CRISPR/Cas9 deletion of MIR155HG in human T cells reduces incidence and severity of acute GVHD in a xenogeneic model.

ABSTRACT: Acute graft-versus-host disease (GVHD) is a major complication of allogeneic hematopoietic cell transplantation (allo-HCT). Using preclinical mouse models of disease, previous work in our laboratory has linked microRNA-155 (miR-155) to the development of acute GVHD. Transplantation of donor T cells from miR-155 host gene (MIR155HG) knockout mice prevented acute GVHD in multiple murine models of disease while maintaining critical graft-versus-leukemia (GVL) response, necessary for relapse prevention. In this study, we used clustered, regularly interspaced, short palindromic repeats (CRISPR)/Cas9 genome editing to delete miR-155 in primary T cells (MIR155HGΔexon3) from human donors, resulting in stable and sustained reduction in expression of miR-155. Using the xenogeneic model of acute GVHD, we show that NOD/SCID/IL2rγnull (NSG) mice receiving MIR155HGΔexon3 human T cells provide protection from lethal acute GVHD compared with mice that received human T cells with intact miR-155. MIR155HGΔexon3 human T cells persist in the recipients displaying decreased proliferation potential, reduced pathogenic T helper-1 cell population, and infiltration into GVHD target organs, such as the liver and skin. Importantly, MIR155HGΔexon3 human T cells retain GVL response significantly improving survival in an in vivo model of xeno-GVL. Altogether, we show that CRISPR/Cas9-mediated deletion of MIR155HG in primary human donor T cells is an innovative approach to generate allogeneic donor T cells that provide protection from lethal GVHD while maintaining robust antileukemic response.

Knockout of the inhibitory receptor TIGIT enhances the antitumor response of ex vivo expanded NK cells and prevents fratricide with therapeutic Fc-active TIGIT antibodies.

BACKGROUND: Inhibitory receptor T-cell Immunoreceptor with Ig and ITIM domains (TIGIT) expressed by Natural Killer (NK) and T cells regulates cancer immunity and has been touted as the next frontier in the development of cancer immunotherapeutics. Although early results of anti-TIGIT and its combinations with antiprogrammed death-ligand 1 were highly exciting, results from an interim analysis of phase III trials are disappointing. With mixed results, there is a need to understand the effects of therapeutic anti-TIGIT on the TIGIT+ immune cells to support its clinical use. Most of the TIGIT antibodies in development have an Fc-active domain, which binds to Fc receptors on effector cells. In mouse models, Fc-active anti-TIGIT induced superior immunity, while Fc receptor engagement was required for its efficacy. NK-cell depletion compromised the antitumor immunity of anti-TIGIT indicating the essential role of NK cells in the efficacy of anti-TIGIT. Since NK cells express TIGIT and Fc-receptor CD16, Fc-active anti-TIGIT may deplete NK cells via fratricide, which has not been studied. METHODS: CRISPR-Cas9-based TIGIT knockout (KO) was performed in expanded NK cells. Phenotypic and transcriptomic properties of TIGIT KO and wild-type (WT) NK cells were compared with flow cytometry, CyTOF, and RNA sequencing. The effect of TIGIT KO on NK-cell cytotoxicity was determined by calcein-AM release and live cell imaging-based cytotoxicity assays. The metabolic properties of TIGIT KO and WT NK cells were compared with a Seahorse analyzer. The effect of the Fc-component of anti-TIGIT on NK-cell fratricide was determined by co-culturing WT and TIGIT KO NK cells with Fc-active and Fc-inactive anti-TIGIT. RESULTS: TIGIT KO increased the cytotoxicity of NK cells against multiple cancer cell lines including spheroids. TIGIT KO NK cells upregulated mTOR complex 1 (mTORC1) signaling and had better metabolic fitness with an increased basal glycolytic rate when co-cultured with cancer cells compared with WT NK cells. Importantly, TIGIT KO prevented NK-cell fratricide when combined with Fc-active anti-TIGIT. CONCLUSIONS: TIGIT KO in ex vivo expanded NK cells increased their cytotoxicity and metabolic fitness and prevented NK-cell fratricide when combined with Fc-active anti-TIGIT antibodies. These fratricide-resistant TIGIT KO NK cells have therapeutic potential alone or in combination with Fc-active anti-TIGIT antibodies to enhance their efficacy.

