq, in-out PCR, and next generation sequencing. KLRC1 -GD2 CAR NK cells display high viability and proliferation, as well as precise cellular targeting and potency against GD2 + human melanoma cells. Notably, KLRC1 -GD2 CAR NK cells overcome HLA-E-based inhibition by HLA-E-expressing, GD2 + melanoma cells. Using a single-step, virus-free genome editing workflow, this study demonstrates the feasibility of precisely disrupting inhibitory signaling within NK cells via CRISPR/Cas9 while expressing a CAR to generate potent allogeneic cell therapies against HLA-E + solid tumors.

Production and characterization of virus-free, CRISPR-CAR T cells capable of inducing solid tumor regression.

BACKGROUND: Chimeric antigen receptor (CAR) T cells have demonstrated high clinical response rates against hematological malignancies (e.g., CD19+ cancers) but have shown limited activity in patients with solid tumors. Recent work showed that precise insertion of a CAR at a defined locus improves treatment outcomes in the context of a CD19 CAR; however, it is unclear if such a strategy could also affect outcomes in solid tumors. Furthermore, CAR manufacturing generally relies on viral vectors for gene delivery, which comprise a complex and resource-intensive part of the manufacturing supply chain. METHODS: Anti-GD2 CAR T cells were generated using CRISPR/Cas9 within 9 days using recombinant Cas9 protein and nucleic acids, without any viral vectors. The CAR was specifically targeted to the T cell receptor alpha constant gene (TRAC). T cell products were characterized at the level of the genome, transcriptome, proteome, and secretome using CHANGE-seq, targeted next-generation sequencing, scRNA-seq, spectral cytometry, and ELISA assays, respectively. Functionality was evaluated in vivo in an NSG™ xenograft neuroblastoma model. RESULTS: In comparison to retroviral CAR T cells, virus-free CRISPR CAR (VFC-CAR) T cells exhibit TRAC-targeted genomic integration of the CAR transgene, elevation of transcriptional and protein characteristics associated with a memory-like phenotype, and low tonic signaling prior to infusion arising in part from the knockout of the T cell receptor. On exposure to the GD2 target antigen, anti-GD2 VFC-CAR T cells exhibit specific cytotoxicity against GD2+ cells in vitro and induce solid tumor regression in vivo. VFC-CAR T cells demonstrate robust homing and persistence and decreased exhaustion relative to retroviral CAR T cells against a human neuroblastoma xenograft model. CONCLUSIONS: This study leverages virus-free genome editing technology to generate CAR T cells featuring a TRAC-targeted CAR, which could inform manufacturing of CAR T cells to treat cancers, including solid tumors.

A Two-Step Bioreactor for Decellularized Lung Epithelialization.

Bioreactors for the reseeding of decellularized lung scaffolds have evolved with various advancements, including biomimetic mechanical stimulation, constant nutrient flow, multi-output monitoring, and large mammal scaling. Although dynamic bioreactors are not new to the field of lung bioengineering, ideal conditions during cell seeding have not been extensively studied or controlled. To address the lack of cell dispersal in traditional seeding methods, we have designed a two-step bioreactor. The first step is a novel system that rotates a seeded lung every 20 min at different angles in a sequence designed to anchor 20% of cells to a particular location based on the known rate of attachment. The second step involves perfusion-ventilation culture to ensure nutrient dispersion and cellular growth. Compared to statically seeded lungs, rotationally seeded lungs had significantly increased dsDNA content and more uniform cellular distribution after perfusion and ventilation had been administered. The addition of this novel seeding system before traditional culture methods will aid in recellularizing the lung and other geometrically complex organs for tissue engineering.

Genome engineering of induced pluripotent stem cells to manufacture natural killer cell therapies.

Natural killer (NK) cells play a crucial role in host immunity by detecting cells that downregulate MHC class I presentation and upregulate stress ligands, as commonly seen in cancers. Current NK therapies using primary NK cells are prone to manufacturing issues related to expansion and storage. Alternative cell sources utilizing immortalized NK cell lines require irradiation and are dependent on systemic IL-2 administration, which has been associated with adverse effects. In contrast, NK cells differentiated from induced pluripotent stem cells (iPSC-NK cells) offer an off-the-shelf alternative that may overcome these bottlenecks. The development of a serum-free and feeder-free differentiation protocol allows for the manufacturing of clinically adaptable iPSC-NK cells that are equally as effective as primary NK cells and the NK-92 cell line for many indications. Moreover, genetic modifications targeting NK-mediated antibody-dependent cellular cytotoxicity capabilities, cytotoxicity, and checkpoint inhibitors may increase the therapeutic potential of iPSC-NK products. This review will highlight the current sources for NK therapies and their respective constraints, discuss recent developments in the manufacturing and genetic engineering of iPSC-NK cells, and provide an overview of ongoing clinical trials using NK cells.

Porcine Lung-Derived Extracellular Matrix Hydrogel Properties Are Dependent on Pepsin Digestion Time.

