Progress Toward a Gene Therapy for Arthritis.

Osteoarthritis (OA) is a highly prevalent, disabling, incurable, and expensive disease that is difficult to treat nonsurgically. The pharmacokinetics of drug delivery to joints are such that it is not possible to target antiarthritic agents, especially biologics, to individual joints with OA at sustained, therapeutic concentrations. More than 30 years ago, we proposed that local, intra-articular gene transfer can overcome this barrier to therapy by engineering articular cells to synthesize antiarthritic gene products endogenously. This article summarizes the progress toward this goal. Initially, a retroviral vector was used to deliver cDNA encoding the interleukin-1 receptor antagonist (IL-1Ra) to the joints of experimental animals. Using an ex vivo strategy, cultures of autologous synovial fibroblasts were genetically modified in cell culture and introduced into joints by means of intra-articular injection. Successful development of this technology led to the first-in-human gene therapy trial for arthritis. This Phase I study targeted metacarpophalangeal joints with rheumatoid arthritis. Although successful, for various reasons, subsequent research targeted OA and used adeno-associated virus as a vector to deliver IL-1Ra by direct in vivo injection into the joint. A Phase I human clinical trial has just been completed successfully in subjects with mid-stage OA of the knee, leading to a Phase Ib study that is in progress.

A nutrigeroscience approach: Dietary macronutrients and cellular senescence.

Cellular senescence, a process in which a cell exits the cell cycle in response to stressors, is one of the hallmarks of aging. Senescence and the senescence-associated secretory phenotype (SASP)-a heterogeneous set of secreted factors that disrupt tissue homeostasis and promote the accumulation of senescent cells-reprogram metabolism and can lead to metabolic dysfunction. Dietary interventions have long been studied as methods to combat age-associated metabolic dysfunction, promote health, and increase lifespan. A growing body of literature suggests that senescence is responsive to diet, both to calories and specific dietary macronutrients, and that the metabolic benefits of dietary interventions may arise in part through reducing senescence. Here, we review what is currently known about dietary macronutrients' effect on senescence and the SASP, the nutrient-responsive molecular mechanisms that may mediate these effects, and the potential for these findings to inform the development of a nutrigeroscience approach to healthy aging.

Guidelines for minimal information on cellular senescence experimentation in vivo.

Cellular senescence is a cell fate triggered in response to stress and is characterized by stable cell-cycle arrest and a hypersecretory state. It has diverse biological roles, ranging from tissue repair to chronic disease. The development of new tools to study senescence in vivo has paved the way for uncovering its physiological and pathological roles and testing senescent cells as a therapeutic target. However, the lack of specific and broadly applicable markers makes it difficult to identify and characterize senescent cells in tissues and living organisms. To address this, we provide practical guidelines called "minimum information for cellular senescence experimentation in vivo" (MICSE). It presents an overview of senescence markers in rodent tissues, transgenic models, non-mammalian systems, human tissues, and tumors and their use in the identification and specification of senescent cells. These guidelines provide a uniform, state-of-the-art, and accessible toolset to improve our understanding of cellular senescence in vivo.

Adoptive transfer of personalized neoantigen-reactive TCR-transduced T cells in metastatic colorectal cancer: phase 2 trial interim results.

Adoptive cell transfer (ACT) with neoantigen-reactive T lymphocytes can mediate cancer regression. Here we isolated unique, personalized, neoantigen-reactive T cell receptors (TCRs) from tumor-infiltrating lymphocytes of patients with metastatic gastrointestinal cancers and incorporated the TCR α and ß chains into gamma retroviral vectors. We transduced autologous peripheral blood lymphocytes and adoptively transferred these cells into patients after lymphodepleting chemotherapy. In a phase 2 single-arm study, we treated seven patients with metastatic, mismatch repair-proficient colorectal cancers who had progressive disease following multiple previous therapies. The primary end point of the study was the objective response rate as measured using RECIST 1.1, and the secondary end points were safety and tolerability. There was no prespecified interim analysis defined in this study. Three patients had objective clinical responses by RECIST criteria including regressions of metastases to the liver, lungs and lymph nodes lasting 4 to 7 months. All patients received T cell populations containing ≥50% TCR-transduced cells, and all T cell populations were polyfunctional in that they secreted IFNγ, GM-CSF, IL-2 and granzyme B specifically in response to mutant peptides compared with wild-type counterparts. TCR-transduced cells were detected in the peripheral blood of five patients, including the three responders, at levels ≥10% of CD3+ cells 1 month post-ACT. In one patient who responded to therapy, ~20% of CD3+ peripheral blood lymphocytes expressed transduced TCRs more than 2 years after treatment. This study provides early results suggesting that ACT with T cells genetically modified to express personalized neoantigen-reactive TCRs can be tolerated and can mediate tumor regression in patients with metastatic colorectal cancers. ClinicalTrials.gov registration: NCT03412877 .