Deletion of Glycogen Synthase Kinase 3 Beta Reprograms NK Cell Metabolism.

Loss of cytotoxicity and defective metabolism are linked to glycogen synthase kinase 3 beta (GSK3ß) overexpression in natural killer (NK) cells from patients with acute myeloid leukemia or from healthy donors after expansion ex vivo with IL-15. Drug inhibition of GSK3ß in these NK cells improves their maturation and cytotoxic activity, but the mechanisms of GSK3ß-mediated dysfunction have not been well studied. Here, we show that expansion of NK cells with feeder cells expressing membrane-bound IL-21 maintained normal GSK3ß levels, allowing us to study GSK3ß function using CRISPR gene editing. We deleted GSK3B and expanded paired-donor knockout and wild-type (WT) NK cells and then assessed transcriptional and functional alterations induced by loss of GSK3ß. Surprisingly, our data showed that deletion of GSK3B did not alter cytotoxicity, cytokine production, or maturation (as determined by CD57 expression). However, GSK3B-KO cells demonstrated significant changes in expression of genes related to rRNA processing, cell proliferation, and metabolic function, suggesting possible metabolic reprogramming. Next, we found that key genes downregulated in GSK3B-KO NK cells were upregulated in GSK3ß-overexpressing NK cells from AML patients, confirming this correlation in a clinical setting. Lastly, we measured cellular energetics and observed that GSK3B-KO NK cells exhibited 150% higher spare respiratory capacity, a marker of metabolic fitness. These findings suggest a role for GSK3ß in regulating NK cell metabolism.

Optimization and validation of CAR transduction into human primary NK cells using CRISPR and AAV.

Human primary natural killer (NK) cells are being widely advanced for cancer immunotherapy. However, methods for gene editing of these cells have suffered low transduction rates, high cell death, and loss of transgene expression after expansion. Here, we developed a highly efficient method for site-specific gene insertion in NK cells using CRISPR (Cas9/RNP) and AAVs. We compared AAV vectors designed to mediate gene insertion by different DNA repair mechanisms, homology arm lengths, and virus concentrations. We then validated the method for site-directed gene insertion of CD33-specific CARs into primary human NK cells. CAR transduction was efficient, its expression remained stable after expansion, and it improved efficacy against AML targets.

CRISPR Gene Editing of Human Primary NK and T Cells for Cancer Immunotherapy.

Antitumor activity of immune cells such as T cells and NK cells has made them auspicious therapeutic regimens for adaptive cancer immunotherapy. Enhancing their cytotoxic effects against malignancies and overcoming their suppression in tumor microenvironment (TME) may improve their efficacy to treat cancers. Clustered, regularly interspaced short palindromic repeats (CRISPR) genome editing has become one of the most popular tools to enhance immune cell antitumor activity. In this review we highlight applications and practicability of CRISPR/Cas9 gene editing and engineering strategies for cancer immunotherapy. In addition, we have reviewed several approaches to study CRISPR off-target effects.

Assessment of Beta-2 Microglobulin Gene Edited Airway Epithelial Stem Cells as a treatment for Sulfur Mustard Inhalation.

Respiratory system damage is the primary cause of mortality in individuals who are exposed to vesicating agents including sulfur mustard (SM). Despite these devastating health complications, there are no fielded therapeutics that are specific for such injuries. Previous studies reported that SM inhalation depleted the tracheobronchial airway epithelial stem cell (TSC) pool and supported the hypothesis, TSC replacement will restore airway epithelial integrity and improve health outcomes for SM-exposed individuals. TSC express Major Histocompatibility Complex (MHC-I) transplantation antigens which increases the chance that allogeneic TSC will be rejected by the patient's immune system. However, previous studies reported that Beta-2 microglobulin (B2M) knockout cells lacked cell surface MHC-I and suggested that B2M knockout TSC would be tolerated as an allogeneic graft. This study used a Cas9 ribonucleoprotein (RNP) to generate B2M-knockout TSC, which are termed Universal Donor Stem Cells (UDSC). Whole genome sequencing identified few off-target modifications and demonstrated the specificity of the RNP approach. Functional assays demonstrated that UDSC retained their ability to self-renew and undergo multilineage differentiation. A preclinical model of SM inhalation was used to test UDSC efficacy and identify any treatment-associated adverse events. Adult male Sprague-Dawley rats were administered an inhaled dose of 0.8 mg/kg SM vapor which is the inhaled LD50 on day 28 post-challenge. On recovery day 2, vehicle or allogeneic Fisher rat UDSC were delivered intravenously (n = 30/group). Clinical parameters were recorded daily, and planned euthanasia occurred on post-challenge days 7, 14, and 28. The vehicle and UDSC treatment groups exhibited similar outcomes including survival and a lack of adverse events. These studies establish a baseline which can be used to further develop UDSC as a treatment for SM-induced airway disease.

