Contribution of VEGF-B-Induced Endocardial Endothelial Cell Lineage in Physiological Versus Pathological Cardiac Hypertrophy.

BACKGROUND: Preclinical studies have shown the therapeutic potential of VEGF-B (vascular endothelial growth factor B) in revascularization of the ischemic myocardium, but the associated cardiac hypertrophy and adverse side effects remain a concern. To understand the importance of endothelial proliferation and migration for the beneficial versus adverse effects of VEGF-B in the heart, we explored the cardiac effects of autocrine versus paracrine VEGF-B expression in transgenic and gene-transduced mice. METHODS: We used single-cell RNA sequencing to compare cardiac endothelial gene expression in VEGF-B transgenic mouse models. Lineage tracing was used to identify the origin of a VEGF-B-induced novel endothelial cell population and adeno-associated virus-mediated gene delivery to compare the effects of VEGF-B isoforms. Cardiac function was investigated using echocardiography, magnetic resonance imaging, and micro-computed tomography. RESULTS: Unlike in physiological cardiac hypertrophy driven by a cardiomyocyte-specific VEGF-B transgene (myosin heavy chain alpha-VEGF-B), autocrine VEGF-B expression in cardiac endothelium (aP2 [adipocyte protein 2]-VEGF-B) was associated with septal defects and failure to increase perfused subendocardial capillaries postnatally. Paracrine VEGF-B led to robust proliferation and myocardial migration of a novel cardiac endothelial cell lineage (VEGF-B-induced endothelial cells) of endocardial origin, whereas autocrine VEGF-B increased proliferation of VEGF-B-induced endothelial cells but failed to promote their migration and efficient contribution to myocardial capillaries. The surviving aP2-VEGF-B offspring showed an altered ratio of secreted VEGF-B isoforms and developed massive pathological cardiac hypertrophy with a distinct cardiac vessel pattern. In the normal heart, we found a small VEGF-B-induced endothelial cell population that was only minimally expanded during myocardial infarction but not during physiological cardiac hypertrophy associated with mouse pregnancy. CONCLUSIONS: Paracrine and autocrine secretions of VEGF-B induce expansion of a specific endocardium-derived endothelial cell population with distinct angiogenic markers. However, autocrine VEGF-B signaling fails to promote VEGF-B-induced endothelial cell migration and contribution to myocardial capillaries, predisposing to septal defects and inducing a mismatch between angiogenesis and myocardial growth, which results in pathological cardiac hypertrophy.

Single-Cell Gene-Regulatory Networks of Advanced Symptomatic Atherosclerosis.

BACKGROUND: While our understanding of the single-cell gene expression patterns underlying the transformation of vascular cell types during the progression of atherosclerosis is rapidly improving, the clinical and pathophysiological relevance of these changes remains poorly understood. METHODS: Single-cell RNA sequencing data generated with SmartSeq2 (≈8000 genes/cell) in 16 588 single cells isolated during atherosclerosis progression in Ldlr-/-Apob100/100 mice with human-like plasma lipoproteins and from humans with asymptomatic and symptomatic carotid plaques was clustered into multiple subtypes. For clinical and pathophysiological context, the advanced-stage and symptomatic subtype clusters were integrated with 135 tissue-specific (atherosclerotic aortic wall, mammary artery, liver, skeletal muscle, and visceral and subcutaneous, fat) gene-regulatory networks (GRNs) inferred from 600 coronary artery disease patients in the STARNET (Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task) study. RESULTS: Advanced stages of atherosclerosis progression and symptomatic carotid plaques were largely characterized by 3 smooth muscle cells (SMCs), and 3 macrophage subtype clusters with extracellular matrix organization/osteogenic (SMC), and M1-type proinflammatory/Trem2-high lipid-associated (macrophage) phenotypes. Integrative analysis of these 6 clusters with STARNET revealed significant enrichments of 3 arterial wall GRNs: GRN33 (macrophage), GRN39 (SMC), and GRN122 (macrophage) with major contributions to coronary artery disease heritability and strong associations with clinical scores of coronary atherosclerosis severity. The presence and pathophysiological relevance of GRN39 were verified in 5 independent RNAseq data sets obtained from the human coronary and aortic artery, and primary SMCs and by targeting its top-key drivers, FRZB and ALCAM in cultured human coronary artery SMCs. CONCLUSIONS: By identifying and integrating the most gene-rich single-cell subclusters of atherosclerosis to date with a coronary artery disease framework of GRNs, GRN39 was identified and independently validated as being critical for the transformation of contractile SMCs into an osteogenic phenotype promoting advanced, symptomatic atherosclerosis.

