CRISPR-mediated TGFBR2 knockout renders human ovarian cancer tumor-infiltrating lymphocytes resistant to TGF-ß signaling.

BACKGROUND: The correlation between elevated T-cell infiltration and improved survival of ovarian cancer (OvCa) patients suggests that endogenous tumor-infiltrating lymphocytes (TIL) possess some degree of antitumor activity that can be harnessed for OvCa immunotherapy. We previously optimized a protocol for ex vivo OvCa TIL expansion for adoptive cell therapy, which is now being tested in a clinical trial at our institution (NCT03610490). Building on this success, we embarked on genetic modification of OvCa TIL to overcome key immunosuppressive factors present in the tumor microenvironment. Here, we present the preclinical optimization of CRISPR/Cas9-mediated knockout of the TGF-ß receptor 2 (TGFBR2) in patient-derived OvCa TIL. METHODS: OvCa TILs were generated from four patients' tumor samples obtained at surgical resection and subjected to CRISPR/Cas9-mediated knockout of TGFBR2 before undergoing a rapid expansion protocol. TGFBR2-directed gRNAs were comprehensively evaluated for their TGFBR2 knockout efficiency and off-target activity. Furthermore, the impact of TGFBR2 knockout on TIL expansion, function, and downstream signaling was assayed. RESULTS: TGFBR2 knockout efficiencies ranging from 59±6% to 100%±0% were achieved using 5 gRNAs tested in four independent OvCa TIL samples. TGFBR2 knockout TIL were resistant to immunosuppressive TGF-ß signaling as evidenced by a lack of SMAD phosphorylation, a lack of global transcriptional changes in response to TGF-ß stimulation, equally strong secretion of proinflammatory cytokines in the presence and absence of TGF-ß, and improved cytotoxicity in the presence of TGF-ß. CRISPR-modification itself did not alter the ex vivo expansion efficiency, immunophenotype, nor the TCR clonal diversity of OvCa TIL. Importantly for clinical translation, comprehensive analysis of CRISPR off-target effects revealed no evidence of off-target activity for our top two TGFBR2-targeting gRNAs. CONCLUSIONS: CRISPR/Cas9-mediated gene knockout is feasible and efficient in patient-derived OvCa TIL using clinically-scalable methods. We achieved efficient and specific TGFBR2 knockout, yielding an expanded OvCa TIL product that was resistant to the immunosuppressive effects of TGF-ß. This study lays the groundwork for clinical translation of CRISPR-modified TIL, providing opportunities for engineering more potent TIL therapies not only for OvCa treatment, but for the treatment of other solid cancers as well.

Applications of CRISPR Genome Editing to Advance the Next Generation of Adoptive Cell Therapies for Cancer.

Adoptive cell therapy (ACT) for cancer shows tremendous potential; however, several challenges preclude its widespread use. These include poor T-cell function in hostile tumor microenvironments, a lack of tumor-specific target antigens, and the high cost and poor scalability of cell therapy manufacturing. Creative genome-editing strategies are beginning to emerge to address each of these limitations, which has initiated the next generation of cell therapy products now entering clinical trials. CRISPR is at the forefront of this revolution, offering a simple and versatile platform for genetic engineering. This review provides a comprehensive overview of CRISPR applications that have advanced ACT. SIGNIFICANCE: The clinical impact of ACT for cancer can be expanded by implementing specific genetic modifications that enhance the potency, safety, and scalability of cellular products. Here we provide a detailed description of such genetic modifications, highlighting avenues to enhance the therapeutic efficacy and accessibility of ACT for cancer. Furthermore, we review high-throughput CRISPR genetic screens that have unveiled novel targets for cell therapy enhancement.

Ultrasound-Stimulated Phase-Change Contrast Agents for Transepithelial Delivery of Macromolecules, Toward Gastrointestinal Drug Delivery.

