BACKGROUND:Recombinant Adeno-Associated Virus (rAAV) gene therapies are now approved for the treatment of certain rare diseases and require specialized knowledge for clinical implementation. OBJECTIVES:Our objective was to develop and evaluate an artificial intelligence (AI) chatbot called 'Gene-Genie' with domain expertise in the implementation of rAAV gene therapies. METHODS:The study was conducted in two phases. The first phase, Gene-Genie development, involved technical scoping, proof-of-concept, and technical testing by a team of pharmacists, medical practitioners, regulatory specialists, and laboratory scientists. In the second phase, Gene-Genie was evaluated using an exploratory pilot pre-post study design, based on feedback from hospital pharmacy leaders before and after using Gene-Genie. The total score on the questionnaire ranged from 0 to 28, with a higher score indicating better perceived knowledge of gene therapy. RESULTS:The AI chatbot was successfully developed in phase 1 to deliver accurate, reliable, and context-aware interactions, aligned with the scope of the intended use case. The chatbot successfully referenced a curated and linked set of resources and avoided off-topic questions and responses. In the second phase, 8 pharmacy leaders tested Gene-Genie and provided feedback on its utility to further improve the chatbot. The mean ± standard deviation score on the perceived knowledge questionnaire was 17.0 ± 5.5 pre and 20.0 ± 2.2 post using Gene-Genie. The majority of participants agreed or strongly agreed (n = 5, 62.5%) that they would recommend Gene-Genie to other hospital Directors of Pharmacy (or similar) to help them with gene therapy implementation. CONCLUSION:We have developed Gene-Genie, which is an AI conversational chatbot that is domain-specific to gene therapy. Exploratory findings suggest that a domain-specific chatbot like Gene-Genie may be useful to those in hospital pharmacy leadership positions to implement gene therapy programs.

Glioblastoma (GBM) is an aggressive primary adult brain tumor that rapidly recurs after standard-of-care treatments, including surgery, chemotherapy and radiotherapy. While immune checkpoint inhibitor therapies have transformed outcomes in many tumor types, particularly when used neoadjuvantly or as a first-line treatment, including in melanoma brain metastases, they have shown limited efficacy in patients with resected or recurrent GBM. The lack of efficacy has been attributed to the scarcity of tumor-infiltrating lymphocytes (TILs), an immunosuppressive tumor microenvironment and low tumor mutation burden typical of GBM tumors, plus exclusion of large molecules from the brain parenchyma. We hypothesized that upfront neoadjuvant combination immunotherapy, administered with disease in situ, could induce a stronger immune response than treatment given after resection or after recurrence. Here, we present a case of newly diagnosed IDH-wild-type, MGMT promoter unmethylated GBM, treated with a single dose of neoadjuvant triplet immunotherapy (anti-programmed cell death protein 1 plus anti-cytotoxic T-lymphocyte protein 4 plus anti-lymphocyte-activation gene 3) followed by maximal safe resection 12 days later. The anti-programmed cell death protein 1 drug was bound to TILs in the resected GBM and there was marked TIL infiltration and activation compared with the baseline biopsy. After 17 months, there is no definitive sign of recurrence. If used first line, before safe maximal resection, checkpoint inhibitors are capable of immune activation in GBM and may induce a response. A clinical trial of first-line neoadjuvant combination checkpoint inhibitor therapy in newly diagnosed GBM is planned (GIANT; trial registration no. NCT06816927 ).