Developing transcriptomic signatures as a biomarker of cellular senescence.

Cellular senescence is a cell fate driven by different types of stress, where damaged cells exit from the cell cycle and, in many cases, develop an inflammatory senescence-associated secretory phenotype (SASP). Senescence has often been linked to driving aging and the onset of multiple diseases conferred by the harmful SASP, which disrupts tissue homeostasis and impairs the regular function of many tissues. This phenomenon was first observed in vitro when fibroblasts halted replication after approximately 50 population doublings. In addition to replication-induced senescence, factors such as DNA damage and oncogene activation can induce cellular senescence both in culture and in vivo. Despite their contribution to aging and disease, identifying senescent cells in vivo has been challenging due to their heterogeneity. Although senescent cells can express the cell cycle inhibitors p16Ink4a and/or p21Cip1 and exhibit SA-ß-gal activity and evidence of a DNA damage response, there is no universal biomarker for these cells, regardless of inducer or cell type. Recent studies have analyzed the transcriptomic characteristics of these cells, leading to the identification of signature gene sets like CellAge, SeneQuest, and SenMayo. Advancements in single-cell and spatial RNA sequencing now allow for analyzing senescent cell heterogeneity within the same tissue and the development of machine learning algorithms, e.g., SenPred, SenSig, and SenCID, to discover cellular senescence using RNA sequencing data. Such insights not only deepen our understanding of the genetic pathways driving cellular senescence, but also promote the development of its quantifiable biomarkers. This review summarizes the current knowledge of transcriptomic signatures of cellular senescence and their potential as in vivo biomarkers.

Religious participation is associated with fewer dementia diagnoses among Black people in the United States.

INTRODUCTION: Black people had the highest prevalence of Alzheimer's disease and related dementias (ADRD) of any racial/ethnic group in the United States (US) as of 2020. As racial disparities in the prevalence of ADRD are being investigated, more evidence is necessary to determine the pathways and mechanisms that either slow ADRD progression or improve quality of life for those affected. Religion/spirituality (R/S) has been shown to affect health outcomes but has rarely been studied as a possible pathway for reducing ADRD risk. Crucially, Black people also report higher levels of R/S than other racial/ethnic groups in the United States. This research asks if R/S affects ADRD risk among Black adults and if any effects persist after controlling for hypertension. METHODS: We conducted a secondary data analysis drawing from the Health and Retirement Study (HRS), a nationally representative longitudinal dataset with an oversampling of Black adults. RESULTS: We used logistic regression analysis to demonstrate how R/S has an ameliorating impact on ADRD risk among Black people, even after controlling for hypertension. Those who never attended religious services had 2.37 higher odds of being diagnosed with ADRD than those who attended more than once a week. Further, as R/S attendance increased, ADRD risk decreased linearly. CONCLUSION: These findings demonstrate the importance that existing cultural networks (e.g., R/S) can have for reducing ADRD burden for Black people and has important implications for the role of R/S in shaping ADRD symptomatology.

Structural characterization and AlphaFold modeling of human T cell receptor recognition of NRAS cancer neoantigens.

T cell receptors (TCRs) that recognize cancer neoantigens are important for anti-cancer immune responses and immunotherapy. Understanding the structural basis of TCR recognition of neoantigens provides insights into their exquisite specificity and can enable design of optimized TCRs. We determined crystal structures of a human TCR in complex with NRAS Q61K and Q61R neoantigen peptides and HLA-A1 MHC, revealing the molecular underpinnings for dual recognition and specificity versus wild-type NRAS peptide. We then used multiple versions of AlphaFold to model the corresponding complex structures, given the challenge of immune recognition for such methods. Interestingly, one implementation of AlphaFold2 (TCRmodel2) was able to generate accurate models of the complexes, while AlphaFold3 also showed strong performance, although success was lower for other complexes. This study provides insights into TCR recognition of a shared cancer neoantigen, as well as the utility and practical considerations for using AlphaFold to model TCR-peptide-MHC complexes.