SIX2 promotes cell plasticity via Wnt/ß-catenin signalling in androgen receptor independent prostate cancer.

The use of androgen receptor (AR) inhibitors in prostate cancer gives rise to increased cellular lineage plasticity resulting in resistance to AR-targeted therapies. In this study, we examined the chromatin landscape of AR-positive prostate cancer cells post-exposure to the AR inhibitor enzalutamide. We identified a novel regulator of cell plasticity, the homeobox transcription factor SIX2, whose motif is enriched in accessible chromatin regions after treatment. Depletion of SIX2 in androgen-independent PC-3 prostate cancer cells induced a switch from a stem-like to an epithelial state, resulting in reduced cancer-related properties such as proliferation, colony formation, and metastasis both in vitro and in vivo. These effects were mediated through the downregulation of the Wnt/ß-catenin signalling pathway and subsequent reduction of nuclear ß-catenin. Collectively, our findings provide compelling evidence that the depletion of SIX2 may represent a promising strategy for overcoming the cell plasticity mechanisms driving antiandrogen resistance in prostate cancer.

Long-term continuous monitoring of arrhythmias in pigs with insertable cardiac monitors.

Arrhythmia detection is essential when assessing the safety of novel drugs and therapies in preclinical studies. Many short-term arrhythmia monitoring methods exist, including non-invasive ECG and Holter. However, there are no reliable, long-term, non-invasive, or minimally invasive methods for cardiac arrhythmia follow-up in large animals that allows free movement with littermates. A long follow-up time is needed when estimating the impact of long-lasting drugs or therapies, such as gene therapy. We evaluated the feasibility and performance of insertable cardiac monitors (ICMs) in pigs for minimally invasive, long-term monitoring of cardiac arrhythmias that allows free movement and species-specific behavior. Multiple implantation sites were tested to assess signal quality. ICMs recognized reliably many different arrhythmias but failed to detect single extrasystoles. They also over-diagnosed T-waves, resulting in oversensing. Muscle activity and natural startles of the animals caused noise, leading to a heterogeneous signal requiring post-recording evaluation. In spite of these shortcomings, the ICMs showed to be very useful for minimally invasive long-term monitoring of cardiac rhythm in pigs.

The Mineralocorticoid Receptors in diabetic kidney disease.

Diabetes mellitus is one of the leading causes of chronic kidney disease and its progression to end-stage kidney disease. Diabetic kidney disease (DKD) is characterized by glomerular hypertrophy, hyperfiltration, inflammation and the onset of albuminuria, together with a progressive reduction in glomerular filtration rate. This progression is further accompanied by tubulointerstitial inflammation and fibrosis. Factors such as genetic predisposition, epigenetic modifications, metabolic derangements, hemodynamic alterations, inflammation, and inappropriate renin-angiotensin-aldosterone system (RAAS) activity contribute to the onset and progression of DKD. In this context, decades of work have focused on glycemic and blood pressure reduction strategies, especially targeting the RAAS to slow disease progression. While much of the work has focused on targeting angiotensin II, emerging data support that the mineralocorticoid receptor (MR) is integral in the development and progression of DKD. Molecular mechanisms linked to the underlying pathophysiological changes derived from MR activation include vascular endothelial, as well as epithelial cell responses, to oxidative stress and inflammation. These responses lead to alterations in the microcirculatory environment, the abnormal release of extracellular vesicles, gut dysbiosis, epithelial-mesenchymal transition, and kidney fibrosis. Herein we present recent experimental and clinical evidence on the MR in DKD onset and progress along with new MR based strategies for the treatment and prevention of DKD.