Background aims: Despite promising results in pre-clinical studies, mesenchymal stromal cells (MSCs) face significant challenges in clinical translation. A scoping review by our group highlighted two key issues contributing to this gap: (i) lack of a clear and consensus definition for MSCs and (ii) under-reporting of critical parameters in MSC clinical studies. To address these issues, we conducted a modified Delphi study to establish and implement a consensus definition for MSCs and develop reporting guidelines for MSC clinical studies. Methods: A steering committee of 22 international experts, including stakeholders from different MSC research fields, participated in the three Delphi rounds. For the first round, to obtain a broad perspective, additional investigators recommended by the steering committee were invited to participate. The first two rounds consisted of online surveys, whereas the third round took the form of a virtual meeting. Participants were asked to rate a series of potential defining characteristics of MSCs and items for reporting guidelines. Consensus was defined as at least 80% of the participants rating the item in the same category of importance. Results: Eighty-seven international participants participated in the first round survey (spring 2023), 17 participants participated in the second online survey (fall 2023) and 15 participants participated in the final virtual consensus meeting (January 2024). For the MSC definition, 20 items were considered and nine reached consensus. Items included terminology (one item), cell marker expression (five items), tissue origin (one item), stemness (one item) and description of critical quality attributes (one item). For the reporting guidelines, with the 28 initial items and the additional items suggested during round 1, a total of 33 items to report were included. This included items on MSC intervention group and control (e.g., MSC product, dose and administration), MSC characteristics (e.g., MSC provenance, "fitness," viability and immune compatibility) and MSC culture conditions (e.g., oxygen environment, culture medium and use of serum). Conclusions: By applying a Delphi method to establish a consensus definition for MSCs and reporting guidelines for MSC-based clinical trials, this work represents a significant advance in improving transparency and reproducibility in the conduct and reporting of MSC research. (c) 2024 International Society for Cell & Gene Therapy. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

Systemic gene therapy using adeno-associated virus (AAV) vectors is approved for the treatment of several genetic disorders, but challenges and toxicities associated with high vector doses remain. We report an alternate receptor for AAV (AAVR2, carboxypeptidase D [CPD]), which is distinct from the multi-serotype AAV receptor (AAVR). AAVR2 enables the transduction of clade E AAVs, including AAV8, and determines an exclusive AAVR-independent transduction pathway for AAV11 and AAV12. We characterized direct binding between the AAV8 capsid and AAVR2 by cryo-electron microscopy (cryo-EM) and identified contact residues. We observed that AAV8 directly binds to the carboxypeptidase-like domain 1 of AAVR2 via its variable region VIII and demonstrated that AAV capsids that lack AAVR2 binding can be bioengineered to engage with AAVR2. Finally, we overexpressed a minimal functional AAVR2 to enhance AAV transduction in vivo. Our study provides insights into AAV biology and clinically deployable solutions to reduce dose-related toxicities associated with AAV vectors.

