Hepatitis B virus particles in serum contain minus strand DNA and degraded pregenomic RNA of variable and inverse lengths.

This study utilized digital PCR to quantify HBV RNA and HBV DNA within three regions of the HBV genome. Analysis of 75 serum samples from patients with chronic infection showed that HBV RNA levels were higher in core than in S and X regions (median 7.20 vs. 6.80 and 6.58 log copies/mL; p < .0001), whereas HBV DNA levels showed an inverse gradient (7.71 vs. 7.73 and 7.77 log copies/mL, p < .001). On average 80% of the nucleic acid was DNA by quantification in core. The core DNA/RNA ratio was associated with viral load and genotype. In individual patients, the relations between RNA levels in core, S and X were stable over time (n = 29; p = .006). The results suggest that pregenomic RNA is completely reverse transcribed to minus DNA in ≈75% of the virus particles, whereas the remaining 25% contain both RNA and DNA of lengths that reflect variable progress of the polymerase.

Stealth transgenes enable CAR-T cells to evade host immune responses.

BACKGROUND: Adoptive cell therapy, such as chimeric antigen receptor (CAR)-T cell therapy, has improved patient outcomes for hematological malignancies. Currently, four of the six FDA-approved CAR-T cell products use the FMC63-based αCD19 single-chain variable fragment, derived from a murine monoclonal antibody, as the extracellular binding domain. Clinical studies demonstrate that patients develop humoral and cellular immune responses to the non-self CAR components of autologous CAR-T cells or donor-specific antigens of allogeneic CAR-T cells, which is thought to potentially limit CAR-T cell persistence and the success of repeated dosing. METHODS: In this study, we implemented a one-shot approach to prevent rejection of engineered T cells by simultaneously reducing antigen presentation and the surface expression of both Classes of the major histocompatibility complex (MHC) via expression of the viral inhibitors of transporter associated with antigen processing (TAPi) in combination with a transgene coding for shRNA targeting class II MHC transactivator (CIITA). The optimal combination was screened in vitro by flow cytometric analysis and mixed lymphocyte reaction assays and was validated in vivo in mouse models of leukemia and lymphoma. Functionality was assessed in an autologous setting using patient samples and in an allogeneic setting using an allogeneic mouse model. RESULTS: The combination of the Epstein-Barr virus TAPi and an shRNA targeting CIITA was efficient and effective at reducing cell surface MHC classes I and II in αCD19 'stealth' CAR-T cells while retaining in vitro and in vivo antitumor functionality. Mixed lymphocyte reaction assays and IFNγ ELISpot assays performed with T cells from patients previously treated with autologous αCD19 CAR-T cells confirm that CAR T cells expressing the stealth transgenes evade allogeneic and autologous anti-CAR responses, which was further validated in vivo. Importantly, we noted anti-CAR-T cell responses in patients who had received multiple CAR-T cell infusions, and this response was reduced on in vitro restimulation with autologous CARs containing the stealth transgenes. CONCLUSIONS: Together, these data suggest that the proposed stealth transgenes may reduce the immunogenicity of autologous and allogeneic cellular therapeutics. Moreover, patient data indicate that repeated doses of autologous FMC63-based αCD19 CAR-T cells significantly increased the anti-CAR T cell responses in these patients.

A qualitative analysis of patient and caregiver experiences with myelomeningocele through online discussion boards.

PURPOSE: Patients and caregivers impacted by myelomeningocele (MMC) use online discussion board forums to create community and share information and concerns about this complex medical condition. We aim to identify the primary concerns expressed on these forums with the goal of understanding gaps in care that may merit investment of resources to improve care received by this population. METHODS: Anonymous posts from online MMC discussion boards were compiled using internet search engines. Posts were then analyzed using an adaptation of the Grounded Theory Method, a three-step system involving open, axial, and selective coding of the data by two independent researchers to identify common themes. RESULTS: Analysis of 400 posts written primarily by parents (n = 342, 85.5%) and patients (n = 45, 11.25%) yielded three overarching themes: questions surrounding quality of life, a lack of support for mothers of children with MMC, and confusion with a complex healthcare system. Many posts revealed concerns about management and well-being with MMC, including posts discussing symptoms and related conditions (n = 299, 75.75%), treatments (n = 259, 65.75%), and emotional aspects of MMC (n = 146, 36.5%). Additionally, families, especially mothers, felt a lack of support in their roles as caregivers. Finally, in 118 posts (29.5%), patients and families expressed frustration with navigating a complex healthcare system and finding specialists whose opinions they trusted. CONCLUSIONS: MMC is a complex medical condition that impacts patients and families in unique ways. Analysis of online discussion board posts identified key themes to be addressed in order to improve the healthcare experiences of those impacted by MMC.

