Modular design of synthetic receptors for programmed gene regulation in cell therapies.

Synthetic biology has established powerful tools to precisely control cell function. Engineering these systems to meet clinical requirements has enormous medical implications. Here, we adopted a clinically driven design process to build receptors for the autonomous control of therapeutic cells. We examined the function of key domains involved in regulated intramembrane proteolysis and showed that systematic modular engineering can generate a class of receptors that we call synthetic intramembrane proteolysis receptors (SNIPRs) that have tunable sensing and transcriptional response abilities. We demonstrate the therapeutic potential of the receptor platform by engineering human primary T cells for multi-antigen recognition and production of dosed, bioactive payloads relevant to the treatment of disease. Our design framework enables the development of fully humanized and customizable transcriptional receptors for the programming of therapeutic cells suitable for clinical translation.

Naturally occurring T cell mutations enhance engineered T cell therapies.

Adoptive T cell therapies have produced exceptional responses in a subset of patients with cancer. However, therapeutic efficacy can be hindered by poor T cell persistence and function1. In human T cell cancers, evolution of the disease positively selects for mutations that improve fitness of T cells in challenging situations analogous to those faced by therapeutic T cells. Therefore, we reasoned that these mutations could be co-opted to improve T cell therapies. Here we systematically screened the effects of 71 mutations from T cell neoplasms on T cell signalling, cytokine production and in vivo persistence in tumours. We identify a gene fusion, CARD11-PIK3R3, found in a CD4+ cutaneous T cell lymphoma2, that augments CARD11-BCL10-MALT1 complex signalling and anti-tumour efficacy of therapeutic T cells in several immunotherapy-refractory models in an antigen-dependent manner. Underscoring its potential to be deployed safely, CARD11-PIK3R3-expressing cells were followed up to 418 days after T cell transfer in vivo without evidence of malignant transformation. Collectively, our results indicate that exploiting naturally occurring mutations represents a promising approach to explore the extremes of T cell biology and discover how solutions derived from evolution of malignant T cells can improve a broad range of T cell therapies.

Toward the clinical development of synthetic immunity to cancer.

Synthetic biology (synbio) tools, such as chimeric antigen receptors (CARs), have been designed to target, activate, and improve immune cell responses to tumors. These therapies have demonstrated an ability to cure patients with blood cancers. However, there are significant challenges to designing, testing, and efficiently translating these complex cell therapies for patients who do not respond or have immune refractory solid tumors. The rapid progress of synbio tools for cell therapy, particularly for cancer immunotherapy, is encouraging but our development process should be tailored to increase translational success. Particularly, next-generation cell therapies should be rooted in basic immunology, tested in more predictive preclinical models, engineered for potency with the right balance of safety, educated by clinical findings, and multi-faceted to combat a range of suppressive mechanisms. Here, we lay out five principles for engineering future cell therapies to increase the probability of clinical impact, and in the context of these principles, we provide an overview of the current state of synbio cell therapy design for cancer. Although these principles are anchored in engineering immune cells for cancer therapy, we posit that they can help guide translational synbio research for broad impact in other disease indications with high unmet need.

Open science, communal culture, and women's participation in the movement to improve science.

Science is undergoing rapid change with the movement to improve science focused largely on reproducibility/replicability and open science practices. This moment of change-in which science turns inward to examine its methods and practices-provides an opportunity to address its historic lack of diversity and noninclusive culture. Through network modeling and semantic analysis, we provide an initial exploration of the structure, cultural frames, and women's participation in the open science and reproducibility literatures (n = 2,926 articles and conference proceedings). Network analyses suggest that the open science and reproducibility literatures are emerging relatively independently of each other, sharing few common papers or authors. We next examine whether the literatures differentially incorporate collaborative, prosocial ideals that are known to engage members of underrepresented groups more than independent, winner-takes-all approaches. We find that open science has a more connected, collaborative structure than does reproducibility. Semantic analyses of paper abstracts reveal that these literatures have adopted different cultural frames: open science includes more explicitly communal and prosocial language than does reproducibility. Finally, consistent with literature suggesting the diversity benefits of communal and prosocial purposes, we find that women publish more frequently in high-status author positions (first or last) within open science (vs. reproducibility). Furthermore, this finding is further patterned by team size and time. Women are more represented in larger teams within reproducibility, and women's participation is increasing in open science over time and decreasing in reproducibility. We conclude with actionable suggestions for cultivating a more prosocial and diverse culture of science.

