No Vacillation on HPV Vaccination.

Evidence of the safety and protective benefits of human papillomavirus virus (HPV) vaccines as an anti-cancer measure is overwhelming. However, vaccine uptake varies widely across countries and falls short of levels needed to achieve population immunity. We highlight policy measures that would help ensure greater worldwide coverage and save lives.

First Approved Kinase Inhibitor for AML.

Activating mutations of FLT3 occur in about 30% of acute myeloid leukemia (AML) cases and are associated with relapse and poor prognosis. Midostaurin is the first drug approved for AML since 2000, and the first multi-kinase inhibitor approved for the FLT3-mutant subtype. To view this Bench to Bedside, open or download the PDF.

Orchestrated intron retention regulates normal granulocyte differentiation.

Intron retention (IR) is widely recognized as a consequence of mis-splicing that leads to failed excision of intronic sequences from pre-messenger RNAs. Our bioinformatic analyses of transcriptomic and proteomic data of normal white blood cell differentiation reveal IR as a physiological mechanism of gene expression control. IR regulates the expression of 86 functionally related genes, including those that determine the nuclear shape that is unique to granulocytes. Retention of introns in specific genes is associated with downregulation of splicing factors and higher GC content. IR, conserved between human and mouse, led to reduced mRNA and protein levels by triggering the nonsense-mediated decay (NMD) pathway. In contrast to the prevalent view that NMD is limited to mRNAs encoding aberrant proteins, our data establish that IR coupled with NMD is a conserved mechanism in normal granulopoiesis. Physiological IR may provide an energetically favorable level of dynamic gene expression control prior to sustained gene translation.

Recent advances in cancer fusion transcript detection.

Extensive investigation of gene fusions in cancer has led to the discovery of novel biomarkers and therapeutic targets. To date, most studies have neglected chromosomal rearrangement-independent fusion transcripts and complex fusion structures such as double or triple-hop fusions, and fusion-circRNAs. In this review, we untangle fusion-related terminology and propose a classification system involving both gene and transcript fusions. We highlight the importance of RNA-level fusions and how long-read sequencing approaches can improve detection and characterization. Moreover, we discuss novel bioinformatic tools to identify fusions in long-read sequencing data and strategies to experimentally validate and functionally characterize fusion transcripts.

SpliceWiz: interactive analysis and visualization of alternative splicing in R.

Alternative splicing (AS) is a crucial mechanism for regulating gene expression and isoform diversity in eukaryotes. However, the analysis and visualization of AS events from RNA sequencing data remains challenging. Most tools require a certain level of computer literacy and the available means of visualizing AS events, such as coverage and sashimi plots, have limitations and can be misleading. To address these issues, we present SpliceWiz, an R package with an interactive Shiny interface that allows easy and efficient AS analysis and visualization at scale. A novel normalization algorithm is implemented to aggregate splicing levels within sample groups, thereby allowing group differences in splicing levels to be accurately visualized. The tool also offers downstream gene ontology enrichment analysis, highlighting ASEs belonging to functional pathways of interest. SpliceWiz is optimized for speed and efficiency and introduces a new file format for coverage data storage that is more efficient than BigWig. Alignment files are processed orders of magnitude faster than other R-based AS analysis tools and on par with command-line tools. Overall, SpliceWiz streamlines AS analysis, enabling reliable identification of functionally relevant AS events for further characterization. SpliceWiz is a Bioconductor package and is also available on GitHub (https://github.com/alexchwong/SpliceWiz).

Detailed DNA methylation characterisation of phyllodes tumours identifies a signature of malignancy and distinguishes phyllodes from metaplastic breast carcinoma.