Drs. Thompson and Kwiatkowski contributed equally. Introduction: Beti-cel gene therapy is a one-time treatment for transfusion-dependent β-thalassemia (TDT) that adds functional copies of the β-globin gene (βT87Q) to address the underlying cause of disease. Here, we report long-term outcomes of participants treated with beti-cel with up to 10 years of follow-up. Methods: After completion of a 2-year phase 1/2 (HGB-204 [NCT01745120]; HGB-205 [NCT02151526]) or phase 3 (HGB-207 [NCT02906202]; HGB-212 [NCT03207009]) beti-cel parent study, treated participants were eligible for the long-term, 13-year follow-up study LTF-303 (NCT02633943). Clinical efficacy, iron homeostasis, health-related quality of life (HRQOL), and safety are reported through last follow-up visit. Key outcomes were also examined by genotype (β0/β0 vs non-β0/β0) and by participant age at enrollment (pediatric: <12 years; adolescent: ≥12 years to <18 years; adult: ≥18 years). Results:As of Feb 2024, 63 participants (median [range] age: 17 [4-35] years) had received beti-cel and subsequently enrolled in LTF-303 (median [range] follow-up: 71.2 [34.5-121.4] months); 2 participants had 10 years of follow-up, 51 (81.0%) participants had at least 5 years of follow-up, and 1 participant withdrew consent after 39 months of follow-up due to personal reasons. Peripheral blood vector copy number (PB VCN) and HbAT87Q levels were stable by month 6 after infusion, sustained across studies up to 10 years, and were higher in phase 3 versus phase 1/2 studies following beti-cel drug product manufacturing optimization, which is similar to the commercial process. At last follow-up, median (range) PB VCN was 0.4 (0.1-4.9) c/dg and 1.6 (0.1-4.8) c/dg in phase 1/2 and phase 3, respectively. Among participants who achieved transfusion independence (TI) in phase 1/2 studies (15/22 [68.2%]), median (range) HbAT87Q was 7.6 (3.9-10.8) g/dL and weighted average hemoglobin (Hb) during TI was 10.24 (9.1-13.1) g/dL. In phase 3 studies, 37/41 (90.2%) participants achieved TI, median (range) HbAT87Q was 9.8 (5.1-14.3) g/dL, and weighted average Hb during TI was 11.24 (9.8-13.9) g/dL. Transduction efficiency, pharmacodynamics, TI rate, and weighted average Hb were similar across genotypes and ages. While iron overload management was at the physician's discretion, 28/37 (78.4%) phase 3 participants who achieved TI are no longer receiving iron-chelation therapy, and of those 28, 22 (78.6%) had liver iron concentration (LIC) <5 mg Fe/g dry weight (dw). Among phase 1/2 and phase 3 participants who achieved and maintained TI and had baseline data, median (range) change from baseline in serum ferritin at month 24 was -907.9 (-5014 to 5539) ng/mL (n=51). At month 60, median (range) change from baseline in serum ferritin and LIC were -1951.0 (-7079 to 1345) ng/mL (n=39) and -2.2 (-20.6 to 9.6) mg Fe/g dw (n=30), respectively, indicating improvement in iron overload. Cardiac T2* remained stable and was >20 msec at last follow-up in all participants who achieved TI, including 2 participants with cardiac T2* ≤20 msec at baseline. Among participants who achieved TI and had HRQOL data, clinically meaningful improvements were reported in Short Form-36 Health Survey Questionnaire (SF-36) scores at month 24 for the mental component summary and at month 36 for physical component summary (mean score increase >2). Participants had sustained SF-36 physical and emotional scores above the normative population mean (50) at month 60. Clinically meaningful improvements in Pediatric Quality of Life Inventory total scores were reported at month 36, and scores remained above the normative population mean (81) at month 60. All 26 participants who achieved TI and completed a questionnaire reported an overall benefit with beti-cel. No beti-cel-related serious adverse events were reported >2 years after infusion through last follow-up. No malignancies, insertional oncogenesis, or vector-derived replication-competent lentivirus were reported. Conclusion: These data provide evidence of the sustained favorable benefit-risk profile of gene addition therapy with beti-cel in participants with TDT and will inform real-world treatment decisions.Beti-cel is a potentially curative gene addition therapy for patients with TDT across genotypes and ages through achievement of durable TI, normal or near-normal Hb, and a favorable long-term safety profile with up to 10 years of follow-up.

CD19 directed chimeric antigen receptor (CAR) T-cell therapy is now standard of care for relapsed/refractory large B-cell non-Hodgkin lymphoma. Despite good overall response rates, many patients still experience disease progression and therefore it is important to predict those at risk of relapse following CAR T-cell therapy. We performed a prospective study using a flow cytometric assay at a single treatment centre to assess early CAR T-cell expansion in vivo 6 – 9 days after CAR-T cell infusion. Early CAR T-cell expansion was used in conjunction with additional clinical risk factors to identify those at higher risk of relapse or treatment failure. Forty-four patients treated with commercial CD19 directed CAR T-cell therapy were included in the study, with a median follow up of 306 days. CAR T-cell expansion of >30 cells/μl was associated with a lower risk of disease progression or death (HR 0.34, p=0.048), but did not correlate with the risk of death alone. Patients who had poor early CAR T-cell expansion (<30 cells/μl) in addition to high lactate dehydrogenase (LDH) had significantly lower median progression-free and overall survival. High LDH alone was not a statistically significant risk factor for death or disease progression and therefore the interaction between CAR T-cell expansion and this clinical risk factor may be important in predicting response. Mean CAR T-cell count was higher in patients with grade 2-4 cytokine release syndrome (54.9 cells/μl) compared to grades 0-1 (25.5 cells/μl, p = 0.01). The methodology of this assay is easily reproducible outside of a clinical trial, allowing for real-life implementation within clinical settings. This study suggests that early assessment of CAR T-cell expansion can assist in identifying patients with poor overall survival who may benefit from early intervention or more intensive monitoring.