Efficacy of Intravesical Nadofaragene Firadenovec for Patients With Bacillus Calmette-Guérin-Unresponsive Nonmuscle-Invasive Bladder Cancer: 5-Year Follow-Up From a Phase 3 Trial.

PURPOSE: Nadofaragene firadenovec-vncg is a nonreplicating adenoviral vector-based gene therapy for bacillus Calmette-Guérin (BCG)-unresponsive carcinoma in situ (CIS) with/without high-grade Ta/T1. We report outcomes following 5 years of planned follow-up. MATERIALS AND METHODS: This open-label phase 3 trial (NCT02773849) enrolled patients with BCG-unresponsive nonmuscle-invasive bladder cancer in 2 cohorts: CIS ± Ta/T1 (CIS; n = 107) and Ta/T1 without CIS (Ta/T1 cohort; n = 50). Patients received 75 mL (3 × 1011 vp/mL) nadofaragene firadenovec intravesically once every 3 months with cystoscopy and cytology assessments, with continued treatment offered to those remaining high grade recurrence-free (HGRF). RESULTS: One hundred fifty-seven patients were enrolled from 33 US sites (n = 151 included in efficacy analyses). Median follow-up was 50.8 months (interquartile range 39.1-60.0), with 27% receiving ≥ 5 instillations and 7.6% receiving treatment for ≥ 57 months. Of patients with CIS 5.8% (95% CI 2.2-12.2) were HGRF at month 57, and 15% (95% CI 6.1-27.8) of patients with high-grade Ta/T1 were HGRF at month 57. Kaplan-Meier-estimated HGRF survival at 57 months was 13% (95% CI 6.9-21.5) and 33% (95% CI 19.5-46.6) in the CIS and Ta/T1 cohorts, respectively. Cystectomy-free survival at month 60 was 49% (95% CI 40.0-57.1): 43% (95% CI 32.2-53.7) in the CIS cohort and 59% (95% CI 43.1-71.4) in the Ta/T1 cohort. Overall survival at 60 months was 80% (71.0, 86.0): 76% (64.6-84.5) and 86% (70.9-93.5) in the CIS and Ta/T1 cohorts, respectively. Only 5 patients (4 with CIS and 1 with Ta/T1) experienced clinical progression to muscle-invasive disease. CONCLUSIONS: At 60 months, nadofaragene firadenovec-vncg allowed bladder preservation in nearly half of the patients and proved to be a safe option for BCG-unresponsive nonmuscle-invasive bladder cancer.

Optimizing lentiviral vector formulation conditions for efficient ex vivo transduction of primary human T cells in chimeric antigen receptor T-cell manufacturing.

BACKGROUND AIMS: Chimeric antigen receptor (CAR) T-cell products are commonly generated using lentiviral vector (LV) transduction. Optimal final formulation buffer (FFB) supporting LV stability during cryostorage is crucial for cost-effective manufacturing. METHODS: To identify the ideal LV FFB composition for ex vivo CAR-T production, primary human T cells were transduced with vesicular stomatitis virus G-protein (VSV-G) -pseudotyped LVs (encoding a reporter gene or an anti-CD19-CAR). The formulations included phosphate-buffered saline (PBS), HEPES, or X-VIVOTM 15, and stabilizing excipients. The functional and viral particle titers and vector copy number were measured after LV cryopreservation and up to 24 h post-thaw incubation. CAR-Ts were produced with LVs in selected FFBs, and the resulting cells were characterized. RESULTS: Post-cryopreservation, HEPES-based FFBs provided higher LV functional titers than PBS and X-VIVOTM 15, and 10% trehalose-20 mM MgCl2 improved LV transduction efficiency in PBS and 50 mM HEPES. Thawed vectors remained stable at +4°C, while a ≤ 25% median decrease in the functional titer occurred during 24 h at room temperature. Tested excipients did not enhance LV post-thaw stability. CAR-Ts produced using LVs cryopreserved in 10% trehalose- or sucrose-20 mM MgCl2 in 50 mM HEPES showed comparable transduction rates, cell yield, viability, phenotype, and in vitro functionality. CONCLUSION: A buffer consisting of 10% trehalose-20 mM MgCl2 in 50 mM HEPES provided a feasible FFB to cryopreserve a VSV-G -pseudotyped LV for CAR-T-cell production. The LVs remained relatively stable for at least 24 h post-thaw, even at ambient temperatures. This study provides insights into process development, showing LV formulation data generated using the relevant target cell type for CAR-T-cell manufacturing.