OptoChamber: A Low-cost, Easy-to-Make, Customizable, and Multi-Chambered Electronic Device for Applying Optogenetic Stimulation to Larval Drosophila melanogaster.

Optogenetics is a powerful tool used to manipulate physiological processes in animals through cell-specific expression of genetically modified channelrhodopsins. In Drosophila melanogaster, optogenetics is frequently used for temporal control of neuronal activation or silencing through light-dependent actuation of cation and anion channelrhodopsins, respectively. The high setup costs and complexity associated with commercially available optogenetic systems prevents many investigators from exploring the use of this technology. We developed a low-cost, customizable, and easy-to-make optogenetics chamber (OptoChamber) and verified its functionality in a robust cellular assay: activity-dependent remodeling of larval motor neurons in Drosophila embryos.

Posterior Transdural Approach for Thoracic Corpectomies in the Setting of Complex Spine Deformity Reconstruction.

BACKGROUND AND OBJECTIVE: There are many surgical approaches for execution of a thoracic corpectomy. In cases of challenging deformity, traditional posterior approaches might not be sufficient to complete the resection of the vertebral body. In this technical note, we describe indications and technique for a transdural multilevel high thoracic corpectomy. METHODS: A 25-year-old man with a history of neurofibromatosis type 1 presented with instrumentation failure after a previous T1-T12 posterior spinal fusion, extensive laminectomy, and tumor resection. The patient presented with progressive back pain, had broad dural ectasia, and a progressive kyphotic rotational and anteriorly translated spinal deformity. To resect the medial-most aspect of the vertebral body, a bilateral extracavitary approach was attempted, but was found insufficient. A transdural approach was subsequently performed. A left paramedian durotomy was made, followed by generous arachnoid dissection, bilateral dentate ligament division, and T4 rootlet sacrifice to mobilize the spinal cord. A ventral durotomy was then made and the ventral dura was reflected over the spinal cord to protect it while drilling. The corpectomy was then completed. The ventral and dorsal durotomies were closed primarily and reinforced with fibrin glue and fibrin sealant patch. The corpectomy defect was filled with nonstructural autograft. RESULTS: The focal kyphosis was corrected with a combination of rod contouring, compression, and in situ bending. During the surgery, the patient had stable neuromonitoring data, and postoperatively had no neurological deficits. On follow-up until 1 year, the patient presented with no signs of cerebrospinal spinal leaks, no motor or sensory deficits, minimal incisional pain, and significantly improved posture. CONCLUSION: Complex high thoracic (T3-5) ventral pathology inaccessible via a bilateral extracavitary approach may be accessed via a transdural approach as opposed to an anterior/lateral transthoracic approach that requires mobilization of cardiovascular structures or scapula.

Patient and caregiver perceptions of Chiari malformation: a qualitative analysis of online discussion boards.

OBJECTIVE: Patients and their caregivers utilize online discussion board forums as a means to seek and exchange information about their or a loved one's condition. It is important for providers to be aware of such concerns and experiences. The goal of this study was to identify the primary concerns expressed on these discussion boards regarding Chiari malformation type I (CM) and to help guide clinicians in understanding patient challenges in the treatment of CM. METHODS: The authors performed thematic analysis of anonymous online discussion board posts as identified through internet search engines. They then adopted a previously developed grounded theory method that utilizes a three-tiered coding and grouping process of posts based on commonly discovered content themes. RESULTS: Analysis of 400 discussion board posts identified four distinct themes raised by CM patients and their caregivers: the path to diagnosis, symptoms experienced, surgical intervention, and high emotional burden. Although each individual experience was unique, the path toward a CM diagnosis was expressed as a journey involving multiple physicians, alternative diagnoses, and feelings of dismissal from providers. The most common reported symptoms included dizziness, headaches, neck and back pain, sensory issues, weakness and paresthesias of the extremities, speech issues, and general fatigue. Additionally, there was an overall sense of uncertainty from patients seeking advice regarding surgical intervention, with users expressing diverse sentiments that included both positive and negative outcomes regarding surgical treatment. Lastly, a wide range of emotions was expressed related to a CM diagnosis, including concern, worry, anxiety, depression, stress, fear, and frustration. CONCLUSIONS: CM is a frequent imaging diagnosis identified in patients presenting with a wide range of symptoms, and as a result this leads to a diverse set of patient experiences. Analysis of CM patient and caregiver discussion boards revealed key themes that clinicians may address when counseling for CM.