Background Pancreatic cancer is an aggressive disease with poor prognosis. The only potentially curative treatment option is surgical resection, however recurrence is common. Biomarkers to detect minimal residual disease, assist with risk stratification, relapse and real time monitoring, are required. Circulating tumor cells (CTCs) are a promising liquid biopsy biomarker for solid tumors. However, their role in monitoring minimal residual disease in pancreatic cancer remains to be determined. Our study aimed to investigate whether detection and enumeration of CTCs could predict recurrence and provide monitoring of disease status. Method Participants planned for Whipple procedure or partial pancreatectomy were enrolled in this prospective pilot study. Intraoperatively, 7.5 mL of portal and peripheral venous blood were collected, and peripheral venous blood was also collected post-surgery. CTC identification and enumeration were performed using the AccuCyte-CyteFinder platform and CellSieve microfiltration. Results Of 29 participants, 20 were confirmed to have epithelial cancer by histopathology, where 15 had pancreatic ductal adenocarcinoma. In those with epithelial cancer, CTCs were detected intraoperatively in 75% of portal venous blood samples, in contrast to 40% detected in peripheral venous blood (median: 6 and 0 per 7.5mL respectively). Only portal venous CTC detection was predictive of pancreatic ductal adenocarcinoma relapse. The positive (> 5) portal venous CTC group had a 6.67 times higher risk of recurring (odds ratio = 20.43, sensitivity = 1.00, specificity = 0.625). Detection of peripheral venous CTCs post-surgery was also correlated with relapse in a small subset of patients. Conclusions If validated, CTCs may provide a prognostic and monitoring biomarker in patients with pancreatic cancer undergoing surgery.

Chromodomain helicase DNA-binding (CHD) enzymes play a pivotal role in genome regulation. They possess highly conserved ATPase domains flanked by poorly characterized and intrinsically disordered N- and C-termini. Using mass spectrometry, we identify dozens of novel protein-protein interactions (PPIs) within the N- and C-termini of human CHD family members. We also define a highly conserved aggregation-prone region (APR) within the C-terminus of CHD4 which is critical for its interaction with the nucleosome remodeling and deacetylase (NuRD), as well as ChAHP (CHD4, activity-dependent neuroprotective protein (ADNP), and HP1γ) complexes. Further analysis reveals a regulatory role for the CHD4 APR in gene transcription during erythrocyte formation. Our results highlight that the N- and C-termini of CHD chromatin remodelers shape protein interaction networks that drive unique transcriptional programs.

The nucleosome remodelling and deacetylase complex (NuRD) plays a key role in chromatin regulation and a wide range of biological processes including development, haemopoiesis, immunity and neurogenesis. Its interaction with tissue-enriched and sequence-specific transcription factors (TFs) leads to distinct functional outputs in the given tissue by targeting a specific set of genes. However, how NuRD dynamically and specifically regulates gene expression in a tissue-specific manner is poorly understood. Here, we refine an N-terminal specific NuRD interaction motif which enables direct engagement with many transcriptional regulatory proteins. Using a series of structural modelling and biochemical techniques, we show that ZNF512B, a poorly characterised neuronal-expressed zinc finger protein, directly binds to the RBBP4 subunit of the NuRD complex. Subsequent knockdown of ZNF512B results in the downregulation of several neural-related molecular pathways suggesting that ZNF512B may play a regulatory role during neurogenesis. We also show that in NTERA-2 neural cells, the expression of ZNF512B is necessary for cell growth and survival, and is markedly enhanced during neural progenitor cell (NPC) differentiation. In summary, our data suggest that ZNF512B might regulate neural-specific transcriptional programs via engagement with the NuRD complex. ### Competing Interest Statement The authors have declared no competing interest.