The gastrointestinal (GI) tract presents a notoriously difficult barrier for macromolecular drug delivery, especially for biologics. Herein, we demonstrate that ultrasound-stimulated phase change contrast agents (PCCAs) can transiently disrupt confluent colorectal adenocarcinoma monolayers and improve the transepithelial transport of a macromolecular model drug. With ultrasound treatment in the presence of PCCAs, we achieved a maximum of 44 ± 15% transepithelial delivery of 70-kDa fluorescein isothiocyanate-dextran, compared with negligible delivery through sham control monolayers. Among all tested rarefactional pressures (300-600 kPa), dextran delivery efficiency was consistently greatest at 300 kPa. To explore this unexpected finding, we quantified stable and inertial cavitation energy generated by various ultrasound exposure conditions. In general, lower pressures resulted in more persistent cavitation activity during the 30-s ultrasound exposures, which may explain the enhanced dextran delivery efficiency. Thus, a unique advantage of using low boiling point PCCAs for this application is that the same low-pressure pulses can be used to induce vaporization and provide maximal delivery.

Oxygen microbubbles improve radiotherapy tumor control in a rat fibrosarcoma model - A preliminary study.

Cancer affects 39.6% of Americans at some point during their lifetime. Solid tumor microenvironments are characterized by a disorganized, leaky vasculature that promotes regions of low oxygenation (hypoxia). Tumor hypoxia is a key predictor of poor treatment outcome for all radiotherapy (RT), chemotherapy and surgery procedures, and is a hallmark of metastatic potential. In particular, the radiation therapy dose needed to achieve the same tumor control probability in hypoxic tissue as in normoxic tissue can be up to 3 times higher. Even very small tumors (<2-3 mm3) comprise 10-30% of hypoxic regions in the form of chronic and/or transient hypoxia fluctuating over the course of seconds to days. We investigate the potential of recently developed lipid-stabilized oxygen microbubbles (OMBs) to improve the therapeutic ratio of RT. OMBs, but not nitrogen microbubbles (NMBs), are shown to significantly increase dissolved oxygen content when added to water in vitro and increase tumor oxygen levels in vivo in a rat fibrosarcoma model. Tumor control is significantly improved with OMB but not NMB intra-tumoral injections immediately prior to RT treatment and effect size is shown to depend on initial tumor volume on RT treatment day, as expected.

An evaluation of the sonoporation potential of low-boiling point phase-change ultrasound contrast agents in vitro.

BACKGROUND: Phase-change ultrasound contrast agents (PCCAs) offer a solution to the inherent limitations associated with using microbubbles for sonoporation; they are characterized by prolonged circulation lifetimes, and their nanometer-scale sizes may allow for passive accumulation in solid tumors. As a first step towards the goal of extravascular cell permeabilization, we aim to characterize the sonoporation potential of a low-boiling point formulation of PCCAs in vitro. METHODS: Parameters to induce acoustic droplet vaporization and subsequent microbubble cavitation were optimized in vitro using high-speed optical microscopy. Sonoporation of pancreatic cancer cells in suspension was then characterized at a range of pressures (125-600 kPa) and pulse lengths (5-50 cycles) using propidium iodide as an indicator molecule. RESULTS: We achieved sonoporation efficiencies ranging from 8 ± 1% to 36 ± 4% (percent of viable cells), as evidenced by flow cytometry. Increasing sonoporation efficiency trended with increasing pulse length and peak negative pressure. CONCLUSIONS: We conclude that PCCAs can be used to induce the sonoporation of cells in vitro, and our results warrant further investigation into the use of PCCAs as extravascular sonoporation agents in vivo.

Therapeutic gas delivery via microbubbles and liposomes.

Gaseous molecules including nitric oxide, hydrogen sulfide, carbon monoxide and oxygen mediate numerous cell signaling pathways and have important physiological roles. Several noble gasses have been shown to elicit biological responses. These bioactive gasses hold great therapeutic potential, however, their controlled delivery remains a significant challenge. Recently, researchers have begun using microbubbles and liposomes to encapsulate such gasses for parenteral delivery. The resultant particles are acoustically active, and ultrasound can be used to stimulate and/or image gas release in a targeted region. This review provides a summary of recent advances in therapeutic gas delivery using microbubbles and liposomes.