Utilization of Augmented Reality Head-Mounted Display for the Surgical Management of Thoracolumbar Spinal Trauma.

Background and Objectives: Augmented reality head-mounted display (AR-HMD) is a novel technology that provides surgeons with a real-time CT-guided 3-dimensional recapitulation of a patient's spinal anatomy. In this case series, we explore the use of AR-HMD alongside more traditional robotic assistance in surgical spine trauma cases to determine their effect on operative costs and perioperative outcomes. Materials and Methods: We retrospectively reviewed trauma patients who underwent pedicle screw placement surgery guided by AR-HMD or robotic-assisted platforms at an academic tertiary care center between 1 January 2021 and 31 December 2022. Outcome distributions were compared using the Mann-Whitney U test. Results: The AR cohort (n = 9) had a mean age of 66 years, BMI of 29.4 kg/m2, Charlson Comorbidity Index (CCI) of 4.1, and Surgical Invasiveness Index (SII) of 8.8. In total, 77 pedicle screws were placed in this cohort. Intra-operatively, there was a mean blood loss of 378 mL, 0.78 units transfused, 398 min spent in the operating room, and a 20-day LOS. The robotic cohort (n = 13) had a mean age of 56 years, BMI of 27.1 kg/m2, CCI of 3.8, and SII of 14.2. In total, 128 pedicle screws were placed in this cohort. Intra-operatively, there was a mean blood loss of 432 mL, 0.46 units transfused units used, 331 min spent in the operating room, and a 10.4-day LOS. No significant difference was found between the two cohorts in any outcome metrics. Conclusions: Although the need to address urgent spinal conditions poses a significant challenge to the implementation of innovative technologies in spine surgery, this study represents an initial effort to show that AR-HMD can yield comparable outcomes to traditional robotic surgical techniques. Moreover, it highlights the potential for AR-HMD to be readily integrated into Level 1 trauma centers without requiring extensive modifications or adjustments.

Mesothelin CAR T Cells Secreting Anti-FAP/Anti-CD3 Molecules Efficiently Target Pancreatic Adenocarcinoma and its Stroma.

PURPOSE: Targeting solid tumors with chimeric antigen receptor (CAR) T cells remains challenging due to heterogenous target antigen expression, antigen escape, and the immunosuppressive tumor microenvironment (TME). Pancreatic cancer is characterized by a thick stroma generated by cancer-associated fibroblasts (CAF), which may contribute to the limited efficacy of mesothelin-directed CAR T cells in early-phase clinical trials. To provide a more favorable TME for CAR T cells to target pancreatic ductal adenocarcinoma (PDAC), we generated T cells with an antimesothelin CAR and a secreted T-cell-engaging molecule (TEAM) that targets CAF through fibroblast activation protein (FAP) and engages T cells through CD3 (termed mesoFAP CAR-TEAM cells). EXPERIMENTAL DESIGN: Using a suite of in vitro, in vivo, and ex vivo patient-derived models containing cancer cells and CAF, we examined the ability of mesoFAP CAR-TEAM cells to target PDAC cells and CAF within the TME. We developed and used patient-derived ex vivo models, including patient-derived organoids with patient-matched CAF and patient-derived organotypic tumor spheroids. RESULTS: We demonstrated specific and significant binding of the TEAM to its respective antigens (CD3 and FAP) when released from mesothelin-targeting CAR T cells, leading to T-cell activation and cytotoxicity of the target cell. MesoFAP CAR-TEAM cells were superior in eliminating PDAC and CAF compared with T cells engineered to target either antigen alone in our ex vivo patient-derived models and in mouse models of PDAC with primary or metastatic liver tumors. CONCLUSIONS: CAR-TEAM cells enable modification of tumor stroma, leading to increased elimination of PDAC tumors. This approach represents a promising treatment option for pancreatic cancer.

Virtual, Augmented, and Mixed Reality Applications for Surgical Rehearsal, Operative Execution, and Patient Education in Spine Surgery: A Scoping Review.