Navigating the prime editing strategy to treat cardiovascular genetic disorders in transforming heart health.

INTRODUCTION: After understanding the genetic basis of cardiovascular disorders, the discovery of prime editing (PE), has opened new horizons for finding their cures. PE strategy is the most versatile editing tool to change cardiac genetic background for therapeutic interventions. The optimization of elements, prediction of efficiency, and discovery of the involved genes regulating the process have not been completed. The large size of the cargo and multi-elementary structure makes the in vivo heart delivery challenging. AREAS COVERED: Updated from recent published studies, the fundamentals of the PEs, their application in cardiology, potentials, shortcomings, and the future perspectives for the treatment of cardiac-related genetic disorders will be discussed. EXPERT OPINION: The ideal PE for the heart should be tissue-specific, regulatable, less immunogenic, high transducing, and safe. However, low efficiency, sup-optimal PE architecture, the large size of required elements, the unclear role of transcriptomics on the process, unpredictable off-target effects, and its context-dependency are subjects that need to be considered. It is also of great importance to see how beneficial or detrimental cell cycle or epigenomic modifier is to bring changes into cardiac cells. The PE delivery is challenging due to the size, multi-component properties of the editors and liver sink.

Insights into Prime Editing Technology: A Deep Dive into Fundamentals, Potentials, and Challenges.

As the most versatile and precise gene editing technology, prime editing (PE) can establish a durable cure for most human genetic disorders. Several generations of PE have been developed based on an editor machine or prime editing guide RNA (pegRNA) to achieve any kind of genetic correction. However, due to the early stage of development, PE complex elements need to be optimized for more efficient editing. Smart optimization of editor proteins as well as pegRNA has been contemplated by many researchers, but the universal PE machine's current shortcomings remain to be solved. The modification of PE elements, fine-tuning of the host genes, manipulation of epigenetics, and blockage of immune responses could be used to reach more efficient PE. Moreover, the host factors involved in the PE process, such as repair and innate immune system genes, have not been determined, and PE cell context dependency is still poorly understood. Regarding the large size of the PE elements, delivery is a significant challenge and the development of a universal viral or nonviral platform is still far from complete. PE versions with shortened variants of reverse transcriptase are still too large to fit in common viral vectors. Overall, PE faces challenges in optimization for efficiency, high context dependency during the cell cycling, and delivery due to the large size of elements. In addition, immune responses, unpredictability of outcomes, and off-target effects further limit its application, making it essential to address these issues for broader use in nonpersonalized gene editing. Besides, due to the limited number of suitable animal models and computational modeling, the prediction of the PE process remains challenging. In this review, the fundamentals of PE, including generations, potential, optimization, delivery, in vivo barriers, and the future landscape of the technology are discussed.

Cell-Specific Targeting of the Endothelium in the Cardiorenal Syndrome.