Gene therapy holds promise for patients with inherited monogenic disorders, cancer, and rare genetic diseases. Naturally occurring adeno-associated virus (AAV) offers a well-suited vehicle for clinical gene transfer due to its lack of significant clinical pathogenicity and amenability to be engineered to deliver therapeutic transgenes in a variety of cell types for long-term sustained expression. AAV has been bioengineered to produce recombinant AAV (rAAV) vectors for many gene therapies that are approved or in late-stage development. However, ongoing challenges hamper wider use of rAAV vector-mediated therapies. These include immunity against rAAV vectors, limited transgene packaging capacity, sub-optimal tissue transduction, potential risks of insertional mutagenesis and vector shedding. This review focuses on aspects of immunity against rAAV, mediated by anti-AAV neutralizing antibodies (NAbs) arising after natural exposure to AAVs or after rAAV vector administration. We provide an in-depth analysis of factors determining AAV seroprevalence and examine clinical approaches to managing anti-AAV NAbs pre- and post-vector administration. Methodologies used to quantify anti-AAV NAb levels and strategies to overcome pre-existing AAV immunity are also discussed. The broad adoption of rAAV vector-mediated gene therapies will require wider clinical appreciation of their current limitations and further research to mitigate their impact.

BACKGROUND AIMS:Several anti-mesothelin (MSLN) chimeric antigen receptor (CAR) T cells are in phase 1/2 clinical trials to treat solid-organ malignancies. The effect of MSLN antigen density on MSLN CAR cytotoxicity against tumor cells has not been examined previously, nor are there data regarding the effect of agents that increase MSLN antigen density on anti-MSLN CAR T cell efficacy.METHODS:MSLN antigen density was measured on a panel of pancreatic cancer and mesothelioma cell lines by flow cytometry. In parallel, the cytotoxicity and specificity of two anti-MSLN CAR T cells (m912 and SS1) were compared against these cell lines using a real-time impedance-based assay. The effect of two MSLN 'sheddase' inhibitors (lanabecestat and TMI-1) that increase MSLN surface expression was also tested in combination with CAR T cells.RESULTS:SS1 CAR T cells were more cytotoxic compared with m912 CAR T cells against cell lines that expressed fewer than ∼170 000 MSLN molecules/cell. A comparison of the m912 and amatuximab (humanized SS1) antibodies identified that amatuximab could detect and bind to lower levels of MSLN on pancreatic cancer and mesothelioma cell lines, suggesting that superior antibody/scFv affinity was the reason for the SS1 CAR's superior cytotoxicity. The cytotoxicity of m912 CAR T cells was improved in the presence of sheddase inhibitors, which increased MSLN antigen density.CONCLUSIONS:These data highlight the value of assessing CAR constructs against a panel of cells expressing varying degrees of target tumor antigen as occurs in human tumors. Furthermore, the problem of low antigen density may be overcome by concomitant administration of drugs that inhibit enzymatic shedding of MSLN.

The rapid proliferation of businesses engaged in direct-to-consumer advertising of unproven stem cell interventions has raised troubling questions about whether government bodies can regulate this health market effectively. Recent developments in Australia and Canada suggest that such fears are unfounded and that targeted regulatory action can have meaningful effects.