Multimodal Facial Aesthetic Treatment on the Appearance of Aging, Social Confidence, and Psychological Well-being: HARMONY Study.

BACKGROUND: A global approach to facial rejuvenation involves multiple treatment modalities. OBJECTIVES: The aim of this study was to evaluate the impact of multimodal facial aesthetic treatment on self-reported psychological and social outcomes. METHODS: HARMONY, a prospective, multicenter, 4-month study, enrolled patients aged 35 to 65 years to receive on-label treatment with a combination of hyaluronic fillers (VYC-20L, HYC-24L, and/or HYC-24L+), onabotulinumtoxinA, and bimatoprost. Fillers were injected on Day 1, with touch-ups performed on Day 14. OnabotulinumtoxinA was injected at Month 3 into glabellar lines and/or crow's feet lines. Patients applied bimatoprost to eyelashes once daily for 17 weeks. Mean change from baseline on FACE-Q Psychological Well-being and Social Confidence Scales, FACE-Q Aging Appearance Appraisal Scale, and FACE-Q Age Appraisal Visual Analog Scale were assessed. RESULTS: Of 100 patients treated, 93 were evaluated at 4 months posttreatment. Significant improvement vs baseline was observed on the FACE-Q Scales for Psychological Well-being (mean change, -19.9; P < 0.00001), Social Confidence (mean change, -18.2; P < 0.00001), and Aging Appearance (mean change, -28.5; P < 0.0001). On average, patients' self-assessed age was 0.1 years older than actual age at baseline and 4.5 years younger at Month 4 (P < 0.001 vs baseline). Forty-two patients experienced adverse events, all mild to moderate. CONCLUSIONS: Multimodal, full facial aesthetic treatment improves patients' self-reported psychological well-being, social confidence, aging appearance, and perceptions of chronologic age.

The Facial Aging Process From the "Inside Out".

The normal course of aging alters the harmonious, symmetrical, and balanced facial features found in youth, not only impacting physical attractiveness but also influencing self-esteem and causing miscommunication of affect based on facial miscues. With this evidence-based paper, the authors aim to provide a comprehensive overview of the latest research on the etiology and progression of facial aging by explaining the aging process from the "inside out," that is, from the bony platform to the skin envelope. A general overview of the changes occurring within each of the main layers of the facial anatomy is presented, including facial skeleton remodeling, fat pad atrophy or repositioning, changes in muscle tone and thickness, and weakening and thinning of the skin. This is followed by an in-depth analysis of specific aging regions by facial thirds (upper, middle, and lower thirds). This review may help aesthetic physicians in the interpretation of the aging process and in prioritizing and rationalizing treatment decisions to establish harmonious facial balance in younger patients or to restore balance lost with age in older patients.

Efficacy, Patient-Reported Outcomes, and Safety for Millennial Subjects Treated With OnabotulinumtoxinA for Moderate to Severe Horizontal Forehead Lines.

BACKGROUND: Millennials (aged 18-34 years) represent a growing segment of the facial aesthetic market. OBJECTIVE: To evaluate investigator-assessed efficacy, patient-reported outcomes (PROs), and safety for millennials versus subjects aged at least 35 years after onabotulinumtoxinA treatment of forehead lines (FHL) across 2 phase 3 studies. METHODS: Eligible subjects with moderate to severe FHL received onabotulinumtoxinA (FHL: 20 U; glabellar lines: 20 U, with/without 24 U in crow's feet line regions) or placebo. All findings were pooled by the age group. RESULTS: Millennials composed 15% of subjects (176/1,178). Day 30 responder rates of at least 1-grade Facial Wrinkle Scale improvement in FHL severity for millennials versus subjects aged 35 years and older were 100% versus 97.8% at maximum eyebrow elevation and 78.4% versus 83.5% at rest, respectively. Responder rates were significantly greater with onabotulinumtoxinA than placebo (p ≤ .015) for both groups through Day 180. Similar trends were observed for achieving none/mild severity. Both age groups reported high satisfaction rates and improved psychological impacts with onabotulinumtoxinA treatment. No new safety signals were detected. CONCLUSION: OnabotulinumtoxinA treatment was well tolerated, and both age groups experienced significant improvements in FHL severity, high satisfaction, and improved psychological impacts after treatment. Millennials reported numerically greater improvements.