BACKGROUND: The vascular endothelium serves as a semi-selective permeable barrier as a conduit for transport of fluid, solutes, and various cell populations between the vessel lumen and tissues. The endothelium thus has a dynamic role in the regulation of coagulation, immune system, lipid and electrolyte transport, as well as neurohumoral influences on vascular tone and end-organ injury to tissues such as the heart and kidney. SUMMARY: Within this framework, pharmacologic strategies for heart and kidney diseases including blood pressure, glycemic control, and lipid reduction provide significant risk reduction, yet certain populations are at risk for substantial residual risk for disease progression and treatment resistance and often have unwanted off-target effects leaving the need for adjunct, alternative targeted therapies. Recent advances in techniques in sequencing and spatial transcriptomics have paved the way for the development of new therapies for targeting heart and kidney disease that include various gene, cell, and nano-based therapies. Cell-specific endothelium-specific targeting of viral vectors will enable their use for the treatment of heart and kidney diseases with gene therapy that can avoid unwanted off-target effects, improve treatment resistance, and reduce residual risk for disease progression. KEY MESSAGES: The vascular endothelium is an important therapeutic target for chronic kidney and cardiovascular diseases. Developing endothelial-specific gene therapies can benefit patients who develop resistance to current treatments.

Deletion of the Murine Ortholog of the Human 9p21.3 Locus Leads to Insulin Resistance and Obesity in Hypercholesterolemic Mice.

The 9p21.3 genomic locus is a hot spot for disease-associated single-nucleotide polymorphisms (SNPs), and its strongest associations are with coronary artery disease (CAD). The disease-associated SNPs are located within the sequence of a long noncoding RNA ANRIL, which potentially contributes to atherogenesis by regulating vascular cell stress and proliferation, but also affects pancreatic ß-cell proliferation. Altered expression of a neighboring gene, CDKN2B, has been also recognized to correlate with obesity and hepatic steatosis in people carrying the risk SNPs. In the present study, we investigated the impact of 9p21.3 on obesity accompanied by hyperlipidemia in mice carrying a deletion of the murine ortholog for the 9p21.3 (Chr4Δ70/Δ70) risk locus in hyperlipidemic Ldlr-/-ApoB100/100 background. The Chr4Δ70/Δ70 mice showed decreased mRNA expression of insulin receptors in white adipose tissue already at a young age, which developed into insulin resistance and obesity by aging. In addition, the Sirt1-Ppargc1a-Ucp2 pathway was downregulated together with the expression of Cdkn2b, specifically in the white adipose tissue in Chr4Δ70/Δ70 mice. These results suggest that the 9p21.3 locus, ANRIL lncRNA, and their murine orthologues may regulate the key energy metabolism pathways in a white adipose tissue-specific manner in the presence of hypercholesterolemia, thus contributing to the pathogenesis of metabolic syndrome.

Caridac vein retroinjections provide an efficient approach for global left ventricular gene transfer with adenovirus and adeno-associated virus.

Heart failure (HF) is a major burden worldwide, and new therapies are urgently needed. Gene therapy is a promising new approach to treat myocardial diseases. However, current cardiac gene delivery methods for producing global myocardial effects have been inefficient. The aim of this study was to develop an endovascular, reproducible, and clinically applicable gene transfer method for global left ventricular (LV) transduction. Domestic pigs (n = 52) were used for the experiments. Global LV myocardium coverage was achieved by three retrograde injections into the three main LV vein branches. The distribution outcome was significantly improved by simultaneous transient occlusions of the corresponding coronary arteries and the main anastomotic veins of the retroinjected veins. The achieved cardiac distribution was visualized first by administering Indian Ink solution. Secondly, AdLacZ (2 × 1012vp) and AAV2-GFP (2 × 1013vg) gene transfers were performed to study gene transduction efficacy of the method. By retrograde injections with simultaneous coronary arterial occlusions, both adenovirus (Ad) and adeno-associated virus (AAV) vectors were shown to deliver an efficient transduction of the LV. We conclude that retrograde injections into the three main LV veins is a potential new approach for a global LV gene transfer.

Translatome profiling reveals Itih4 as a novel smooth muscle cell-specific gene in atherosclerosis.