Hydrogels derived from decellularized lungs are promising materials for tissue engineering in the development of clinical therapies and for modeling the lung extracellular matrix (ECM) in vitro. Characterizing and controlling the resulting physical, biochemical, mechanical, and biologic properties of decellularized ECM (dECM) after enzymatic solubilization and gelation are thus of key interest. As the role of enzymatic pepsin digestion in effecting these properties has been understudied, we investigated the digestion time-dependency on key parameters of the resulting ECM hydrogel. Using resolubilized, homogenized decellularized pig lung dECM as a model system, significant time-dependent changes in protein concentration, turbidity, and gelation potential were found to occur between the 4 and 24 h digestion time points, and plateauing with longer digestion times. These results correlated with qualitative scanning electron microscopy images and quantitative analysis of hydrogel interconnectivity and average fiber diameter. Interestingly, the time-dependent changes in the storage modulus tracked with the hydrogel interconnectivity results, while the Young's modulus values were more closely related to average fiber size at each time point. The structural and biochemical alterations correlated with significant changes in metabolic activity of several representative lung cells seeded onto the hydrogels with progressive decreases in cell viability and alterations in morphology observed in cells cultured on hydrogels produced with dECM digested for >12 and up to 72 h of digestion. These studies demonstrate that 12 h pepsin digest of pig lung dECM provides an optimal balance between desirable physical ECM hydrogel properties and effects on lung cell behaviors.

Laminin-driven Epac/Rap1 regulation of epithelial barriers on decellularized matrix.

Decellularized tissues offer a unique tool for developing regenerative biomaterials or in vitro platforms for the study of cell-extracellular matrix (ECM) interactions. One main challenge associated with decellularized lung tissue is that ECM components can be stripped away or altered by the detergents used to remove cellular debris. Without characterizing the composition of lung decellularized ECM (dECM) and the cellular response caused by the altered composition, it is difficult to utilize dECM for regeneration and specifically, engineering the complexities of the alveolar-capillary barrier. This study takes steps towards uncovering if dECM must be enhanced with lost ECM proteins to achieve proper epithelial barrier formation. To achieve this, the epithelial barrier function was assessed on dECM coatings with and without the systematic addition of several key basement membrane proteins. After comparing barrier function on collagen I, fibronectin, laminin, and dECM in varying combinations as an in vitro coating, the alveolar epithelium exhibited superior barrier function when dECM was supplemented with laminin as evidenced by trans-epithelial electrical resistance (TEER) and permeability assays. Increased barrier resistance with laminin addition was associated with upregulation of Claudin-18, E-cadherin, and junction adhesion molecule (JAM)-A, and stabilization of zonula occludens (ZO)-1 at junction complexes. The Epac/Rap1 pathway was observed to play a role in the ECM-mediated barrier function determined by protein expression and Epac inhibition. These findings revealed potential ECM coatings and molecular therapeutic targets for improved regeneration with decellularized scaffolds. STATEMENT OF SIGNIFICANCE: Efforts to produce a transplantable organ-scale biomaterial for lung regeneration has not been entirely successful to date, due to incomplete cell-cell junction formation, ultimately leading to severe edema in vivo. To fully understand the process of alveolar junction formation on ECM-derived biomaterials, this research has characterized and tailored decellularized ECM (dECM) to mitigate reductions in barrier strength or cell attachment caused by abnormal ECM compositions or detergent damage to dECM. These results indicate that laminin-driven Epac signaling plays a vital role in the stabilization of the alveolar barrier. Addition of laminin or Epac agonists during alveolar regeneration can reduce epithelial permeability within bioengineered lungs.

Electrospun Decellularized Lung Matrix Scaffold for Airway Smooth Muscle Culture.

Chronic respiratory disease affects many people worldwide with little known about the intricate mechanisms driving the pathology, making it difficult to develop novel therapies. Improving the understanding of airway smooth muscle and extracellular matrix (ECM) interactions is key to developing treatments for this leading cause of death. With currently no relevant or controllable in vivo or in vitro models to investigate cell-ECM interactions in the small airways, the development of a biomimetic in vitro model with cell attachment, signaling, and organization is needed. The goal of this study was to create a biologically and structurally relevant in vitro model of small airway smooth muscle. In order to achieve this goal, a scaffold was engineered from synthetic poly-l-lactic acid (PLLA) and decellularized pig lung ECM (PLECM). PLECM scaffolds have improved physical characteristics over synthetic scaffolds, by exhibiting a significant decrease in the elastic modulus and an increase in hydrophilicity. Histological staining and SDS-PAGE showed that essential proteins or protein fragments found in natural ECM were present after processing. Human bronchial smooth muscle cells (HBSMCs) seeded onto PLECM 3D scaffolds formed confluent layers and maintained a contractile phenotype, as demonstrated by the organized arrangement of actin filaments within the cell and expected contractile protein expression of calponin 1. HBSMCs cultured on electrospun PLECM scaffold also increased alpha-1 type 1 collagen compared to those cultured on PLLA scaffolds. In summary, this research demonstrates that a PLLA/PLECM composite electrospun mat is a promising tool to produce an in vitro model of the airway with the potential for a better understanding of bronchiole smooth muscle behavior in diseased or normal states.