Phyllodes tumours (PTs) are rare fibroepithelial lesions of the breast that are classified as benign, borderline, or malignant. As little is known about the molecular underpinnings of PTs, current diagnosis relies on histological examination. However, accurate classification is often difficult, particularly for distinguishing borderline from malignant PTs. Furthermore, PTs can be misdiagnosed as other tumour types with shared histological features, such as fibroadenoma and metaplastic breast cancers. As DNA methylation is a recognised hallmark of many cancers, we hypothesised that DNA methylation could provide novel biomarkers for diagnosis and tumour stratification in PTs, whilst also allowing insight into the molecular aetiology of this otherwise understudied tumour. We generated whole-genome methylation data using the Illumina EPIC microarray in a novel PT cohort (n = 33) and curated methylation microarray data from published datasets including PTs and other potentially histopathologically similar tumours (total n = 817 samples). Analyses revealed that PTs have a unique methylome compared to normal breast tissue and to potentially histopathologically similar tumours (metaplastic breast cancer, fibroadenoma and sarcomas), with PT-specific methylation changes enriched in gene sets involved in KRAS signalling and epithelial-mesenchymal transition. Next, we identified 53 differentially methylated regions (DMRs) (false discovery rate < 0.05) that specifically delineated malignant from non-malignant PTs. The top DMR in both discovery and validation cohorts was hypermethylation at the HSD17B8 CpG island promoter. Matched PT single-cell expression data showed that HSD17B8 had minimal expression in fibroblast (putative tumour) cells. Finally, we created a methylation classifier to distinguish PTs from metaplastic breast cancer samples, where we revealed a likely misdiagnosis for two TCGA metaplastic breast cancer samples. In conclusion, DNA methylation alterations are associated with PT histopathology and hold the potential to improve our understanding of PT molecular aetiology, diagnostics, and risk stratification. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Dynamic changes in RNA m6A and 5 hmC influence gene expression programs during macrophage differentiation and polarisation.

RNA modifications are essential for the establishment of cellular identity. Although increasing evidence indicates that RNA modifications regulate the innate immune response, their role in monocyte-to-macrophage differentiation and polarisation is unclear. While m6A has been widely studied, other RNA modifications, including 5 hmC, remain poorly characterised. We profiled m6A and 5 hmC epitranscriptomes, transcriptomes, translatomes and proteomes of monocytes and macrophages at rest and pro- and anti-inflammatory states. Transcriptome-wide mapping of m6A and 5 hmC reveals enrichment of m6A and/or 5 hmC on specific categories of transcripts essential for macrophage differentiation. Our analyses indicate that m6A and 5 hmC modifications are present in transcripts with critical functions in pro- and anti-inflammatory macrophages. Notably, we also discover the co-occurrence of m6A and 5 hmC on alternatively-spliced isoforms and/or opposing ends of the untranslated regions (UTR) of mRNAs with key roles in macrophage biology. In specific examples, RNA 5 hmC controls the decay of transcripts independently of m6A. This study provides (i) a comprehensive dataset to interrogate the role of RNA modifications in a plastic system (ii) a resource for exploring different layers of gene expression regulation in the context of human monocyte-to-macrophage differentiation and polarisation, (iii) new insights into RNA modifications as central regulators of effector cells in innate immunity.

Multiyear Factor VIII Expression after AAV Gene Transfer for Hemophilia A.

BACKGROUND: The goal of gene therapy for patients with hemophilia A is to safely impart long-term stable factor VIII expression that predictably ameliorates bleeding with the use of the lowest possible vector dose. METHODS: In this phase 1-2 trial, we infused an investigational adeno-associated viral (AAV) vector (SPK-8011) for hepatocyte expression of factor VIII in 18 men with hemophilia A. Four dose cohorts were enrolled; the lowest-dose cohort received a dose of 5 × 1011 vector genomes (vg) per kilogram of body weight, and the highest-dose cohort received 2 × 1012 vg per kilogram. Some participants received glucocorticoids within 52 weeks after vector administration either to prevent or to treat a presumed AAV capsid immune response. Trial objectives included evaluation of the safety and preliminary efficacy of SPK-8011 and of the expression and durability of factor VIII. RESULTS: The median safety observation period was 36.6 months (range, 5.5 to 50.3). A total of 33 treatment-related adverse events occurred in 8 participants; 17 events were vector-related, including 1 serious adverse event, and 16 were glucocorticoid-related. Two participants lost all factor VIII expression because of an anti-AAV capsid cellular immune response that was not sensitive to immune suppression. In the remaining 16 participants, factor VIII expression was maintained; 12 of these participants were followed for more than 2 years, and a one-stage factor VIII assay showed no apparent decrease in factor VIII activity over time (mean [±SD] factor VIII activity, 12.9±6.9% of the normal value at 26 to 52 weeks when the participants were not receiving glucocorticoids vs. 12.0±7.1% of the normal value at >52 weeks after vector administration; 95% confidence interval [CI], -2.4 to 0.6 for the difference between matched pairs). The participants had a 91.5% reduction (95% CI, 88.8 to 94.1) in the annualized bleeding rate (median rate, 8.5 events per year [range, 0 to 43.0] before vector administration vs. 0.3 events per year [range, 0 to 6.5] after vector administration). CONCLUSIONS: Sustained factor VIII expression in 16 of 18 participants who received SPK-8011 permitted discontinuation of prophylaxis and a reduction in bleeding episodes. No major safety concerns were reported. (Funded by Spark Therapeutics and the National Heart, Lung, and Blood Institute; ClinicalTrials.gov numbers, NCT03003533 and NCT03432520.).

