Purpose:Intravesical interferon-alpha (IFNα) gene therapy has shown promise in treating BCG-unresponsive non-muscle invasive bladder cancer (NMIBC). Ongoing work in our lab aims to further improve its treatment efficacy by identifying resistance mechanisms and deploying targeted combination treatment strategies. Experimental design:We performed end-tumor RNA-seq analysis of MB49 murine tumors treated with IFNα gene therapy, identifying the ErbB pathway as a resistance mechanism. We consequently hypothesized that a combination treatment involving an ErbB pathway blocker and IFNα could yield improved outcomes. MB49 cells were treated in vitro with lentiviral IFNα (LV-IFNα) gene therapy, with/without Afatinib, a pan-ErbB inhibitor, and cell viability and migration assays were performed. Next, in vivo studies were conducted in the syngeneic MB49 orthotopic murine bladder cancer model. The mice were randomized into 5 treatment groups (n=10 each): saline (Ctrl), LV-Ctrl, oral Afatinib monotherapy, intravesical LV-IFNα monotherapy, and the experimental intravesical LV-IFNα + oral Afatinib combination therapy. Overall survival (OS) and drug toxicity were assessed. Results:Combination therapy significantly reduced MB49 cell viability in vitro compared to all other treatment conditions (mean relative ATPase activity at 72 h for the combination treatment was 4%, compared to 100%, 26%, and 28% for Ctrl, LV-IFNα, and Afatinib, respectively, p<0.001). This additive effect on cell viability appeared to be driven by a combination of early-cytostatic and late-cytolytic effects. The combination treatment also markedly inhibited cell migration (mean migrated cells/10x Boyden chamber assay at 36 h were: 92.3 for the combination therapy and 631.0, 600.4, and 270.3 for Ctrl, LV-IFNα, and Afatinib, respectively, p<0.001). Finally, the in vivo studies demonstrated improved OS with combination therapy (median OS was 49 d in the combination group vs 15, 29, and 26 d in Ctrl, LV-IFNα, and Afatinib groups, respectively, Log-rank p<0.001). No mice in the combination therapy group died of drug toxicity. Conclusions:Our preliminary findings suggest that the ErbB pathway may serve as a clinically significant resistance mechanism to intravesical IFNα gene therapy, and when targeted concurrently, may improve treatment efficacy.

Natural killer (NK) cell-based therapies are a promising approach in cancer, but their efficacy is limited by impaired effector function and tumor-intrinsic resistance. To systematically identify therapeutic strategies that target both sides of the cancer-immune interface, we designed a multimodal immunopharmacologic screening platform comprising high-throughput co-culture drug screens, cytokine secretome profiling, single-cell perturbation screens, and genome-scale CRISPR screening, followed by validation in biobanked patient-derived models. Applying the platform across five blood cancer types, we identified protein kinase C (PKC) activation to simultaneously increase effector cytotoxicity and cytokine secretion through transcriptomic rewiring, and tumor susceptibility to NK cell killing through tumor-intrinsic PKC-δ. In patient samples, PKC activation sensitized NK-resistant leukemic progenitors to NK cell killing. In addition, NEDD8 inhibition enhanced NK function and shifted tumor TNF signaling towards pro-apoptotic pathways. Our platform provides a systematic approach to identify drugs rewiring both sides of the cancer-immune interface to circumvent tumor immune resistance.

HIF-prolyl hydroxylase inhibitors are used to treat anemia in chronic kidney disease. These drugs stabilize hypoxia-inducible factors HIF-1α and HIF-2α, which activate erythropoiesis and iron metabolism pathways. Clinically approved HIF-PHIs including roxadustat and molidustat exhibit distinct molecular structures and selectivity profiles, yet their HIF-independent effects remain poorly understood. Here we show that roxadustat and molidustat modulate mitochondrial function, oxidative stress, lysosomal activity, and lipid accumulation, resulting in distinct cellular phenotypes in HIF-null cells. Notably, roxadustat exhibited anti-proliferative and anti-angiogenic activity in HIF-null cells, contradicting expectations of VEGF-driven angiogenesis via HIF stabilization. RNA sequencing and pathway analysis revealed compound-specific off-target gene regulation affecting cellular processes beyond canonical hypoxia responses including energy metabolism and immune signaling. These findings illuminate mechanisms underlying potential adverse effects- such as thrombosis- and identify alternative therapeutic pathways, providing a framework for optimizing HIF-PHI safety profiles and expanding their clinical applications in oncology and metabolic disorders.