Neoantigen-specific stimulation of tumor-infiltrating lymphocytes enables effective TCR isolation and expansion while preserving stem-like memory phenotypes.

BACKGROUND: Tumor-infiltrating lymphocytes (TILs) targeting neoantigens can effectively treat a selected set of metastatic solid cancers. However, harnessing TILs for cancer treatments remains challenging because neoantigen-reactive T cells are often rare and exhausted, and ex vivo expansion can further reduce their frequencies. This complicates the identification of neoantigen-reactive T-cell receptors (TCRs) and the development of TIL products with high reactivity for patient treatment. METHODS: We tested whether TILs could be in vitro stimulated against neoantigens to achieve selective expansion of neoantigen-reactive TILs. Given their prevalence, mutant p53 or RAS were studied as models of human neoantigens. An in vitro stimulation method, termed "NeoExpand", was developed to provide neoantigen-specific stimulation to TILs. 25 consecutive patient TILs from tumors harboring p53 or RAS mutations were subjected to NeoExpand. RESULTS: We show that neoantigenic stimulation achieved selective expansion of neoantigen-reactive TILs and broadened the neoantigen-reactive CD4+ and CD8+ TIL clonal repertoire. This allowed the effective isolation of novel neoantigen-reactive TCRs. Out of the 25 consecutive TIL samples, neoantigenic stimulation enabled the identification of 16 unique reactivities and 42 TCRs, while conventional TIL expansion identified 9 reactivities and 14 TCRs. Single-cell transcriptome analysis revealed that neoantigenic stimulation increased neoantigen-reactive TILs with stem-like memory phenotypes expressing IL-7R, CD62L, and KLF2. Furthermore, neoantigenic stimulation improved the in vivo antitumor efficacy of TILs relative to the conventional OKT3-induced rapid TIL expansion in p53-mutated or KRAS-mutated xenograft mouse models. CONCLUSIONS: Taken together, neoantigenic stimulation of TILs selectively expands neoantigen-reactive TILs by frequencies and by their clonal repertoire. NeoExpand led to improved phenotypes and functions of neoantigen-reactive TILs. Our data warrant its clinical evaluation. TRIAL REGISTRATION NUMBER: NCT00068003, NCT01174121, and NCT03412877.

Clinical validation of C12FDG as a marker associated with senescence and osteoarthritic phenotypes.

Chronic conditions associated with aging have proven difficult to prevent or treat. Senescence is a cell fate defined by loss of proliferative capacity and the development of a pro-inflammatory senescence-associated secretory phenotype comprised of cytokines/chemokines, proteases, and other factors that promotes age-related diseases. Specifically, an increase in senescent peripheral blood mononuclear cells (PBMCs), including T cells, is associated with conditions like frailty, rheumatoid arthritis, and bone loss. However, it is unknown if the percentage of senescent PBMCs associated with age-associated orthopedic decline could be used for potential diagnostic or prognostic use in orthopedics. Here, we report senescent cell detection using the fluorescent compound C12FDG to quantify PBMCs senescence across a large cohort of healthy and osteoarthritic patients. There is an increase in the percent of circulating C12FDG+ PBMCs that is commensurate with increases in age and senescence-related serum biomarkers. Interestingly, C12FDG+ PBMCs and T cells also were found to be elevated in patients with mild to moderate osteoarthritis, a progressive joint disease that is strongly associated with inflammation. The percent of C12FDG+ PBMCs and age-related serum biomarkers were decreased in a small subgroup of study participants taking the senolytic drug fisetin. These results demonstrate quantifiable measurements in a large group of participants that could create a composite score of healthy aging sensitive enough to detect changes following senolytic therapy and may predict age-related orthopedic decline. Detection of peripheral senescence in PBMCs and subsets using C12FDG may be clinically useful for quantifying cellular senescence and determining how and if it plays a pathological role in osteoarthritic progression.

Cognitive Decline and Other Late-Stage Neurologic Complications in Cockayne Syndrome.