Mesothelin antigen density influences anti-mesothelin chimeric antigen receptor T cell cytotoxicity.

BACKGROUND AIMS: Several anti-mesothelin (MSLN) chimeric antigen receptor (CAR) T cells are in phase 1/2 clinical trials to treat solid-organ malignancies. The effect of MSLN antigen density on MSLN CAR cytotoxicity against tumor cells has not been examined previously, nor are there data regarding the effect of agents that increase MSLN antigen density on anti-MSLN CAR T cell efficacy. METHODS: MSLN antigen density was measured on a panel of pancreatic cancer and mesothelioma cell lines by flow cytometry. In parallel, the cytotoxicity and specificity of two anti-MSLN CAR T cells (m912 and SS1) were compared against these cell lines using a real-time impedance-based assay. The effect of two MSLN 'sheddase' inhibitors (lanabecestat and TMI-1) that increase MSLN surface expression was also tested in combination with CAR T cells. RESULTS: SS1 CAR T cells were more cytotoxic compared with m912 CAR T cells against cell lines that expressed fewer than ∼170 000 MSLN molecules/cell. A comparison of the m912 and amatuximab (humanized SS1) antibodies identified that amatuximab could detect and bind to lower levels of MSLN on pancreatic cancer and mesothelioma cell lines, suggesting that superior antibody/scFv affinity was the reason for the SS1 CAR's superior cytotoxicity. The cytotoxicity of m912 CAR T cells was improved in the presence of sheddase inhibitors, which increased MSLN antigen density. CONCLUSIONS: These data highlight the value of assessing CAR constructs against a panel of cells expressing varying degrees of target tumor antigen as occurs in human tumors. Furthermore, the problem of low antigen density may be overcome by concomitant administration of drugs that inhibit enzymatic shedding of MSLN.

Increased chromatin accessibility facilitates intron retention in specific cell differentiation states.

Dynamic intron retention (IR) in vertebrate cells is of widespread biological importance. Aberrant IR is associated with numerous human diseases including several cancers. Despite consistent reports demonstrating that intrinsic sequence features can help introns evade splicing, conflicting findings about cell type- or condition-specific IR regulation by trans-regulatory and epigenetic mechanisms demand an unbiased and systematic analysis of IR in a controlled experimental setting. We integrated matched mRNA sequencing (mRNA-Seq), whole-genome bisulfite sequencing (WGBS), nucleosome occupancy methylome sequencing (NOMe-Seq) and chromatin immunoprecipitation sequencing (ChIP-Seq) data from primary human myeloid and lymphoid cells. Using these multi-omics data and machine learning, we trained two complementary models to determine the role of epigenetic factors in the regulation of IR in cells of the innate immune system. We show that increased chromatin accessibility, as revealed by nucleosome-free regions, contributes substantially to the retention of introns in a cell-specific manner. We also confirm that intrinsic characteristics of introns are key for them to evade splicing. This study suggests an important role for chromatin architecture in IR regulation. With an increasing appreciation that pathogenic alterations are linked to RNA processing, our findings may provide useful insights for the development of novel therapeutic approaches that target aberrant splicing.

High mesothelin expression by immunohistochemistry predicts improved survival in pleural mesothelioma.

AIMS: Mesothelin (MSLN) is a cancer-associated antigen that is overexpressed in malignancies such as mesothelioma, pancreatic and ovarian cancer. It is also a target for novel personalised therapies, including antibodies, antibody-drug conjugates and chimeric antigen receptor T cells. Immunohistochemistry may predict those who would best respond to anti-mesothelin therapies and guide decisions in therapeutic strategy. This study aimed to assess the intensity and distribution of MSLN immunostaining in mesothelioma, and to determine the prognostic value of MSLN expression by histochemical-score (H-score). METHODS AND RESULTS: The MN1 anti-MSLN antibody was used to stain a formalin-fixed paraffin-embedded tissue microarray of histologically confirmed mesothelioma from 75 consecutive patients who had undergone pleurectomy with or without decortication. MSLN positivity, the staining intensity, distribution of staining and H-score were evaluated. The correlation of H-score with prognosis was investigated. Sixty-six per cent of epithelioid tumours were MSLN-positive (with expression in > 5% tumour cells). Of MSLN-expressing epithelioid tumours, 70.4% had moderate (2+) or strong (3+) intensity MSLN immunostaining, although only 37% of samples had staining in ≥ 50% of tumour cells. In multivariate analysis, MSLN H-score as a continuous variable and an H-score ≥ 33 were independent predictors of improved survival (P = 0.04 and P < 0.001, respectively). CONCLUSIONS: MSLN expression was more heterogenous in epithelioid mesothelioma than reported previously. Therefore, it would be appropriate to perform an immunohistochemical assessment of MSLN expression to stratify and assess patient suitability for mesothelin-targeted personalised therapies, such as chimeric antigen receptor T cells.