Immune responses against recombinant adeno-associated virus (rAAV) are one of the major obstacles in gene therapy. We investigated the potential of Programmed Death 1 ligands 1 and 2 (PD-L1/2) to protect AAV-transduced cells from immunological clearance. Ligand compatibility for co-delivery was first evaluated using two transgenes, VEGF-B186 and muSEAP, separated from PD-L1/2 by a self-cleaving P2A peptide. After proper cleavage and biological activity of the co-produced proteins were demonstrated in vitro, the effect of PD-L1/2 co-expression on muSEAP production and persistence was studied in naïve and vector pre-immunized mice. Vectors (rAAV6-muSEAP, rAAV6-muSEAP-PD-L1, or rAAV6-muSEAP-PD-L2) were injected into two sites of the gastrocnemius muscle at a total dose of 1×1010 vg. Co-delivery of PD-L1, particularly, significantly enhanced muSEAP secretion into the bloodstream up to 12 weeks despite elevated anti-AAV6 responses in pre-immunized mice. muSEAP secretion increased 33.3- and 31.4-fold with the co-delivery of PD-L1, while the increase was only 5.6- and 9.3-fold in the muSEAP control group at 5 and 12 weeks, respectively. Ligand-treated pre-immunized animals also had less T-cell infiltration into the treated muscle compared to naïve animals. In summary, co-delivery of PD-L1/2 alongside a transgene represents a promising strategy for achieving sustained gene expression in individuals pre-exposed to AAV.

Heart failure is the leading cause of hospitalization globally, burdening healthcare systems and the economy. Heart failure is a multifactorial cardiac syndrome, where the heart fails to maintain sufficient cardiac output to support body function. As the population ages, heart failure rates will increase. Current treatments, such as medications and surgery, are not suitable for all patients, creating a need for alternative therapies. Gene therapy offers promising new approaches, with therapeutic angiogenesis, regenerative strategies, and calcium ion regulation as key targets. SERCA2a plays a critical role in calcium regulation, and clinical trials have focused on its potential as a therapeutic agent. VEGF-B, which specifically targets the myocardium, also regulates myocardial metabolism and SERCA2a activity. Although clinical trials have been conducted, results have not consistently replicated pre-clinical success. This review summarizes the current state of gene therapy for heart failure, including therapeutic agents, vectors, delivery methods, and preclinical models.

HIF-prolyl hydroxylase inhibitors (HIF-PHIs) are used to treat anemia in chronic kidney disease. These drugs stabilize hypoxia-inducible factors HIF-1α and HIF-2α, which activate erythropoiesis and iron metabolism pathways. Clinically approved HIF-PHIs such as roxadustat and molidustat exhibit distinct molecular structures and selectivity profiles, yet their HIF-independent effects remain poorly understood. Here we generated HIF1A/EPAS1 double-knockout cells to systematically dissect non-canonical HIF-PHI actions. Roxadustat and molidustat induced distinct phenotypic alterations and transcriptional reprogramming in HIF-null cells, demonstrating substantial HIF-independent effects. Notably, roxadustat exhibited anti-angiogenic activity in HIF-null cells, contradicting expectations of VEGF-driven angiogenesis via HIF stabilization. RNA sequencing and pathway analysis revealed compound-specific off-target gene regulation affecting cellular processes beyond canonical hypoxia responses including mitochondrial function, lipid metabolism, and immune signaling. These findings illuminate mechanisms underlying potential adverse effects-such as thrombosis- and identify alternative therapeutic pathways, providing a framework for optimizing HIF-PHI safety profiles and expanding their clinical applications in oncology and metabolic disorders.