AIMS: Vascular smooth muscle cells (SMCs) and their derivatives are key contributors to the development of atherosclerosis. However, studying changes in SMC gene expression in heterogeneous vascular tissues is challenging due to the technical limitations and high cost associated with current approaches. In this paper, we apply translating ribosome affinity purification sequencing to profile SMC-specific gene expression directly from tissue. METHODS AND RESULTS: To facilitate SMC-specific translatome analysis, we generated SMCTRAP mice, a transgenic mouse line expressing enhanced green fluorescent protein (EGFP)-tagged ribosomal protein L10a (EGFP-L10a) under the control of the SMC-specific αSMA promoter. These mice were further crossed with the atherosclerosis model Ldlr-/-, ApoB100/100 to generate SMCTRAP-AS mice and used to profile atherosclerosis-associated SMCs in thoracic aorta samples of 15-month-old SMCTRAP and SMCTRAP-AS mice. Our analysis of SMCTRAP-AS mice showed that EGFP-L10a expression was localized to SMCs in various tissues, including the aortic wall and plaque. The TRAP fraction demonstrated high enrichment of known SMC-specific genes, confirming the specificity of our approach. We identified several genes, including Cemip, Lum, Mfge8, Spp1, and Serpina3, which are known to be involved in atherosclerosis-induced gene expression. Moreover, we identified several novel genes not previously linked to SMCs in atherosclerosis, such as Anxa4, Cd276, inter-alpha-trypsin inhibitor-4 (Itih4), Myof, Pcdh11x, Rab31, Serpinb6b, Slc35e4, Slc8a3, and Spink5. Among them, we confirmed the SMC-specific expression of Itih4 in atherosclerotic lesions using immunofluorescence staining of mouse aortic roots and spatial transcriptomics of human carotid arteries. Furthermore, our more detailed analysis of Itih4 showed its link to coronary artery disease through the colocalization of genome-wide association studies, splice quantitative trait loci (QTL), and protein QTL signals. CONCLUSION: We generated a SMC-specific TRAP mouse line to study atherosclerosis and identified Itih4 as a novel SMC-expressed gene in atherosclerotic plaques, warranting further investigation of its putative function in extracellular matrix stability and genetic evidence of causality.

DPYSL5 is highly expressed in treatment-induced neuroendocrine prostate cancer and promotes lineage plasticity via EZH2/PRC2.

Treatment-induced neuroendocrine prostate cancer (t-NEPC) is a lethal subtype of castration-resistant prostate cancer resistant to androgen receptor (AR) inhibitors. Our study unveils that AR suppresses the neuronal development protein dihydropyrimidinase-related protein 5 (DPYSL5), providing a mechanism for neuroendocrine transformation under androgen deprivation therapy. Our unique CRPC-NEPC cohort, comprising 135 patient tumor samples, including 55 t-NEPC patient samples, exhibits a high expression of DPYSL5 in t-NEPC patient tumors. DPYSL5 correlates with neuroendocrine-related markers and inversely with AR and PSA. DPYSL5 overexpression in prostate cancer cells induces a neuron-like phenotype, enhances invasion, proliferation, and upregulates stemness and neuroendocrine-related markers. Mechanistically, DPYSL5 promotes prostate cancer cell plasticity via EZH2-mediated PRC2 activation. Depletion of DPYSL5 decreases proliferation, induces G1 phase cell cycle arrest, reverses neuroendocrine phenotype, and upregulates luminal genes. In conclusion, DPYSL5 plays a critical role in regulating prostate cancer cell plasticity, and we propose the AR/DPYSL5/EZH2/PRC2 axis as a driver of t-NEPC progression.

ciRS-7 and miR-7 regulate ischemia-induced neuronal death via glutamatergic signaling.

Brain functionality relies on finely tuned regulation of gene expression by networks of non-coding RNAs (ncRNAs) such as the one composed by the circular RNA ciRS-7 (also known as CDR1as), the microRNA miR-7, and the long ncRNA Cyrano. We describe ischemia-induced alterations in the ncRNA network both in vitro and in vivo and in transgenic mice lacking ciRS-7 or miR-7. Our data show that cortical neurons downregulate ciRS-7 and Cyrano and upregulate miR-7 expression during ischemia. Mice lacking ciRS-7 exhibit reduced lesion size and motor impairment, while the absence of miR-7 alone results in increased ischemia-induced neuronal death. Moreover, miR-7 levels in pyramidal excitatory neurons regulate neurite morphology and glutamatergic signaling, suggesting a potential molecular link to the in vivo phenotype. Our data reveal the role of ciRS-7 and miR-7 in modulating ischemic stroke outcome, shedding light on the pathophysiological function of intracellular ncRNA networks in the brain.