OnabotulinumtoxinA for Simultaneous Treatment of Upper Facial Lines: Subject-Reported Satisfaction and Impact From a Phase 3 Study.

BACKGROUND: Patient-reported outcomes are increasingly recognized as important measures of treatment benefit. OBJECTIVE: To evaluate subject-reported satisfaction and impact outcomes with onabotulinumtoxinA treatment in neurotoxin-naive adults with forehead lines (FHL), glabellar lines (GL), and crow's feet lines (CFL). METHODS: This Phase 3 study randomized 787 subjects to onabotulinumtoxinA 64 U (FHL 20 U, GL 20 U, and CFL 24 U), 40 U (FHL 20 U, GL 20 U, and CFL placebo), or placebo in double-blind Period 1. Subjects could receive up to 2 additional 64 U treatments in open-label Period 2. Patient-reported outcomes were assessed using the validated Facial Line Satisfaction Questionnaire (FLSQ) and 11-item Facial Line Outcomes (FLO-11) Questionnaire. RESULTS: The proportion of subjects mostly or very satisfied was significantly greater with onabotulinumtoxinA 64 U and 40 U versus placebo (87.9% and 81.4% vs 3.2%; p < .0001). Responder rates on FLSQ Impact Domain, FLO-11 Items 1, 4, 5, and total score were significantly greater with onabotulinumtoxinA versus placebo on Day 30 (p < .0001). Responder rates favoring onabotulinumtoxinA in Period 1 were maintained with repeated onabotulinumtoxinA 64 U treatment in Period 2. CONCLUSION: OnabotulinumtoxinA treatment was associated with high subject satisfaction and significant improvements in appearance-related psychological and emotional impacts.

Efficacy, Patient-Reported Outcomes, and Safety in Male Subjects Treated With OnabotulinumtoxinA for Improvement of Moderate to Severe Horizontal Forehead Lines.

BACKGROUND: Men represent a growing segment of the facial aesthetic market. OBJECTIVE: To evaluate investigator-assessed efficacy, patient-reported outcomes, and safety after onabotulinumtoxinA treatment of forehead lines (FHL) in men. METHODS: Subjects with moderate to severe FHL received onabotulinumtoxinA (frontalis: 20 U; glabellar complex: 20 U, with/without 24 U in crow's feet regions) or placebo in 6-month, double-blind periods of 2 pivotal trials. Results for men were pooled. RESULTS: Men comprised 12% (140/1,178) of subjects. Day 30 male responder rates for achieving at least 1-grade Facial Wrinkle Scale (FWS) improvement at maximum eyebrow elevation and at rest were 98.2% and 93.3%, respectively; a significant difference in responder rates was maintained versus placebo (p < .05) through Day 150. Despite men having proportionately more severe FHL at baseline, 81.8% and 79.8% achieved Day 30 FWS ratings of none or mild at maximum eyebrow elevation and at rest, respectively (p < .05); significance versus placebo was maintained through Day 120. Men reported high satisfaction rates and improved psychological impacts. No new safety signals were detected. CONCLUSION: Standard dosing and administration of onabotulinumtoxinA significantly improved static and dynamic FHL appearance, despite men having proportionately more severe FHL at baseline. Men reported high satisfaction and appearance-related psychological impact improvements.

Pooled Subject-Reported Outcomes From 2 Phase 3 Studies of OnabotulinumtoxinA for Simultaneous Treatment of Forehead and Glabellar Lines.