The need for safe, allogeneic cell therapies for cancer is driving a growing interest in CAR-NK-based therapies, which, unlike CAR-T cell therapies, offer the potential for off-the-shelf administration. Lentiviruses pseudotyped with vesicular stomatitis virus glycoprotein G (VSV-G) are commonly used for genetic modification of cell therapy products. Their use in NK cells, however, is limited by low transduction efficiency. This study explores the complexities of NK cell transduction using lentiviral vectors pseudotyped with VSV-G. We demonstrate that efficient transduction depends on multiple factors, such as NK cell activation, domain organization of CAR construct, lentivirus pseudotype selection, and the use of transduction enhancers. By optimizing these elements, we achieved effective transduction, facilitating the use of VSV-G-pseudotyped lentiviral vectors for therapeutic NK cell production. Our optimized workflow comprises NK cell activation with interleukins, followed by transduction with an NK-cell-specific CAR construct using VSV-G-pseudotyped LVs in the presence of BX795 and retronectin, resulting in excellent transduction efficiency (91% using GFP and 80% using CAR) without compromising NK cell phenotype, growth, or cytotoxicity. This allows for the use of a widely used gene transfer vector with an excellent safety record for producing therapeutic NK cell products.

We present a protocol to evaluate natural killer (NK)-tumor interactions using an ex ovo chick embryo system. We describe procedures to culture GFP-labeled SH-SY5Y neuroblastoma cells, expand and Hoechst-label human NK cells, and perform sequential intracardiac injections. We use fluorescence imaging to visualize NK and tumor cell distribution in vivo and live imaging to assess interactions in vitro. This approach provides a rapid and accessible platform to quantify NK cell activity in a living vertebrate model.

Immune checkpoint inhibitors (ICI), such as antibodies against Programmed Death 1 (PD-1), are widely used to treat different types of cancer. Unfortunately, only a small subset of patients benefits from such treatments, and the systemic administration of ICIs can cause severe immune-related adverse events. To overcome these hurdles, we performed an in vitro proof-of-concept investigation of recombinant adenoviral vectors encoding different variants of secreted PD-1 (sPD-1), differing in size, cysteine mutations, oligomerization, and effector functions. First, eight sPD-1 variants were screened, followed by the generation of recombinant adenoviruses with four of the highest-performing sPD-1 constructs based on qualitative analysis of sPD-1 secretion and ligand binding. The secretion of sPD-1 was analyzed using immunoblotting, while binding to PD-L1 and PD-L2 ligands was assessed using a pull-down assay and ELISA. In addition, the inhibition of PD-1:PD-L1 interaction was studied using cell-based signaling bioassays. It was demonstrated that the Fc-fusion sPD-1 variants resulted in the highest sPD-1 yields and were the most efficient in ligand binding. In particular, the single-chain Fc-fusion sPD-1 constructs were the most potent variants, as they effectively blocked PD-1-mediated signaling in T cells in two different coculture assays. The results of this in vitro proof-of-concept study indicate that stable and well-secreted sPD-1 constructs have significant potential for site-specific immune gene therapy.

Background/Objectives: Lentiviral vectors (LVs) are most commonly pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G), which lends LVs a wide tropism as it uses the low-density lipoprotein receptor (LDLR) as the main receptor for cell entry. In some gene therapy and research applications, however, alternative pseudotypes can be useful. In this work, we characterized LVs pseudotyped with lymphocytic choriomeningitis virus (LCMV) glycoprotein, particularly in gene transfer to an LDLR-deficient mouse strain used to model cardiovascular disease, Ldlr-/-ApoB100/100. Methods: LCMV-LVs were used in vitro to test their transduction efficiency across a variety of cell types. In vivo, the gene transfer efficiency, LV-specific immune responses and biodistribution of VSV-G-LVs and LCMV-LVs were compared after systemic gene transfer. Results: In vitro, LCMV-LVs transduced all tested cell types at high efficiency without the use of transduction enhancers. In vivo, VSV-G-LVs showed a higher gene transfer efficiency at the same LV dose, but increasing the LCMV-LV dose enhanced the measured vector copy numbers. With both pseudotypes, most of the vector accrued in the liver, but with LCMV-LVs, a larger portion of the measured vector copies were found in the lungs. VSV-G-LVs also generated a higher titer of LV-specific IgG antibodies. The gene transfer efficiency of LCMV-LVs was affected by the mouse diet, with a high-fat diet decreasing the transduction. Conclusions: LCMV-LVs can be used as a substitute for VSV-G-LVs if an alternative pseudotype is required; however, they may require the use of a higher LV dose.