Background and Objectives: Cockayne syndrome (CS) is an ultra-rare, autosomal recessive, premature aging disorder characterized by impaired growth, neurodevelopmental delays, neurodegeneration, polyneuropathy, and other multiorgan system complications. The anatomic aspects of CS neurodegeneration have long been known from postmortem examinations and MRI studies, but the clinical features of this neurodegeneration are not well characterized, especially at later stages of the disease. Methods: This was a retrospective observational study in which individuals with CS who survived beyond 18 years were ascertained at 3 centers in the United States, France, and the United Kingdom. Medical records were examined to determine the frequencies and features of the following neurologic complications: neurocognitive/neuropsychiatric decline (8 symptoms), tremors, neuropathy, seizures, and strokes. Results: Among 18 individuals who met inclusion criteria, all but one (94.4%) experienced at least one symptom of neurocognitive/neuropsychiatric decline, with most individuals experiencing at least half of those symptoms. Most participants experienced tremors and peripheral neuropathy, with a few experiencing seizures and strokes. For individuals with available data, 100.0% were reported to have gait ataxia and neuroimaging showed that 85.7% had generalized cerebral atrophy on MRI while 78.6% had white matter changes. Discussion: Symptoms of neurocognitive/neuropsychiatric decline are nearly universal in our cohort of adults with CS, suggesting that these individuals are at risk of developing neurocognitive/neuropsychiatric decline, with symptoms related to but not specific to dementia. Considering the prominent role of DNA repair defects in CS disease mechanisms and emerging evidence for increased DNA damage in neurodegenerative disease, impaired genome maintenance may be a shared pathway underlying multiple forms of neurocognitive/neuropsychiatric decline. Components of the DNA damage response mechanism may bear further study as potential therapeutic targets that could alleviate neurocognitive/neuropsychiatric symptoms in CS and other neurodegenerative disorders.

Integrative Analyses of Tumor and Peripheral Biomarkers in the Treatment of Advanced Renal Cell Carcinoma.

The phase III JAVELIN Renal 101 trial demonstrated prolonged progression-free survival (PFS) in patients (N = 886) with advanced renal cell carcinoma treated with first-line avelumab + axitinib (A+Ax) versus sunitinib. We report novel findings from integrated analyses of longitudinal blood samples and baseline tumor tissue. PFS was associated with elevated lymphocyte levels in the sunitinib arm and an abundance of innate immune subsets in the A+Ax arm. Treatment with A+Ax led to greater T-cell repertoire modulation and less change in T-cell numbers versus sunitinib. In the A+Ax arm, patients with tumors harboring mutations in ≥2 of 10 previously identified PFS-associated genes (double mutants) had distinct circulating and tumor-infiltrating immunologic profiles versus those with wild-type or single-mutant tumors, suggesting a role for non-T-cell-mediated and non-natural killer cell-mediated mechanisms in double-mutant tumors. We provide evidence for different immunomodulatory mechanisms based on treatment (A+Ax vs. sunitinib) and tumor molecular subtypes. SIGNIFICANCE: Our findings provide novel insights into the different immunomodulatory mechanisms governing responses in patients treated with avelumab (PD-L1 inhibitor) + axitinib or sunitinib (both VEGF inhibitors), highlighting the contribution of tumor biology to the complexity of the roles and interactions of infiltrating immune cells in response to these treatment regimens. This article is featured in Selected Articles from This Issue, p. 384.

Senotherapeutic drug treatment ameliorates chemotherapy-induced cachexia.

Cachexia is a debilitating skeletal muscle wasting condition for which we currently lack effective treatments. In the context of cancer, certain chemotherapeutics cause DNA damage and cellular senescence. Senescent cells exhibit chronic activation of the transcription factor NF-κB, a known mediator of the proinflammatory senescence-associated secretory phenotype (SASP) and skeletal muscle atrophy. Thus, targeting NF-κB represents a logical therapeutic strategy to alleviate unintended consequences of genotoxic drugs. Herein, we show that treatment with the IKK/NF-κB inhibitor SR12343 during a course of chemotherapy reduces markers of cellular senescence and the SASP in liver, skeletal muscle, and circulation and, correspondingly, attenuates features of skeletal muscle pathology. Lastly, we demonstrate that SR12343 mitigates chemotherapy-induced reductions in body weight, lean mass, fat mass, and muscle strength. These findings support senescent cells as a promising druggable target to counteract the SASP and skeletal muscle wasting in the context of chemotherapy.