Proangiogenic Growth Factor Therapy for the Treatment of Refractory Angina: A Meta-analysis.

Background: Refractory angina (RFA; limiting angina despite optimal medical therapy) is a growing, global problem, with limited treatment options. Therefore, we conducted a systematic review of randomized controlled trials (RCTs) to evaluate the effect of proangiogenic growth factor therapy (in the form of vascular growth factors delivered either as recombinant proteins or gene therapy) in patients with RFA ineligible for revascularization. Methods: We performed a meta-analysis (PROSPERO: CRD42018107283) of RCTs as per the Preferred Reporting Items for Systematic Reviews and Meta-Analyses methodology. A comprehensive search of the PubMed, CENTRAL, Embase, Cochrane, ClinicalTrials.gov and Google Scholar databases, as well as scientific session abstracts, were performed. The pooled outcomes included major adverse cardiac events (MACE), mortality, myocardial perfusion, and indices of angina severity (Canadian Cardiovascular Society angina class [CCS] and exercise tolerance). A prespecified subgroup analysis was performed for delivery method, vector, and protein type. The standardized mean difference (SMD) or odds ratio (OR) was calculated to assess relevant outcomes. We assessed heterogeneity using the χ2 and I2 tests. Results: We included 16 RCTs involving 1607 patients (1052 received proangiogenic growth factor therapy and 555 received a placebo or optimal medical therapy). Our analysis showed a significant decreased risk of MACE (OR, 0.72; 95% confidence interval [CI], 0.55-0.93) and significantly improved CCS class (SMD, -0.55; 95% CI, -1.10 to 0.00), but not mortality (OR, 0.66; 95% CI, 0.28-1.54) or exercise tolerance (SMD, 0.47; 95% CI, -0.14 to 1.09), in treated patients compared to those in the control group. Conclusions: Proangiogenic growth factor therapy is a promising treatment option for RFA, with beneficial effects seen on MACE and CCS class. The results of ongoing trials are needed before it can be considered for clinical practice.

Using the Jurkat reporter T cell line for evaluating the functionality of novel chimeric antigen receptors.

Background: T cells that are genetically modified with chimeric antigen receptor (CAR) hold promise for immunotherapy of cancer. Currently, there are intense efforts to improve the safety and efficacy of CAR T cell therapies against liquid and solid tumors. Earlier we designed a novel CAR backbone (FiCAR) where the spacer is derived from immunoglobulin (Ig) -like domains of the signal-regulatory protein alpha (SIRPα). However, the analysis of novel CAR using primary T cells is slow and laborious. Methods: To explore the versatility of the CAR backbone, we designed a set of variant FiCARs with different spacer lengths and targeting antigens. To expedite the analysis of the novel CARs, we transduced the FiCAR genes using lentiviruses into Jurkat reporter T cells carrying fluorescent reporter genes. The expression of fluorescent markers in response to FiCAR engagement with targets was analyzed by flow cytometry, and cytotoxicity was evaluated using killing assays. Furthermore, the killing mechanisms that are employed by FiCAR-equipped Jurkat T cells were investigated by flow cytometry, and the intracellular pathways involved in signaling by FiCAR were analyzed by phosphoproteomic analysis using mass spectrometry. Results: Seven different CARs were designed and transduced into Jurkat reporter cells. We show that the SIRPα derived FiCARs can be detected by flow cytometry using the SE12B6A4 antibody recognizing SIRPα. Furthermore, FiCAR engagement leads to robust activation of NFκß and NFAT signaling, as demonstrated by the expression of the fluorescent reporter genes. Interestingly, the Jurkat reporter system also revealed tonic signaling by a HER-2 targeting FiCAR. FiCAR-equipped Jurkat T cells were cytotoxic in cocultures with target cells and target cell engagement lead to an upregulation of CD107a on the Jurkat reporter T cell surface. Phosphoproteomic analyses confirmed signal transduction via the intracellular CD28/CD3ζ sequences upon the interaction of the FiCAR1 with its antigen. In addition, downstream signaling of CD3ζ/ZAP70- SLP-76-PLCγ, PI3K-AKT-NFκB pathways and activation of NFAT and AP-1 were observed. Conclusion: We conclude that the FiCAR backbone can be shortened and lengthened at will by engineering it with one to three SIRPα derived Ig-like domains, and the FiCARs are functional when equipped with different single chain variable fragment target binding domains. The Jurkat reporter system expedites the analysis of novel CARs as to their expression, signaling function, evaluation of tonic signaling issues and cytotoxic activity.