Vertebrates rely on a network of blood vessels to meet organ demands for oxygen and nutrients. While endothelial cells are known to transport excess nutrients to white adipose tissue (WAT) for energy storage, how their metabolic state impacts this process remains unclear. Here, we identify MYCT1 as a conserved, pan-endothelial protein essential for WAT expansion. Endothelial-specific MYCT1 deletion limited WAT expansion independently of angiogenesis, adipogenesis, or systemic metabolic parameters. Mechanistically, MYCT1 interacted with the transmembrane endolysosomal proteins IFITM2/3 to restrict nutrient consumption by the vascular barrier. Loss of MYCT1 caused IFITM2/3 accumulation in early endosomes, promoting excessive endolysosomal degradation and mTORC1 hyperactivation, limiting the WAT energy storage capacity. Notably, endothelial-specific mTORC1 activation through TSC1 deletion phenocopied the fat storage defects of MYCT1 deficiency. Our findings establish the MYCT1-IFITM2/3 complex as endothelial metabolic checkpoint regulating systemic energy storage. Targeting MYCT1-IFITM2/3 may offer new therapeutic options for obesity and metabolic disorders.

Cardiac magnetic resonance imaging (MRI) is a gold standard to assess functional and anatomical properties of the living heart. Inflammation changes the myocardial tissue and, furthermore, MR relaxation properties. Continuous-wave (CW) longitudinal rotating frame relaxation time (T1ρ) mapping has been used to assess myocardial fibrosis and inflammation. Conventional T2 relaxation time is a known marker of edema in the myocardium. In this study, we assessed myocardial inflammation after viral infection in a mouse heart using CW-T1ρ and T2 relaxation times. Adenoviral human vascular endothelial growth factor-A165 (AdVEGF-A165) and empty control adenoviral vector with cytomegalovirus promoter (AdCMV) gene transfers were used to induce inflammation in the mouse myocardium. In vivo CW-T1ρ and T2 relaxation time measurements were performed in both groups (AdVEGF-A165 and AdCMV) after -1-, 1-, 3-, 7-, 14-, 21-, and 28-day post-injection. The inflammation associated with gene transfer was verified by hematoxylin and eosin staining after 14-day post-injection. One day after AdVEGF-A165 and AdCMV injections and inflammatory reactions, CW-T1ρ showed a significant increase, which stayed increased as a function of time. T2 also increased significantly after both injections and inflammatory reactions as compared to before injections. Contrast difference between inflammation and remote areas was visually observed in both groups in CW-T1ρ and T2 maps. Hematoxylin and eosin staining revealed the area of inflammation after Ad injection in both groups after 14-day post-injection. This study showed that both acute and chronic phases of the inflammatory reaction in mouse myocardium caused by myocardial adenoviral injections were associated with increased CW-T1ρ and T2 relaxation time constants. Furthermore, the inflammatory reaction can be followed up with rotating frame and conventional relaxation time mappings.

IntroductionIntravesical interferon-alpha (IFNα) gene therapy is approved by the FDA for BCG-unresponsive non-muscle invasive bladder cancer (NMIBC). Identifying resistance mechanisms and deploying targeted combination treatment strategies is a rational approach to improving treatment responses. We identified the ErbB pathway as a resistance mechanism to IFNα gene therapy, and we hypothesized that combination treatment with an ErbB pathway blocker and IFN-α could improve outcomes in resistant tumors.MethodsMurine bladder cancer cells were treated in vitro with lentiviral IFNα (LV-IFNα) gene therapy, with/without afatinib (Afa), a pan-ErbB inhibitor, and cell viability and migration assays were performed. In vivo studies were conducted in a syngeneic MB49 orthotopic murine bladder cancer model, and mice were randomized into five treatment groups and treated with single treatments: Ctrl (vehicle), LV-Ctrl, LV-IFNα, and combination treatments with LV-Ctrl/Afa or LV-IFNα/Afa.ResultsCombination therapy with LV-IFNα/Afa significantly reduced MB49 cell viability in vitro compared to all other treatment conditions. This additive effect on cell viability appeared to be driven by a combination of early-cytostatic and late-cytolytic effects. The combination treatment also markedly inhibited cell migration. Finally, the in vivo studies demonstrated improved overall survival (OS) with LV-IFNα/Afa (median OS was 49 days in the LV-IFNα/Afa group vs. 15, 14, 29, and 26 days in Ctrl, LV-Ctrl, LV-IFNα, and LV-Ctrl/Afa groups, respectively; log-rank p < 0.001).ConclusionOur findings suggest that the ErbB pathway may serve as a clinically actionable resistance mechanism to intravesical IFNα gene therapy and, when targeted concurrently, it may improve treatment efficacy.