Effects of structured home-based exercise training on circulating endothelial progenitor cells and endothelial function in patients with intermittent claudication.

INTRODUCTION: Exercise training improves walking capacity in patients with intermittent claudication (IC). Endothelial progenitor cells (EPCs), endothelial microparticles (EMPs), and endothelial dysfunction could play a role in this process. METHODS: We measured EPCs and EMPs in a group of 60 patients with IC, and in a control group of 20 individuals without IC, before a treadmill test and 2, 24, and 48 hours after the test. Thirty patients with IC were randomly assigned to perform a 12-week home-based exercise training program. The EPC count, flow-mediated dilation (FMD) of the brachial artery, pain-free walking time (PFWT), and maximum walking time (MWT) were measured at the baseline and after the exercise training program. RESULTS: In patients with IC, EMPs significantly increased 2 hours after the treadmill test, whereas EPCs significantly increased after 24 hours. Among the subjects assigned to complete the training program, we observed a significant increase in the number of EPCs after 12 weeks, as well as an improvement in FMD, PFWT, and MWT. A significant correlation between the variation of EPCs, FMD, and MWT was found. The increase of EPCs and FMD were independent determinants of the walking capacity improvement, without significant interaction. CONCLUSION: Our results suggest that EPCs mobilization contributes to the improvement of walking capacity in patients with IC undergoing structured physical training. A number of different, partly independent, mechanisms are involved in this process, and our results highlight the potential role of EMPs release and endothelial function improvement. ClinicalTrials.gov Identifier: NCT04302571.

Genetic and epigenetic modification of human primary NK cells for enhanced antitumor activity.

Cancer immunotherapy using genetically modified immune cells such as those expressing chimeric antigen receptors has shown dramatic outcomes in patients with refractory and relapsed malignancies. Natural killer (NK) cells as a member of the innate immune system, possessing both anticancer (cytotoxic) and proinflammatory (cytokine) responses to cancers and rare off-target toxicities have great potential for a wide range of cancer therapeutic settings. Therefore, improving NK cell antitumor activity through genetic modification is of high interest in the field of cancer immunotherapy. However, gene manipulation in primary NK cells has been challenging because of broad resistance to many genetic modification methods that work well in T cells. Here we review recent successful approaches for genetic and epigenetic modification of NK cells including epigenetic remodeling, transposons, mRNA-mediated gene delivery, lentiviruses, and CRISPR gene targeting.

CD38 deletion of human primary NK cells eliminates daratumumab-induced fratricide and boosts their effector activity.

Multiple myeloma (MM) is a plasma cell neoplasm that commonly expresses CD38. Daratumumab (DARA), a human monoclonal antibody targeting CD38, has significantly improved the outcome of patients with relapsed or refractory MM, but the response is transient in most cases. Putative mechanisms of suboptimal efficacy of DARA include downregulation of CD38 expression and overexpression of complement inhibitory proteins on MM target cells as well as DARA-induced depletion of CD38high natural killer (NK) cells resulting in crippled antibody-dependent cellular cytotoxicity (ADCC). Here, we tested whether maintaining NK cell function during DARA therapy could maximize DARA-mediated ADCC against MM cells and deepen the response. We used the CRISPR/Cas9 system to delete CD38 (CD38KO) in ex vivo expanded peripheral blood NK cells. These CD38KO NK cells were completely resistant to DARA-induced fratricide, showed superior persistence in immune-deficient mice pretreated with DARA, and enhanced ADCC activity against CD38-expressing MM cell lines and primary MM cells. In addition, transcriptomic and cellular metabolic analysis demonstrated that CD38KO NK cells have unique metabolic reprogramming with higher mitochondrial respiratory capacity. Finally, we evaluated the impact of exposure to all-trans retinoic acid (ATRA) on wild-type NK and CD38KO NK cell function and highlighted potential benefits and drawbacks of combining ATRA with DARA in patients with MM. Taken together, these findings provide proof of concept that adoptive immunotherapy using ex vivo expanded CD38KO NK cells has the potential to boost DARA activity in MM.