Background and Objectives: Advances in virtual reality (VR), augmented reality (AR), and mixed reality (MR) technologies have resulted in their increased application across many medical specialties. VR's main application has been for teaching and preparatory roles, while AR has been mostly used as a surgical adjunct. The objective of this study is to discuss the various applications and prospects for VR, AR, and MR specifically as they relate to spine surgery. Materials and Methods: A systematic review was conducted to examine the current applications of VR, AR, and MR with a focus on spine surgery. A literature search of two electronic databases (PubMed and Scopus) was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). The study quality was assessed using the MERSQI score for educational research studies, QUACS for cadaveric studies, and the JBI critical appraisal tools for clinical studies. Results: A total of 228 articles were identified in the primary literature review. Following title/abstract screening and full-text review, 46 articles were included in the review. These articles comprised nine studies performed in artificial models, nine cadaveric studies, four clinical case studies, nineteen clinical case series, one clinical case-control study, and four clinical parallel control studies. Teaching applications utilizing holographic overlays are the most intensively studied aspect of AR/VR; the most simulated surgical procedure is pedicle screw placement. Conclusions: VR provides a reproducible and robust medium for surgical training through surgical simulations and for patient education through various platforms. Existing AR/MR platforms enhance the accuracy and precision of spine surgeries and show promise as a surgical adjunct.

Genetic retargeting of E3 ligases to enhance CAR T cell therapy.

Chimeric antigen receptor (CAR) T cell therapies are medical breakthroughs in cancer treatment. However, treatment failure is often caused by CAR T cell dysfunction. Additional approaches are needed to overcome inhibitory signals that limit anti-tumor potency. Here, we developed bifunctional fusion "degrader" proteins that bridge one or more target proteins and an E3 ligase complex to enforce target ubiquitination and degradation. Conditional degradation strategies were developed using inducible degrader transgene expression or small molecule-dependent E3 recruitment. We further engineered degraders to block SMAD-dependent TGFß signaling using a domain from the SARA protein to target both SMAD2 and SMAD3. SMAD degrader CAR T cells were less susceptible to suppression by TGFß and demonstrated enhanced anti-tumor potency in vivo. These results demonstrate a clinically suitable synthetic biology platform to reprogram E3 ligase target specificity for conditional, multi-specific endogenous protein degradation, with promising applications including enhancing the potency of CAR T cell therapy.

Chemical genetic control of cytokine signaling in CAR-T cells using lenalidomide-controlled membrane-bound degradable IL-7.

CAR-T cell therapy has emerged as a breakthrough therapy for the treatment of relapsed and refractory hematologic malignancies. However, insufficient CAR-T cell expansion and persistence is a leading cause of treatment failure. Exogenous or transgenic cytokines have great potential to enhance CAR-T cell potency but pose the risk of exacerbating toxicities. Here we present a chemical-genetic system for spatiotemporal control of cytokine function gated by the off-patent anti-cancer molecular glue degrader drug lenalidomide and its analogs. When co-delivered with a CAR, a membrane-bound, lenalidomide-degradable IL-7 fusion protein enforced a clinically favorable T cell phenotype, enhanced antigen-dependent proliferative capacity, and enhanced in vivo tumor control. Furthermore, cyclical pharmacologic combined control of CAR and cytokine abundance enabled the deployment of highly active, IL-7-augmented CAR-T cells in a dual model of antitumor potency and T cell hyperproliferation.

Anti-TACI single and dual-targeting CAR T cells overcome BCMA antigen loss in multiple myeloma.

Chimeric Antigen Receptor (CAR) T cells directed to B cell maturation antigen (BCMA) mediate profound responses in patients with multiple myeloma, but most patients do not achieve long-term complete remissions. In addition, recent evidence suggests that high-affinity binding to BCMA can result in on-target, off-tumor activity in the basal ganglia and can lead to fatal Parkinsonian-like disease. Here we develop CAR T cells against multiple myeloma using a binder to targeting transmembrane activator and CAML interactor (TACI) in mono and dual-specific formats with anti-BCMA. These CARs have robust, antigen-specific activity in vitro and in vivo. We also show that TACI RNA expression is limited in the basal ganglia, which may circumvent some of the toxicities recently reported with BCMA CARs. Thus, single-targeting TACI CARs may have a safer toxicity profile, whereas dual-specific BCMA-TACI CAR T cells have potential to avoid the antigen escape that can occur with single-antigen targeting.

Virtual Assessment as a Way to Reduce Help-seeking Barriers in Older Adults With Subjective Cognitive Decline.