Hypertension is a major contributor to cardiovascular diseases, being the most common comorbidity and the biggest risk factor in heart failure with preserved ejection fraction. Angiotensin II (Ang II) is a known hypertension and heart failure inducer in mice, but its role in the causality in phenotype development remains unclear. Here, hypertension was induced with low (LowA) or high (HighA) pressor doses of Ang II in mice. Both LowA and HighA groups demonstrated equal levels of hypertension with aortic dilatation and decreased aortic wall strain, but only HighA developed left ventricular hypertrophy with advanced cardiac dysfunction, demonstrating the hypertension-independent effects of Ang II on myocardial remodeling. Alterations in electrical conductivity occurred similarly in both groups, with prominent ECG waveform aberrations. The study demonstrates two distinct hypertensive heart disease phenotypes induced by Ang II, providing a valuable preclinical framework that emphasizes the critical role of Ang II in diastolic dysfunction and vascular remodeling beyond its effects on the regulation of blood pressure.

ABSTRACT Natural killer (NK) cells are increasingly recognized as a versatile therapeutic platform, yet their translation is hindered by limited ex vivo proliferation. Feeder cells serve as robust stimulatory component supplying activating signals required to initiate large-scale NK cell expansion. Here, using bench-scale cultures, we evaluated how distinct engineered K562-based feeder cells influence NK cell proliferation, phenotype maintenance, potential for activation, and post-cryopreservation function. Across conditions, feeder-based systems consistently enabled superior, up to 500-fold higher NK cell yield compared to feeder-free system. Variants incorporating membrane-bound costimulatory and cytokine cues yielded the most favorable balance between expansion and functional preservation. Simple adjustments to cryopreservation, including high-density-freezing and centrifuge-free-thawing, further supported NK cell recovery. Together, these findings highlight feeder cells as essential upstream reagents for effective NK cell bioproduction and provide foundational biological insights to guide the rational design and validation of future scalable NK cell manufacturing platforms. GRAPHICAL ABSTRACT

Cardiovascular diseases are the leading cause of death worldwide due to population growth and aging. Myocardial infarct is one of the most crucial cardiovascular diseases. Acute myocardial infarct is conventionally imaged with magnetic resonance imaging (MRI) with T2 mapping due to its sensitivity related to the correlation times of edema and free water molecules. Chronic myocardial infarction, which contains fibrosis and scar tissue, is conventionally imaged with MRI by using contrast agents since contrast agent washout from fibrosis and scar tissue is delayed compared to myocardium. Rotating frame relaxation times T1ρ and T2ρ mappings were developed to provide robust measurements with relatively wide B1 and B0 ranges for these quantities. Since rotating frame methods are sensitive to slow molecular motions, these methods owe potential to characterize both acute and chronic myocardial infarctions. In this study, rotating frame relaxation time mappings were applied to image acute (2 h) and subchronic (7 days after occlusion) myocardial infarcts in in vivo and ex vivo mouse models without using contrast agents. The in vivo imaging protocol contained adiabatic T1ρ and adiabatic T2ρ, both with hyperbolic secant (HS) 1 and 4 pulses, continuous wave T1ρ and conventional T2, together with cine imaging. Mice were imaged 2 h and 7 days after myocardial infarction. Mice were sacrificed at the 2-h or 7-day time point. Ex vivo measurements contained adiabatic T1ρ and adiabatic T2ρ with HS1 and HS4 pulses, continuous wave T1ρ, T1, and T2. After MRI studies, mouse hearts were fixed, and myocardial infarcts were verified using dystrophin and hematoxylin and eosin histology stainings. A clear difference between infarcted and normal myocardium was visible at the 2-h time point in rotating frame relaxation time mapping. Relative relaxation time difference in adiabatic T2ρ with HS4 pulse showed the significant differences between MI and control hearts in vivo. In addition, the results of adiabatic T1ρ with both HS1 and HS4 pulses and continuous wave T1ρ measurements showed significant differences between MI and control hearts at both time points in both in vivo and ex vivo measurements. This study shows that rotating frame relaxation time mappings have the potential to be noninvasive MR diagnostic markers for acute and subchronic myocardial infarcts.