The 4-1BBζ costimulatory domain in chimeric antigen receptors enhances CD8+ T-cell functionality following T-cell receptor stimulation.

BACKGROUND: Chimeric antigen receptor (CAR) T-cells have revolutionized the treatment of CD19- and B-cell maturation antigen-positive haematological malignancies. However, the effect of a CAR construct on the function of T-cells stimulated via their endogenous T-cell receptors (TCRs) has yet to be comprehensively investigated. METHODS: Experiments were performed to systematically assess TCR signalling and function in CAR T-cells using anti-mesothelin human CAR T-cells as a model system. CAR T-cells expressing the CD28 or 4-1BB costimulatory endodomains were manufactured and compared to both untransduced T-cells and CAR T-cells with a non-functional endodomain. These cell products were treated with staphylococcal enterotoxin B to stimulate the TCR, and in vitro functional assays were performed by flow cytometry. RESULTS: Increased proliferation, CD69 expression and IFNγ production were identified in CD8+ 4-1BBζ CAR T-cells compared to control untransduced CD8+ T-cells. These functional differences were associated with higher levels of phosphorylated ZAP70 after stimulation. In addition, these functional differences were associated with a differing immunophenotype, with a more than two-fold increase in central memory cells in CD8+ 4-1BBζ CAR T-cell products. CONCLUSION: Our data indicate that the 4-1BBζ CAR enhances CD8+ TCR-mediated function. This could be beneficial if the TCR targets epitopes on malignant tissues or infectious agents, but detrimental if the TCR targets autoantigens.

International Society for Cell & Gene Therapy Position Paper: Key considerations to support evidence-based cell and gene therapies and oppose marketing of unproven products.

The field of regenerative medicine, including cellular immunotherapies, is on a remarkable growth trajectory. Dozens of cell-, tissue- and gene-based products have received marketing authorization worldwide while hundreds-to-thousands are either in preclinical development or under clinical investigation in phased clinical trials. However, the promise of regenerative therapies has also given rise to a global industry of direct-to-consumer offerings of prematurely commercialized cell and cell-based products with unknown safety and efficacy profiles. Since its inception, the International Society for Cell & Gene Therapy Committee on the Ethics of Cell and Gene Therapy has opposed the premature commercialization of unproven cell- and gene-based interventions and supported the development of evidence-based advanced therapy products. In the present Guide, targeted at International Society for Cell & Gene Therapy members, we analyze this industry, focusing in particular on distinctive features of unproven cell and cell-based products and the use of tokens of scientific legitimacy as persuasive marketing devices. We also provide an overview of reporting mechanisms for patients who believe they have been harmed by administration of unapproved and unproven products and suggest practical strategies to address the direct-to-consumer marketing of such products. Development of this Guide epitomizes our continued support for the ethical and rigorous development of cell and cell-based products with patient safety and therapeutic benefit as guiding principles.

CTCF as a regulator of alternative splicing: new tricks for an old player.

Three decades of research have established the CCCTC-binding factor (CTCF) as a ubiquitously expressed chromatin organizing factor and master regulator of gene expression. A new role for CTCF as a regulator of alternative splicing (AS) has now emerged. CTCF has been directly and indirectly linked to the modulation of AS at the individual transcript and at the transcriptome-wide level. The emerging role of CTCF-mediated regulation of AS involves diverse mechanisms; including transcriptional elongation, DNA methylation, chromatin architecture, histone modifications, and regulation of splicing factor expression and assembly. CTCF thereby appears to not only co-ordinate gene expression regulation but contributes to the modulation of transcriptomic complexity. In this review, we highlight previous discoveries regarding the role of CTCF in AS. In addition, we summarize detailed mechanisms by which CTCF mediates AS regulation. We propose opportunities for further research designed to examine the possible fate of CTCF-mediated alternatively spliced genes and associated biological consequences. CTCF has been widely acknowledged as the 'master weaver of the genome'. Given its multiple connections, further characterization of CTCF's emerging role in splicing regulation might extend its functional repertoire towards a 'conductor of the splicing orchestra'.