Subjective cognitive decline (SCD), a potential early marker for neurodegenerative disease such as Alzheimer's disease, is common among older adults. Although it is often regarded as a personal health concern, most individuals with SCD do not seek help from a health care professional. Help-seeking (HS) is a complex, individualized process with significant life-course implications, and older adults often face several barriers to HS across personal, socioeconomic, and cultural domains. The pandemic exacerbated these barriers by imposing additional limitations on in-person care. In response, virtual assessment became a popular method to conduct remote care. We provide a narrative review of the challenges and triumphs that came with the transition from in-person, pen-paper cognitive assessments to virtual cognitive assessments. In addition, we address the impact virtual assessment had in tackling barriers that previously limited individuals with SCD from formal HS. We argue that virtual cognitive assessment helps alleviate health access barriers to HS (e.g., cost, transportation, and physician availability) and allows individuals with different coping styles to undergo assessment within more convenient environments. We hope the findings presented in this review inform health care practice, public education, and future research targeted towards the use of virtual assessment to facilitate HS in older adults with SCD.

Evaluating the state of non-invasive imaging biomarkers for traumatic brain injury.

Non-invasive imaging biomarkers are useful for prognostication in patients with traumatic brain injury (TBI) at high risk for morbidity with invasive procedures. The authors present findings from a scoping review discussing the pertinent biomarkers. Embase, Ovid-MEDLINE, and Scopus were queried for original research on imaging biomarkers for prognostication of TBI in adult patients. Two reviewers independently screened articles, extracted data, and evaluated risk of bias. Data was synthesized and confidence evaluated with the linked evidence according to the Grades of Recommendation, Assessment, Development, and Evaluation (GRADE) approach. Our search yielded 3104 unique citations, 44 of which were included in this review. Study populations varied in TBI severity, as defined by Glasgow Coma Scale (GCS), including: mild (n=9), mild and moderate (n=3), moderate and severe (n=7), severe (n=6), and all GCS scores (n=17). Diverse imaging modalities were used for prognostication, predominantly computed tomography (CT) only (n=11), magnetic resonance imaging (MRI) only (n=9), and diffusion tensor imaging (DTI) (N=9). The biomarkers included diffusion coefficient mapping, metabolic characteristics, optic nerve sheath diameter, T1-weighted signal changes, cortical cerebral blood flow, axial versus extra-axial lesions, T2-weighted gradient versus spin echo, translocator protein levels, and trauma imaging of brainstem areas. The majority (93%) of studies identified that the imaging biomarker of interest had a statistically significant prognostic value; however, these are based on a very low to low level of quality of evidence. No study directly compared the effects on specific TBI treatments on the temporal course of imaging biomarkers. The current literature is insufficient to make a strong recommendation about a preferred imaging biomarker for TBI, especially considering GRADE criteria revealing low quality of evidence. Rigorous prospective research of imaging biomarkers of TBI is warranted to improve the understanding of TBI severity.

Neuromodulation of Neural Oscillations in Health and Disease.

Using EEG and local field potentials (LFPs) as an index of large-scale neural activities, research has been able to associate neural oscillations in different frequency bands with markers of cognitive functions, goal-directed behavior, and various neurological disorders. While this gives us a glimpse into how neurons communicate throughout the brain, the causality of these synchronized network activities remains poorly understood. Moreover, the effect of the major neuromodulatory systems (e.g., noradrenergic, cholinergic, and dopaminergic) on brain oscillations has drawn much attention. More recent studies have suggested that cross-frequency coupling (CFC) is heavily responsible for mediating network-wide communication across subcortical and cortical brain structures, implicating the importance of neurotransmitters in shaping coordinated actions. By bringing to light the role each neuromodulatory system plays in regulating brain-wide neural oscillations, we hope to paint a clearer picture of the pivotal role neural oscillations play in a variety of cognitive functions and neurological disorders, and how neuromodulation techniques can be optimized as a means of controlling neural network dynamics. The aim of this review is to showcase the important role that neuromodulatory systems play in large-scale neural network dynamics, informing future studies to pay close attention to their involvement in specific features of neural oscillations and associated behaviors.

Tandem chimeric antigen receptor (CAR) T cells targeting EGFRvIII and IL-13Rα2 are effective against heterogeneous glioblastoma.