Genetic variation in the 9p21.3 chromosomal region has one of the strongest associations known for coronary artery disease (CAD) that often leads to myocardial infarction (MI). This risk locus encodes a long noncoding RNA, ANRIL, which has been suggested to regulate the neighboring cyclin‐dependent kinase inhibitors 2A and B ( Cdkn2A/B ), the key regulators of cell proliferation. In this study, we aimed to clarify the role of the 9p21.3 risk locus in acute and chronic myocardial ischemia in mice. Mice carrying a deletion equivalent to the human CAD risk interval (Chr4 Δ70kb/Δ70kb ) and wild type mice were exposed to MI and followed until 5 days or 4 weeks. In the wild type mice, expression of a lncRNA, Ak148321, was increased after MI, and Cdkn2a was upregulated in chronic ischemia. Chr4 Δ70kb/Δ70kb downregulated both Cdkn2a/b , but this did not affect the survival or cardiac pathology after MI. These results suggest that the 9p21.3 locus is activated in response to cardiac ischemia. However, deficiency in the risk locus does not play a role in the cardiac pathophysiology in mice, supporting the studies suggesting the risk locus being more involved in the development of CAD, rather than the subsequent MI.

Introduction Nadofaragene firadenovec is a novel recombinant adenoviral vector-based intravesical gene therapeutic that delivers IFNA2 to urothelial cells, and was approved for the treatment of BCG-unresponsive NMIBC in 2022. Posttreatment biopsy of patients enrolled on the trial revealed relative PD1 and PD-L1 overexpression in tumor-infiltrating lymphocytes of nonresponders. In vivo orthotopic models replicated these findings, demonstrating PD1 overexpression in tumor-infiltrating T lymphocytes of mice following intravesical adenoviral-IFN treatment. Hence, we sought to examine the treatment efficacy of combination therapy with lentiviral (LV)-IFNα and PD-1 blockade in a murine bladder cancer model. We then examined the IFN-signaling and immune mechanisms responsible for the therapeutic effect of the combination therapy. Methods GFP/luciferase-labeled MB49 cells were administered intravesically into C57BL/6 female mice, and tumor growth was confirmed using bioluminescence imaging. Mice were randomized into one of six groups, including four treatment groups (LV-IFNα alone, anti-PD-1 alone, LV-Ctrl + anti-PD-1 and LV-IFNα + anti-PD-1), and two control groups (no treatment and LV-Ctrl). LV was delivered intravesically and anti-PD-1 mAB was administered via an intraperitoneal injection twice weekly. Mice were sacrificed when moribund or at the end of the 9-week study period. Bladder weights were recorded ex vivo and morphologic examination of specimens was performed to assess presence or absence of tumor. Overall survival (OS) was determined using Kaplan-Meier analysis. To assess changes induced by the combination therapy, we compared the gene signatures of 3 groups using RNAseq of tumors from the same murine model 72 hours post-treatment: 1) PD1 vs Ctrl, 2) LV-IFN vs LV-Ctrl, and 3) LV-IFN/PD1 vs LV-Ctrl/PD1. Results Mice treated with combination therapy (LV-IFNα + anti-PD-1) had the longest survival (log-rank p<0.0001). Bioluminescence decreased amongst all treatment groups compared to controls at week 2; however, durable treatment effect was noted only in the combination mice (week 3 and beyond). At necropsy, bladder tumors were evident on gross morphologic examination in all control mice, whereas no tumors were identified in 14%, 71%, and 86% of mice in the LV-IFNα, anti-PD-1 and LV-IFNα + anti-PD-1 groups, respectively (p<0.0001). IFN pathway and immune-related pathways were significantly upregulated in the combination group compared to either treatment alone. IFN pathways included cytokine storm, IL-1, and IL-6 signaling, among others. Immunological gene sets activated by combination treatment included those from both innate and adaptive immune pathways. Importantly, crosstalk between different immune compartments (such as dendritic cells and natural killer cells or lymphoid and non-lymphoid cells) was upregulated following treatment. Conclusions Combination therapy with LV-IFNα and anti-PD-1 antibody effectively eradicated bladder tumors in immune-competent mice and improved OS. Additionally, combination therapy resulted in upregulation of IFN and immune-related pathways. These findings suggest a therapeutic role for immune checkpoint inhibitors in BCG-unresponsive NMIBC patients treated with nadofaragene treatment.