BACKGROUND: Understanding the subjects' perspective is critical for successfully treating upper facial lines. OBJECTIVE: To understand subjects' self-perception and overall satisfaction after onabotulinumtoxinA treatment for forehead and glabellar lines. METHODS: This analysis pooled data from two 12-month, pivotal phase 3 studies in which toxin-naive subjects received onabotulinumtoxinA 40 U or placebo for treatment of upper facial lines. OnabotulinumtoxinA was administered as 0.1-mL injections at 10 prespecified sites (frontalis: 20 U; glabellar complex: 20 U). Each study used 3 reliable and validated patient-reported outcome instruments to evaluate subject satisfaction and appearance-related psychological effects: the Facial Line Satisfaction Questionnaire (FLSQ), the Facial Line Outcomes (FLO-11) Questionnaire, and the Self-Perception of Age (SPA) Questionnaire. In total, data for 865 subjects (608, onabotulinumtoxinA 40 U; 257, placebo) were analyzed. RESULTS: Treatment with onabotulinumtoxinA 40 U resulted in significant and sustained improvements across all pooled FLO-11 items and FLSQ items compared with placebo. SPA results demonstrated that a significant proportion of subjects in the pooled analysis felt they looked younger after treatment than at baseline (all, p < .0001 vs placebo). CONCLUSION: This study demonstrates a high level of treatment satisfaction and significantly improved appearance-related psychological outcomes among toxin-naive subjects after onabotulinumtoxinA 40 U treatment.

OnabotulinumtoxinA for Treatment of Forehead and Glabellar Lines: Subject-Reported Satisfaction and Impact From a Phase 3 Double-Blind Study.

BACKGROUND: Patient-reported outcomes are important measures of treatment benefit in facial aesthetic medicine. OBJECTIVE: Evaluate prespecified subject-reported satisfaction and impact outcomes with onabotulinumtoxinA treatment of forehead lines (FHL) and glabellar lines (GL). METHODS: The study randomized (3:1) 391 adults with moderate to severe FHL and GL to onabotulinumtoxinA (FHL, 20 U; GL, 20 U) or placebo in double-blind period 1 (days 0-180); subjects could receive up to 2 additional onabotulinumtoxinA treatments in open-label period 2. Patient-reported outcomes were assessed using the validated Facial Line Satisfaction Questionnaire (FLSQ) and the 11-item Facial Line Outcomes (FLO-11) Questionnaire. RESULTS: The proportion of subjects mostly or very satisfied with treatment was significantly greater with onabotulinumtoxinA than with placebo (90.3% vs 1.0%; p < .0001). Responder rates on FLSQ Impact Domain (73.9% vs 18.9%), FLO-11 Item 1 (85.4% vs 3.6%), Item 4 (77.2% vs 11.2%), Item 5 (83.5% vs 7.8%), and total score (86.0% vs 6.9%) were significantly greater with onabotulinumtoxinA than with placebo on Day 30 (p < .0001). Responder rates favoring onabotulinumtoxinA in Period 1 were generally maintained with repeated treatment during Period 2. CONCLUSION: Subjects were highly satisfied with onabotulinumtoxinA treatment and reported significant improvements in appearance-related psychological and emotional impacts of their facial lines.

Racial and Ethnic Differences in Self-Assessed Facial Aging in Women: Results From a Multinational Study.

BACKGROUND: Racial/ethnic variations in skin structure and function may contribute to differential manifestations of facial aging in various races/ethnicities. OBJECTIVE: To examine self-assessed differences in facial aging in women by race/ethnicity and Fitzpatrick skin phototypes. METHODS: Women aged 18 to 75 years in the United States, Canada, the United Kingdom, and Australia compared their features against photonumeric rating scales depicting degrees of severity for 10 facial aging characteristics. Impact of race/ethnicity (black, Hispanic, Asian, and Caucasian) and skin phototypes on severity was assessed. RESULTS: In total, 3,267 women completed the study. Black women reported the least severe facial aging; Caucasian women reported the most severe facial aging, with Asian and Hispanic women falling between these groups. Similarly, women with a skin phototype V/VI reported lesser aging severity than women with phototypes I through IV. More than 30% of black women did not report the presence of moderate/severe aging of facial areas until 60 to 79 years; most Hispanics and Asians did not report moderate/severe facial aging until 50 to 69 years and Caucasians, 40 to 59 years. CONCLUSION: In this diverse sample, black women reported less severe aging of facial features compared with Hispanic, Asian, and Caucasian women. These results were supported by Fitzpatrick skin phototype analyses.

The YscE/YscG chaperone and YscF N-terminal sequences target YscF to the Yersinia pestis type III secretion apparatus.