Background: Chimeric antigen receptor (CAR) T cells have achieved remarkable responses in patients with hematological malignancies; however, the potential of this therapeutic platform for solid tumors like glioblastoma (GBM) has been limited, due in large part to the targeting of single antigens in a heterogeneous disease. Strategies that allow CAR T cells to engage multiple antigens concomitantly may broaden therapeutic responses and mitigate the effects of immune escape. Methods: Here we have developed a novel, dual-specific, tandem CAR T (TanCART) cell with the ability to simultaneously target both EGFRvIII and IL-13Rα2, two well-characterized tumor antigens that are frequently found on the surface of GBM cells but completely absent from normal brain tissues. We employed both standard immunological assays and multiple orthotopic preclinical models including patient-derived xenograft to demonstrate efficacy of this approach against heterogeneous tumors. Results: Tandem CAR T cells displayed enhanced cytotoxicity in vitro against heterogeneous GBM populations, including patient-derived brain tumor cultures (P < .05). Compared to CAR T cells targeting single antigens, dual antigen engagement through the tandem construct was necessary to achieve long-term, complete, and durable responses in orthotopic murine models of heterogeneous GBM, including patient-derived xenografts (P < .05). Conclusions: We demonstrate that TanCART is effective against heterogeneous tumors in the brain. These data lend further credence to the development of multi-specific CAR T cells in the treatment of GBM and other cancers.

Distinct cellular dynamics associated with response to CAR-T therapy for refractory B cell lymphoma.

Chimeric antigen receptor (CAR)-T cell therapy has revolutionized the treatment of hematologic malignancies. Approximately half of patients with refractory large B cell lymphomas achieve durable responses from CD19-targeting CAR-T treatment; however, failure mechanisms are identified in only a fraction of cases. To gain new insights into the basis of clinical response, we performed single-cell transcriptome sequencing of 105 pretreatment and post-treatment peripheral blood mononuclear cell samples, and infusion products collected from 32 individuals with large B cell lymphoma treated with either of two CD19 CAR-T products: axicabtagene ciloleucel (axi-cel) or tisagenlecleucel (tisa-cel). Expansion of proliferative memory-like CD8 clones was a hallmark of tisa-cel response, whereas axi-cel responders displayed more heterogeneous populations. Elevations in CAR-T regulatory cells among nonresponders to axi-cel were detected, and these populations were capable of suppressing conventional CAR-T cell expansion and driving late relapses in an in vivo model. Our analyses reveal the temporal dynamics of effective responses to CAR-T therapy, the distinct molecular phenotypes of CAR-T cells with differing designs, and the capacity for even small increases in CAR-T regulatory cells to drive relapse.

Nuclear entry and egress of parvoviruses.

Parvoviruses are small non-enveloped single-stranded DNA viruses, which depend on host cell nuclear transcriptional and replication machinery. After endosomal exposure of nuclear localization sequence and a phospholipase A2 domain on the capsid surface, and escape into the cytosol, parvovirus capsids enter the nucleus. Due to the small capsid diameter of 18-26 nm, intact capsids can potentially pass into the nucleus through nuclear pore complexes (NPCs). This might be facilitated by active nuclear import, but capsids may also follow an alternative entry pathway that includes activation of mitotic factors and local transient disruption of the nuclear envelope. The nuclear entry is followed by currently undefined events of viral genome uncoating. After genome release, viral replication compartments are initiated and infection proceeds. Parvoviral genomes replicate during cellular S phase followed by nuclear capsid assembly during virus-induced S/G2 cell cycle arrest. Nuclear egress of capsids occurs upon nuclear envelope degradation during apoptosis and cell lysis. An alternative pathway for nuclear export has been described using active transport through the NPC mediated by the chromosome region maintenance 1 protein, CRM1, which is enhanced by phosphorylation of the N-terminal domain of VP2. However, other alternative but not yet uncharacterized nuclear export pathways cannot be excluded.

Non-cleavable hinge enhances avidity and expansion of CAR-T cells for acute myeloid leukemia.

Chimeric antigen receptor (CAR) T cell therapy is effective in lymphoid malignancies, but there has been limited data in myeloid cancers. Here, we start with a CD27-based CAR to target CD70 ("native") in acute myeloid leukemia (AML), and we find modest efficacy in vivo, consistent with prior reports. We then use orthogonal approaches to increase binding on both the tumor and CAR-T cell sides of the immune synapse: a pharmacologic approach (azacitidine) to increase antigen density of CD70 in myeloid tumors, and an engineering approach to stabilize binding of the CAR to CD70. To accomplish the latter, we design a panel of hinge-modified regions to mitigate cleavage of the extracellular portion of CD27. Our CD8 hinge and transmembrane-modified CD70 CAR-T cells are less prone to cleavage, have enhanced binding avidity, and increased expansion, leading to more potent in vivo activity. This enhanced CD70-targeted CAR is a promising candidate for further clinical development.