Strategies to improve safety profile of AAV vectors.

Adeno-associated virus (AAV) vectors are currently used in four approved gene therapies for Leber congenital amaurosis (Luxturna), spinal muscular atrophy (Zolgensma), aromatic L-amino acid decarboxylase deficiency (Upstaza) and Haemophilia A (Roctavian), with several more therapies being investigated in clinical trials. AAV gene therapy has long been considered extremely safe both in the context of immunotoxicity and genotoxicity, but recent tragic deaths in the clinical trials for X-linked myotubular myopathy and Duchenne's muscular dystrophy, together with increasing reports of potential hepatic oncogenicity in animal models have prompted re-evaluation of how much trust we can place on the safety of AAV gene therapy, especially at high doses. In this review we cover genome and capsid engineering strategies that can be used to improve safety of the next generation AAV vectors both in the context of immunogenicity and genotoxicity and discuss the gaps that need filling in our current knowledge about AAV vectors.

Novel modular chimeric antigen receptor spacer for T cells derived from signal regulatory protein alpha Ig-like domains.

Background: T cells equipped with chimeric antigen receptors (CAR) have shown remarkable efficacy in targeting B lineage malignancies. Improvement of the CAR structure is needed, however, with a view to developing flexibly modifiable spacers that are inert in interactions with unwanted cells. Specifically, binding to cells carrying receptors for IgG's crystallizable fragment (FcR), that recognize IgG-derived domains in CARs is to be avoided. Methods: Two novel CARs targeting the CD19 antigen where the IgG1-CH2 and -CH3 domains were replaced with Ig-like domains from signal-regulatory protein α (SIRPα) were designed in silico. An IgG1-based CAR and a CAR lacking both SIRPα and IgG1 domains were used as comparators. The phenotype and memory phenotype of the expanded cells were analyzed by flow cytometry, and CAR T cell activation and cytotoxic efficacy were assessed in co-culture experiments in response to CD19+ target cells. Unwanted interactions with FcR-expressing myeloid cells were interrogated in co-culture assays with THP-1 monocytic cells. Results: T cells carrying the novel SIRPα-based CARs enacted potent in vitro cytotoxicity against CD19 positive B-lineage leukemia cells, comparable to traditional IgG1-based CAR T cells. Co-culture of IgG1-based CAR T cells with FcR-expressing THP-1 monocytic cells led to prominent cell surface expression of CD69 on T cells together with production of Interleukin (IL)-2 and Interferon-γ, and production of IL-1ß, indicating activation of the T cells and monocytes, respectively. Longer co-culture led to killing of the monocytes. No signs of T cell nor monocyte activation were detected in co-cultures of SIRPα-based CAR T cells with THP-1 cells. Arming T cells with the SIRPα-based CARs favored differentiation towards CD4+ phenotype during expansion, while the effects on memory phenotype of the T cells were equivalent between the SIRPα- and IgG1-based CARs. In a pilot experiment, T cells modified with one of the SIRPα-based CARs showed dose dependent leukemia cell control. Conclusion: The novel SIRPα based spacers offer a suitable backbone for developing chimeric antigen receptors that evade the off-target binding to FcR while the cells retain a favorable memory phenotype and efficient cytotoxicity, establishing a promising candidate for future in vivo and clinical testing.

Epigenetics and atherosclerosis

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