The needle structures of type III secretion (T3S) systems are formed by the secretion and polymerization of a needle subunit protein, YscF in Yersinia pestis. A subset of T3S systems employ unique heterodimeric chaperones, YscE and YscG in Y. pestis, to prevent the polymerization of needle subunits within the bacterial cell. We demonstrate that the YscE/YscG chaperone is also required for stable YscF expression and for secretion of YscF. Overexpression of a functional maltose-binding protein (MBP)-YscG hybrid protein stabilized cytoplasmic YscF but YscF was not secreted in the absence of YscE. Furthermore, a YscE mutant protein was identified that functioned with YscG to stabilize cytosolic YscF; however, YscF was not secreted. These findings confirm a role for the YscE/YscG chaperone in YscF secretion and suggest that YscE may have a specific role in this process. Recent studies have shown that YscF deleted of its N-terminal 15 residues is still secreted and functional, suggesting that YscF may not require an N-terminal secretion signal. However, we demonstrate that YscF contains an N-terminal secretion signal and that a functional N-terminal signal is required for YscF secretion.

Tie1 deficiency induces endothelial-mesenchymal transition.

Endothelial-mesenchymal transition (EndMT) has a significant role in embryonic heart formation and in various pathologies. However, the molecular mechanisms that regulate EndMT induction remain to be elucidated. We show that suppression of receptor tyrosine kinase Tie1 but not Tie2 induces human endothelial cells to undergo EndMT and that Slug deficiency reverts this process. We find that Erk1/2, Erk5 and Akt cascades control Slug promoter activity induced by Tie1 deficiency. Interestingly, EndMT is present in human pancreatic tumour. We propose that EndMT associated with Tie1 downregulation participates in the pathological development of stroma observed in tumours.

Combining the antianginal drug perhexiline with chemotherapy induces complete pancreatic cancer regression in vivo.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the human cancers with the poorest prognosis. Interestingly, we found that mitochondrial respiration in primary human PDAC cells depends mainly on the fatty acid oxidation (FAO) to meet basic energy requirements. Therefore, we treated PDAC cells with perhexiline, a well-recognized FAO inhibitor used in cardiac diseases. Some PDAC cells respond efficiently to perhexiline, which acts synergistically with chemotherapy (gemcitabine) in vitro and in two xenografts in vivo. Importantly, perhexiline in combination with gemcitabine induces complete tumor regression in one PDAC xenograft. Mechanistically, this co-treatment causes energy and oxidative stress promoting apoptosis but does not exert inhibition of FAO. Yet, our molecular analysis indicates that the carnitine palmitoyltransferase 1C (CPT1C) isoform is a key player in the response to perhexiline and that patients with high CPT1C expression have better prognosis. Our study reveals that repurposing perhexiline in combination with chemotherapy is a promising approach to treat PDAC.

Enhanced clinical assessment of hematologic malignancies through routine paired tumor and normal sequencing.

Genomic profiling of hematologic malignancies has augmented our understanding of variants that contribute to disease pathogenesis and supported development of prognostic models that inform disease management in the clinic. Tumor only sequencing assays are limited in their ability to identify definitive somatic variants, which can lead to ambiguity in clinical reporting and patient management. Here, we describe the MSK-IMPACT Heme cohort, a comprehensive data set of somatic alterations from paired tumor and normal DNA using a hybridization capture-based next generation sequencing platform. We highlight patterns of mutations, copy number alterations, and mutation signatures in a broad set of myeloid and lymphoid neoplasms. We also demonstrate the power of appropriate matching to make definitive somatic calls, including in patients who have undergone allogeneic stem cell transplant. We expect that this resource will further spur research into the pathobiology and clinical utility of clinical sequencing for patients with hematologic neoplasms.

Creating an Interdisciplinary Curriculum within the Undergraduate Arts and Sciences through Agar Art.