Astaxanthin and meclizine extend lifespan in UM-HET3 male mice; fisetin, SG1002 (hydrogen sulfide donor), dimethyl fumarate, mycophenolic acid, and 4-phenylbutyrate do not significantly affect lifespan in either sex at the doses and schedules used.

In genetically heterogeneous (UM-HET3) mice produced by the CByB6F1 × C3D2F1 cross, the Nrf2 activator astaxanthin (Asta) extended the median male lifespan by 12% (p = 0.003, log-rank test), while meclizine (Mec), an mTORC1 inhibitor, extended the male lifespan by 8% (p = 0.03). Asta was fed at 1840 ± 520 (9) ppm and Mec at 544 ± 48 (9) ppm, stated as mean ± SE (n) of independent diet preparations. Both were started at 12 months of age. The 90th percentile lifespan for both treatments was extended in absolute value by 6% in males, but neither was significant by the Wang-Allison test. Five other new agents were also tested as follows: fisetin, SG1002 (hydrogen sulfide donor), dimethyl fumarate, mycophenolic acid, and 4-phenylbutyrate. None of these increased lifespan significantly at the dose and method of administration tested in either sex. Amounts of dimethyl fumarate in the diet averaged 35% of the target dose, which may explain the absence of lifespan effects. Body weight was not significantly affected in males by any of the test agents. Late life weights were lower in females fed Asta and Mec, but lifespan was not significantly affected in these females. The male-specific lifespan benefits from Asta and Mec may provide insights into sex-specific aspects of aging.

Phenotypic signatures of circulating neoantigen-reactive CD8+ T cells in patients with metastatic cancers.

Circulating T cells from peripheral blood (PBL) can provide a rich and noninvasive source for antitumor T cells. By single-cell transcriptomic profiling of 36 neoantigen-specific T cell clones from 6 metastatic cancer patients, we report the transcriptional and cell surface signatures of antitumor PBL-derived CD8+ T cells (NeoTCRPBL). Comparison of tumor-infiltrating lymphocyte (TIL)- and PBL-neoantigen-specific T cells revealed that NeoTCRPBL T cells are low in frequency and display less-dysfunctional memory phenotypes relative to their TIL counterparts. Analysis of 100 antitumor TCR clonotypes indicates that most NeoTCRPBL populations target the same neoantigens as TILs. However, NeoTCRPBL TCR repertoire is only partially shared with TIL. Prediction and testing of NeoTCRPBL signature-derived TCRs from PBL of 6 prospective patients demonstrate high enrichment of clonotypes targeting tumor mutations, a viral oncogene, and patient-derived tumor. Thus, the NeoTCRPBL signature provides an alternative source for identifying antitumor T cells from PBL of cancer patients, enabling immune monitoring and immunotherapies.

Failure to repair endogenous DNA damage in ß-cells causes adult-onset diabetes in mice.

Age is the greatest risk factor for the development of type 2 diabetes mellitus (T2DM). Age-related decline in organ function is attributed to the accumulation of stochastic damage, including damage to the nuclear genome. Islets of T2DM patients display increased levels of DNA damage. However, whether this is a cause or consequence of the disease has not been elucidated. Here, we asked if spontaneous, endogenous DNA damage in ß-cells can drive ß-cell dysfunction and diabetes, via deletion of Ercc1, a key DNA repair gene, in ß-cells. Mice harboring Ercc1-deficient ß-cells developed adult-onset diabetes as demonstrated by increased random and fasted blood glucose levels, impaired glucose tolerance, and reduced insulin secretion. The inability to repair endogenous DNA damage led to an increase in oxidative DNA damage and apoptosis in ß-cells and a significant loss of ß-cell mass. Using electron microscopy, we identified ß-cells in clear distress that showed an increased cell size, enlarged nuclear size, reduced number of mature insulin granules, and decreased number of mitochondria. Some ß-cells were more affected than others consistent with the stochastic nature of spontaneous DNA damage. Ercc1-deficiency in ß-cells also resulted in loss of ß-cell function as glucose-stimulated insulin secretion and mitochondrial function were impaired in islets isolated from mice harboring Ercc1-deficient ß-cells. These data reveal that unrepaired endogenous DNA damage is sufficient to drive ß-cell dysfunction and provide a mechanism by which age increases the risk of T2DM.