Background and objective: Urinary tumor DNA (utDNA) profiling identifies mutations associated with urothelial carcinoma and can be used to detect minimal residual disease (MRD). We evaluate the utility of utDNA profiling to predict treatment failure in bacillus Methods: Urine was collected from participants prior to induction (n = 32) and at their 3-mo evaluation (n = 18) in the parallel-arm, phase 2 study (NCT01687244) of nadofaragene firadenovec. The UroAmp MRD assay (Convergent Genomics, South San Francisco, CA, USA) was used to perform utDNA testing. Risk of HG NMIBC recurrence was determined using two algorithm versions, and recurrence-free survival (RFS) was assessed using a Kaplan-Meier analysis. Key findings and limitations: TP53, TERT, PIK3CA, ARID1A, PLEKHS1, ELF3, and ERBB2 were the most prevalently mutated genes. With pretreatment urine, the validated MRD algorithm resulted in 12-mo RFS of 56% for negative and 22% for positive patients (p = 0.097). The experimental, enhanced algorithm classified two additional patients as positive, giving RFS of 71% for negative and 20% for positive patients (p = 0.012). With 3-mo urine, both algorithms gave RFS of 100% for negative and 38% for positive patients (p = 0.038). Longitudinal utDNA testing classified patients as negative (7%), complete responders (13%), partial responders (27%), unresponsive (20%), and expanding (33%). Conclusions and clinical implications: Urinary MRD testing after nadofaragene firadenovec induction provided statistically significant prognostication of recurrence among phase 2 trial participants.

Vascular endothelial growth factor B186 (VEGF-B186), a ligand for VEGF receptor 1 (VEGFR1) and neuropilin (NRP), promotes vascular growth in healthy and ischemic myocardium. However, the mechanisms and signaling of VEGF-B186 to support angiogenesis have remained unclear. We studied the effects of VEGF-B186 and its variant, VEGF-B186R127S, which cannot bind to NRPs, using VEGFR1 tyrosine kinase knockout (TK-/-) mice to explore the mechanism of VEGF-B186 in promoting vascular growth. Ultrasound-guided adenoviral VEGF-B186, VEGF-B186R127S, and control vector gene transfers were performed into VEGFR1 TK-/- mice hearts. In vitro studies in cardiac endothelial cells and further validation in normal and ischemic pig hearts, as well as in wild-type mice, were conducted. Both VEGF-B186 forms promoted vascular growth in VEGFR1 TK-/- mouse heart and increased the expression of proangiogenic and hematopoietic factors. Unlike VEGF-A, VEGF-B186 forms induced endoplasmic reticulum (ER) stress via the upregulation of Binding immunoglobulin Protein (BiP) as well as ER stress sensors (ATF6, PERK, IRE1α) through ITGAV and ITGA5 integrins, newly identified receptors for VEGF-B, activating the unfolded protein response (UPR) through XBP1. VEGFR1 and NRP are not essential for VEGF-B186-induced vascular growth. Instead, VEGF-B186 can stimulate cardiac regeneration through RGD-binding integrins and ER stress, suggesting a novel mechanism of action for VEGF-B186.