Evidence-based studies on the benefits of integrating STEM into the arts are limited; however, some suggest that it can lead to improved scientific literacy and new approaches for artistic scholarship. Unfortunately, undergraduate education often creates disciplinary silos where the two are not integrated. Here, we discuss a unique collaboration between professors in the art and biology departments. Our goal was to integrate science into art courses using an agar art activity. We hypothesized that art students could effectively learn microbiology laboratory techniques and use them as novel tools for artistic practice. The activity was integrated into two to four sessions of introductory and advanced art courses over four semesters. After learning aseptic technique to culture bacteria, the students were supplied with a variety of media and bacterial strains and tasked with recreating a famous artist's drawing or using their own artistic concept. Student learning was assessed using a rubric to evaluate their art and demonstrate that the learning outcomes were met. Improvement in aesthetic, conception, and technical proficiency in handling the bacteria were demonstrated when comparing their first attempt at creating agar art to their second. Advanced art students earned higher scores than introductory students; however, the average scores for all students were "proficient" or above suggesting that the learning outcomes were met. The art was externally evaluated through American Society for Microbiology's (ASM's) Agar Art Contest and each time, at least one of our student artworks was chosen as a finalist for the People's Choice Award, providing validation of the success of our collaboration.

Complete Genome Sequences of Genamy16 and NovaSharks, Two Gordonia rubripertincta Bacteriophages Isolated from Soil in Southeastern Florida.

We report on two actinobacteriophages, Genamy16 and NovaSharks, that were isolated from soil in Florida using Gordonia rubripertincta NRRL B-16540. The genomes of both phages are ~65,000 bp, with similar GC contents, and, based on gene content similarity to phages in the Actinobacteriophage Database, were assigned to phage cluster DV.

Pooled screening of CAR T cells identifies diverse immune signaling domains for next-generation immunotherapies.

Chimeric antigen receptors (CARs) repurpose natural signaling components to retarget T cells to refractory cancers but have shown limited efficacy in persistent, recurrent malignancies. Here, we introduce "CAR Pooling," a multiplexed approach to rapidly identify CAR designs with clinical potential. Forty CARs with signaling domains derived from a range of immune cell lineages were evaluated in pooled assays for their ability to stimulate critical T cell effector functions during repetitive stimulation that mimics long-term tumor antigen exposure. Several domains were identified from the tumor necrosis factor (TNF) receptor family that have been primarily associated with B cells. CD40 enhanced proliferation, whereas B cell-activating factor receptor (BAFF-R) and transmembrane activator and CAML interactor (TACI) promoted cytotoxicity. These functions were enhanced relative to clinical benchmarks after prolonged antigen stimulation, and CAR T cell signaling through these domains fell into distinct states of memory, cytotoxicity, and metabolism. BAFF-R CAR T cells were enriched for a highly cytotoxic transcriptional signature previously associated with positive clinical outcomes. We also observed that replacing the 4-1BB intracellular signaling domain with the BAFF-R signaling domain in a clinically validated B cell maturation antigen (BCMA)-specific CAR resulted in enhanced activity in a xenotransplant model of multiple myeloma. Together, these results show that CAR Pooling is a general approach for rapid exploration of CAR architecture and activity to improve the efficacy of CAR T cell therapies.

SynNotch CAR circuits enhance solid tumor recognition and promote persistent antitumor activity in mouse models.

The first clinically approved engineered chimeric antigen receptor (CAR) T cell therapies are remarkably effective in a subset of hematological malignancies with few therapeutic options. Although these clinical successes have been exciting, CAR T cells have hit roadblocks in solid tumors that include the lack of highly tumor-specific antigens to target, opening up the possibility of life-threatening "on-target/off-tumor" toxicities, and problems with T cell entry into solid tumor and persistent activity in suppressive tumor microenvironments. Here, we improve the specificity and persistent antitumor activity of therapeutic T cells with synthetic Notch (synNotch) CAR circuits. We identify alkaline phosphatase placental-like 2 (ALPPL2) as a tumor-specific antigen expressed in a spectrum of solid tumors, including mesothelioma and ovarian cancer. ALPPL2 can act as a sole target for CAR therapy or be combined with tumor-associated antigens such as melanoma cell adhesion molecule (MCAM), mesothelin, or human epidermal growth factor receptor 2 (HER2) in synNotch CAR combinatorial antigen circuits. SynNotch CAR T cells display superior control of tumor burden when compared to T cells constitutively expressing a CAR targeting the same antigens in mouse models of human mesothelioma and ovarian cancer. This was achieved by preventing CAR-mediated tonic signaling through synNotch-controlled expression, allowing T cells to maintain a long-lived memory and non-exhausted phenotype. Collectively, we establish ALPPL2 as a clinically viable cell therapy target for multiple solid tumors and demonstrate the multifaceted therapeutic benefits of synNotch CAR T cells.