Distinct secretomes in p16- and p21- positive senescent cells across tissues.

Senescent cells drive age-related tissue dysfunction via the induction of a chronic senescenceassociated secretory phenotype (SASP). The cyclin-dependent kinase inhibitors p21Cip1 and p16Ink4a have long served as markers of cellular senescence. However, their individual roles remain incompletely elucidated. Thus, we conducted a comprehensive examination of multiple single-cell RNA sequencing (scRNA-seq) datasets spanning both murine and human tissues during aging. Our analysis revealed that p21Cip1 and p16Ink4a transcripts demonstrate significant heterogeneity across distinct cell types and tissues, frequently exhibiting a lack of co-expression. Moreover, we identified tissue-specific variations in SASP profiles linked to p21Cip1 or p16Ink4a expression. Our study underscores the extraordinary diversity of cellular senescence and the SASP, emphasizing that these phenomena are inherently cell- and tissue-dependent. However, a few SASP factors consistently contribute to a shared "core" SASP. These findings highlight the need for a more nuanced investigation of senescence across a wide array of biological contexts.

The regulation of Insulin/IGF-1 signaling by miR-142-3p associated with human longevity.

MicroRNAs (miRNAs) have been demonstrated to modulate life span in the invertebrates C. elegans and Drosophila by targeting conserved pathways of aging, such as insulin/IGF-1 signaling (IIS). However, a role for miRNAs in modulating human longevity has not been fully explored. Here we investigated novel roles of miRNAs as a major epigenetic component of exceptional longevity in humans. By profiling the miRNAs in B-cells from Ashkenazi Jewish centenarians and 70-year-old controls without a longevity history, we found that the majority of differentially expressed miRNAs were upregulated in centenarians and predicted to modulate the IIS pathway. Notably, decreased IIS activity was found in B cells from centenarians who harbored these upregulated miRNAs. miR-142-3p, the top upregulated miRNA, was verified to dampen the IIS pathway by targeting multiple genes including GNB2, AKT1S1, RHEB and FURIN . Overexpression of miR-142-3p improved the stress resistance under genotoxicity and induced the impairment of cell cycle progression in IMR90 cells. Furthermore, mice injected with a miR-142-3p mimic showed reduced IIS signaling and improved longevity-associated phenotypes including enhanced stress resistance, improved diet/aging-induced glucose intolerance, and longevity-associated change of metabolic profile. These data suggest that miR-142-3p is involved in human longevity through regulating IIS-mediated pro-longevity effects. This study provides strong support for the use of miR-142-3p as a novel therapeutic to promote longevity or prevent aging/aging-related diseases in human.

Using patient-derived tumor organoids from common epithelial cancers to analyze personalized T-cell responses to neoantigens.

Adoptive cell transfer of tumor-infiltrating lymphocytes (TIL) can mediate durable complete responses in some patients with common epithelial cancers but does so infrequently. A better understanding of T-cell responses to neoantigens and tumor-related immune evasion mechanisms requires having the autologous tumor as a reagent. We investigated the ability of patient-derived tumor organoids (PDTO) to fulfill this need and evaluated their utility as a tool for selecting T-cells for adoptive cell therapy. PDTO established from metastases from patients with colorectal, breast, pancreatic, bile duct, esophageal, lung, and kidney cancers underwent whole exomic sequencing (WES), to define mutations. Organoids were then evaluated for recognition by autologous TIL or T-cells transduced with cloned T-cell receptors recognizing defined neoantigens. PDTO were also used to identify and clone TCRs from TIL targeting private neoantigens and define those tumor-specific targets. PDTO were successfully established in 38/47 attempts. 75% were available within 2 months, a timeframe compatible with screening TIL for clinical administration. These lines exhibited good genetic fidelity with their parental tumors, especially for mutations with higher clonality. Immunologic recognition assays demonstrated instances of HLA allelic loss not found by pan-HLA immunohistochemistry and in some cases WES of fresh tumor. PDTO could also be used to show differences between TCRs recognizing the same antigen and to find and clone TCRs recognizing private neoantigens. PDTO can detect tumor-specific defects blocking T-cell recognition and may have a role as a selection tool for TCRs and TIL used in adoptive cell therapy.