Immunotherapies for pediatric cancer: current landscape and future perspectives.

The advent of immunotherapy has revolutionized how we manage and treat cancer. While the majority of immunotherapy-related studies performed to date have focused on adult malignancies, a handful of these therapies have also recently found success within the pediatric space. In this review, we examine the immunotherapeutic agents that have achieved the approval of the US Food and Drug Administration for treating childhood cancers, highlighting their development, mechanisms of action, and the lessons learned from the seminal clinical trials that ultimately led to their approval. We also shine a spotlight on several emerging immunotherapeutic modalities that we believe are poised to have a positive impact on the treatment of pediatric malignancies in the near future.

Adipose tissue-derived WNT5A regulates vascular redox signaling in obesity via USP17/RAC1-mediated activation of NADPH oxidases.

Obesity is associated with changes in the secretome of adipose tissue (AT), which affects the vasculature through endocrine and paracrine mechanisms. Wingless-related integration site 5A (WNT5A) and secreted frizzled-related protein 5 (SFRP5), adipokines that regulate noncanonical Wnt signaling, are dysregulated in obesity. We hypothesized that WNT5A released from AT exerts endocrine and paracrine effects on the arterial wall through noncanonical RAC1-mediated Wnt signaling. In a cohort of 1004 humans with atherosclerosis, obesity was associated with increased WNT5A bioavailability in the circulation and the AT, higher expression of WNT5A receptors Frizzled 2 and Frizzled 5 in the human arterial wall, and increased vascular oxidative stress due to activation of NADPH oxidases. Plasma concentration of WNT5A was elevated in patients with coronary artery disease compared to matched controls and was independently associated with calcified coronary plaque progression. We further demonstrated that WNT5A induces arterial oxidative stress and redox-sensitive migration of vascular smooth muscle cells via Frizzled 2-mediated activation of a previously uncharacterized pathway involving the deubiquitinating enzyme ubiquitin-specific protease 17 (USP17) and the GTPase RAC1. Our study identifies WNT5A and its downstream vascular signaling as a link between obesity and vascular disease pathogenesis, with translational implications in humans.

Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 Gene Editing Technique in Xenotransplantation.

Genetically modified pigs have been considered favorable resources in xenotransplantation. Microinjection of randomly integrating transgenes into zygotes, somatic cell nuclear transfer, homologous recombination, zinc finger nucleases, transcription activator-like effector nucleases, and most recently, clustered regularly interspaced short palindromic repeats-cas9 (CRISPR/Cas9) are the techniques that have been used to generate these animals. Here, we provide an overview of the CRISPR approaches that have been used to modify genes which are vital in improving xenograft survival rate, including cytidine monophosphate-N-acetylneuraminic acid hydroxylase, B1,4N-acetylgalactosaminyltransferase, isoglobotrihexosylceramide synthase, class I MHC, von Willebrand factor, C3, and porcine endogenous retroviruses. In addition, we will mention the importance of potential candidate genes which could be targeted using CRISPR/Cas9.

Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins.

CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging in primary NK cells. For example, transgene delivery using lentiviral or retroviral transduction resulted in a limited yield of genetically-engineered NK cells due to substantial procedure-associated NK cell apoptosis. We describe here a DNA-free method for genome editing of human primary and expanded NK cells using Cas9 ribonucleoprotein complexes (Cas9/RNPs). This method allowed efficient knockout of the TGFBR2 and HPRT1 genes in NK cells. RT-PCR data showed a significant decrease in gene expression level, and a cytotoxicity assay of a representative cell product suggested that the RNP-modified NK cells became less sensitive to TGFß. Genetically modified cells could be expanded post-electroporation by stimulation with irradiated mbIL21-expressing feeder cells.