Dynamic intron retention modulates gene expression in the monocytic differentiation pathway.

Monocytes play a crucial role in maintaining homeostasis and mediating a successful innate immune response. They also act as central players in diverse pathological conditions, thus making them an attractive therapeutic target. Within the bone marrow, monocytes arise from a committed precursor termed Common Monocyte Progenitor (cMoP). However, molecular mechanisms that regulate the differentiation of cMoP to various monocytic subsets remain unclear. Herein, we purified murine myeloid precursors for deep poly-A-enriched RNA sequencing to understand the role of alternative splicing in the development and differentiation of monocytes under homeostasis. Our analyses revealed intron retention to be the major alternative splicing mechanism involved in the monocyte differentiation cascade, especially in the differentiation of Ly6Chi monocytes to Ly6Clo monocytes. Furthermore, we found that the intron retention of key genes involved in the differentiation of murine Ly6Chi to Ly6Clo monocytes was also conserved in humans. Our data highlight the unique role of intron retention in the regulation of the monocytic differentiation pathway.

Towards resolution of the intron retention paradox in breast cancer.

BACKGROUND: After many years of neglect in the field of alternative splicing, the importance of intron retention (IR) in cancer has come into focus following landmark discoveries of aberrant IR patterns in cancer. Many solid and liquid tumours are associated with drastic increases in IR, and such patterns have been pursued as both biomarkers and therapeutic targets. Paradoxically, breast cancer (BrCa) is the only tumour type in which IR is reduced compared to adjacent normal breast tissue. METHODS: In this study, we have conducted a pan-cancer analysis of IR with emphasis on BrCa and its subtypes. We explored mechanisms that could cause aberrant and pathological IR and clarified why normal breast tissue has unusually high IR. RESULTS: Strikingly, we found that aberrantly decreasing IR in BrCa can be largely attributed to normal breast tissue having the highest occurrence of IR events compared to other healthy tissues. Our analyses suggest that low numbers of IR events in breast tumours are associated with poor prognosis, particularly in the luminal B subtype. Interestingly, we found that IR frequencies negatively correlate with cell proliferation in BrCa cells, i.e. rapidly dividing tumour cells have the lowest number of IR events. Aberrant RNA-binding protein expression and changes in tissue composition are among the causes of aberrantly decreasing IR in BrCa. CONCLUSIONS: Our results suggest that IR should be considered for therapeutic manipulation in BrCa patients with aberrantly low IR levels and that further work is needed to understand the cause and impact of high IR in other tumour types.

Mapping oncogenic protein interactions for precision medicine.

Normal protein-protein interactions (normPPIs) occur with high fidelity to regulate almost every physiological process. In cancer, this highly organised and precisely regulated network is disrupted, hijacked or reprogrammed resulting in oncogenic protein-protein interactions (oncoPPIs). OncoPPIs, which can result from genomic alterations, are a hallmark of many types of cancers. Recent technological advances in the field of mass spectrometry (MS)-based interactomics, structural biology and drug discovery have prompted scientists to identify and characterise oncoPPIs. Disruption of oncoPPI interfaces has become a major focus of drug discovery programs and has resulted in the use of PPI-specific drugs clinically. However, due to several technical hurdles, studies to build a reference oncoPPI map for various cancer types have not been undertaken. Therefore, there is an urgent need for experimental workflows to overcome the existing challenges in studying oncoPPIs in various cancers and to build comprehensive reference maps. Here, we discuss the important hurdles for characterising oncoPPIs and propose a three-phase multidisciplinary workflow to identify and characterise oncoPPIs. Systematic identification of cancer-type-specific oncogenic interactions will spur new opportunities for PPI-focused drug discovery projects and precision medicine.

Establishing a robust chimeric antigen receptor T-cell therapy program in Australia: the Royal Prince Alfred Hospital experience.