Cancer-associated macrophage-like cells (CAMLs) are myeloid-lineage cells associated with cancer-derived material that are detectable in the blood. In addition to circulating tumor cells, CAMLs are a promising liquid biopsy biomarker which may assist with prognostication for patient stratification and monitoring response to chemotherapy and radiotherapy in solid tumors. CAMLs have been detected in blood samples from patients with various tumors including lung, pancreas, breast, oesophageal, and colorectal cancers, and to date have not been detected in healthy individuals. However, the optimal method of detection, their origin, function in the circulation, and ultimate utility have not been fully elucidated. This review provides an overview of CAML-related studies and explores their future potential to guide clinical decision-making.

Phyllodes tumours (PTs) are rare fibroepithelial lesions of the breast that are classified as benign, borderline, or malignant. As little is known about the molecular underpinnings of PTs, current diagnosis relies on histological examination. However, accurate classification is often difficult, particularly for distinguishing borderline from malignant PTs. Furthermore, PTs can be misdiagnosed as other tumour types with shared histological features, such as fibroadenoma and metaplastic breast cancers. As DNA methylation is a recognised hallmark of many cancers, we hypothesised that DNA methylation could provide novel biomarkers for diagnosis and tumour stratification in PTs, whilst also allowing insight into the molecular aetiology of this otherwise understudied tumour. We generated whole-genome methylation data using the Illumina EPIC microarray in a novel PT cohort (n = 33) and curated methylation microarray data from published datasets including PTs and other potentially histopathologically similar tumours (total n = 817 samples). Analyses revealed that PTs have a unique methylome compared to normal breast tissue and to potentially histopathologically similar tumours (metaplastic breast cancer, fibroadenoma and sarcomas), with PT-specific methylation changes enriched in gene sets involved in KRAS signalling and epithelial-mesenchymal transition. Next, we identified 53 differentially methylated regions (DMRs) (false discovery rate < 0.05) that specifically delineated malignant from non-malignant PTs. The top DMR in both discovery and validation cohorts was hypermethylation at the HSD17B8 CpG island promoter. Matched PT single-cell expression data showed that HSD17B8 had minimal expression in fibroblast (putative tumour) cells. Finally, we created a methylation classifier to distinguish PTs from metaplastic breast cancer samples, where we revealed a likely misdiagnosis for two TCGA metaplastic breast cancer samples. In conclusion, DNA methylation alterations are associated with PT histopathology and hold the potential to improve our understanding of PT molecular aetiology, diagnostics, and risk stratification. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Abstract Introduction The majority of pancreatic cancer patients are diagnosed with advanced, metastatic disease. Biomarkers to detect pancreatic cancer at earlier stages could drastically improve the poor survival rates. Circulating tumor cells (CTCs) are tumor cells that disseminate or are shed into the vascular system and have been detected in patients at pre-malignant stages. However, detecting these rare cells accurately and reliably remains a challenge. The AccuCyte-CyteFinder system uses density-based enrichment of blood nucleated cells followed by detection of CTCs using fluorescence staining, whole slide scanning and automated image analysis. This study aimed to examine whether CTCs could be used to identify patients with pancreatic lesions likely to malignant. Methods 50 patients undergoing an endoscopic ultrasound procedure for a pancreatic mass were recruited at the Royal Prince Alfred Hospital, Sydney, Australia or Chris O’Brien Lifehouse, Sydney, Australia. Peripheral blood was collected prior to the procedure and processed for CTCs on the AccuCyte-CyteFinder platform using a CK+ EpCAM+ CD45- staining panel. Results 28% (14/50) were found to have an intraductal papillary mucinous neoplasm (IPMN), 10% (5/50) pancreatic ductal adenocarcinoma (PDAC), 6% (3/50) pancreatic neuroendocrine tumor (PNET), 42% (21/50) non-neoplastic pancreatic cyst, and 14% (7/50) normal (no mass found). CTCs were highest in the PDAC group with an average of 5 cells/ml, followed by the IPMN (3 cells/ml), pancreatic cyst (2.5 cells/ml), and PNET (0 cells/ml). 86% (12/14) of IPMN patients had detectable CTCs. 2 IPMN patients with CTC counts of more than 5 cells/ml resulted in a resected sample having high-grade dysplasia while 3 IPMN patients with no/low CTC counts resulted in a resected sample having low-grade dysplasia. There was no correlation with the size of the pancreatic mass and CTC number. Conclusions CTCs could be detected in patients with pre-malignant pancreatic cancer and could determine malignancy. CTCs could potentially be used to detect pancreatic cancer early however, larger patient numbers and longer follow-up are required to validate these findings. Citation Format: Dannel Yeo, Vera Klemm, Payal Saxena, Althea Bastian, Heidi Strauss, Charbel Sandroussi, Sara Wahlroos, Peter Grimison, John E.J. Rasko. Detection of circulating tumor cells for the early detection of pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C011.