>himeric antigen receptor (CAR) T-cell therapy is a novel approved cancer treatment that has shown remarkable efficacy in the treatment of patients with relapsed leukemia and lymphoma. Implementation of CAR T-cell therapy in a hospital setting requires careful and detailed planning because of the complexities in delivering this specialist service. A multi-disciplinary approach with dedicated funding is required to meet clinical, scientific, logistic and regulatory requirements. Tisagenlecleucel was the first approved CAR T-cell therapy in Australia. The treatment has been made available to Australian patients in specialist public hospitals through federal and state funding. Royal Prince Alfred Hospital (RPAH) is one of Australia's oldest tertiary referral public health care institutions and was approved for the provision of CAR T-cell therapy service in 2019. A multi-disciplinary clinical program has been established for the collection and cryopreservation of donor cells shipped for manufacturing as well as for the receipt, storage and administration of CAR T-cell therapy and patient management. The program encompasses a Therapeutic Goods Administration-accredited apheresis unit and a state-of-the-art facility for cell processing, cryopreservation and storage. The program's clinical expertise extends to hematology, oncology, intensive care, pharmacy, neurology and radiology services with direct experience in managing patients receiving CAR T-cell therapies. The introduction of CAR T-cell therapies at RPAH was a complex undertaking facilitated by the existing infrastructure and clinical expertise.

Sprouty and Spred temporally regulate ERK1/2-signaling to suppress TGFß-induced lens EMT.

Epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) principally contributes to the pathogenesis of fibrotic cataract. Sprouty (Spry) and Spred proteins are receptor tyrosine kinase (RTK) antagonists that can regulate RTK-mediated signaling pathways, such as the MAPK/ERK1/2-signaling pathway. The present study examines the ability of Spry and Spred to inhibit TGFß-induced EMT in LECs. LECs explanted from postnatal-day-21 Wistar rats were transduced with adenoviral vectors coding for Spry1, Spry2 or Spred2, and subsequently treated with or without TGFß2. Immunofluorescent labeling of explants for the epithelial membrane marker ß-catenin, and the mesenchymal marker alpha-smooth muscle actin (α-sma), were used to characterize the progression of EMT. Western blotting was used to quantify levels of α-sma and ERK1/2-signaling. Overexpression of Spry or Spred in LECs was sufficient to suppress EMT in response to TGFß, including a block to cell elongation, ß-catenin delocalization and α-sma accumulation. Spry and Spred were also shown to significantly block ERK1/2 phosphorylation for up to 18 h of TGFß treatment but did not impair the earlier activation of ERK1/2 at 20 min. These findings suggest that Spry and Spred may not directly impact ERK1/2-signaling activated by the serine/threonine kinase TGFß receptor, but may selectively target later ERK1/2-signaling driven by downstream RTK-mediated signaling. Taken together, our data establish Spry and Spred antagonists as potent negative regulators of TGFß-induced EMT that can regulate ERK1/2-signaling in a temporal manner. A greater understanding of how Spry and Spred regulate the complex signaling interactions that underlie TGFß-induced EMT will be essential to facilitate the development of novel therapeutics for different pathologies driven by EMT, including fibrotic forms of cataract.

Locoregional delivery of CAR-T cells in the clinic.

Cellular therapies utilizing T cells expressing chimeric antigen receptors (CARs) have garnered significant interest due to their clinical success in hematological malignancies. Unfortunately, this success has not been replicated in solid tumors, with only a small fraction of patients achieving complete responses. A number of obstacles to effective CAR-T cell therapy in solid tumors have been identified including tumor antigen heterogeneity, poor T cell fitness and persistence, inefficient trafficking and inability to penetrate into the tumor, immune-related adverse events due to on-target/off-tumor toxicity, and the immunosuppressive tumor microenvironment. Many preclinical studies have focused on improvements to CAR design to try to overcome some of these hurdles. However, a growing body of work has also focused on the use of local and/or regional delivery of CAR-T cells as a means to overcome poor T cell trafficking and inefficient T cell penetration into tumors. Most trials that incorporate locoregional delivery of CAR-T cells have targeted tumors of the central nervous system - repurposing an Ommaya/Rickham reservoir for repeated delivery of cells directly to the tumor cavity or ventricles. Hepatic artery infusion is another technique used for locoregional delivery to hepatic tumors. Locoregional delivery theoretically permits increased numbers of CAR-T cells within the tumor while reducing the risk of immune-related systemic toxicity. Studies to date have been almost exclusively phase I. The growing body of evidence indicates that locoregional delivery of CAR-T cells is both safe and feasible. This review focuses specifically on the use of locoregional delivery of CAR-T cells in clinical trials.