The first completed clinical trial of induced pluripotent stem cell (iPS cell)-derived cells was conducted in 15 participants with steroid-resistant acute graft-versus-host disease. After intravenous infusion of mesenchymal stromal cells (CYP-001 derived from a clone of human iPS cells), we reported the safety, tolerability and efficacy within the primary evaluation period at day 100. We now report results at the 2-year follow-up: 9 of 15 (60%) participants survived, which compares favorably with previously reported outcomes in studies of steroid-resistant acute graft-versus-host disease. Causes of death were complications commonly observed in recipients of allogeneic hematopoietic stem cell transplantation, and not considered by the investigators to be related to CYP-001 treatment. There were no serious adverse events, tumors or other safety concerns related to CYP-001. In conclusion, systemic delivery of iPS cell-derived cells was safe and well tolerated over 2 years of follow-up, with sustained outcomes up to 2 years after the first infusion. ClinicalTrials.gov registration: NCT02923375 .

BACKGROUND Fidanacogene elaparvovec, an adeno-associated virus (AAV) gene-therapy vector for hemophilia B containing a high-activity human factor IX variant (FIX-R338L/FIX-Padua), was associated with sustained factor IX activity in a phase 1-2a study. METHODS We conducted a phase 3 open-label study of fidanacogene elaparvovec at a dose of 5x10(11) vector genome copies per kilogram of body weight. Men 18 to 65 years of age with hemophilia B and a factor IX level of 2% or less were eligible for screening if they had received at least 6 months of therapy with prophylactic factor IX concentrate. The primary end point, tested for noninferiority, was the annualized bleeding rate (treated and untreated bleeding episodes) from week 12 to month 15 after treatment with fidanacogene elaparvovec as compared with the prophylaxis lead-in period. Superiority, additional efficacy end points, and safety were also assessed. RESULTS Of 316 men who underwent screening for the lead-in study, 204 (64.6%) were not eligible; 188 (59.5%) of those were ineligible owing to the presence of anti-AAV neutralizing antibodies. Of the 45 participants who received fidanacogene elaparvovec, 44 completed at least 15 months of follow-up. The annualized rate of bleeding for all bleeding episodes decreased by 71%, from 4.42 (95% confidence interval [CI], 1.80 to 7.05) at baseline to 1.28 (95% CI, 0.57 to 1.98) after gene therapy, a treatment difference of -3.15 episodes (95% CI, -5.46 to -0.83; P=0.008). This result shows the noninferiority and superiority of fidanacogene elaparvovec to prophylaxis. At 15 months, the mean factor IX activity was 26.9% (median, 22.9%; range, 1.9 to 119.0) by one-stage SynthASil assay. A total of 28 participants (62%) received glucocorticoids for increased aminotransferase levels or decreased factor IX levels (or both) starting between 11 and 123 days. No infusion-related serious adverse events, thrombotic events, development of factor IX inhibitors, or malignant conditions were observed. CONCLUSIONS Fidanacogene elaparvovec was superior to prophylaxis for the treatment of participants with hemophilia B, leading to reduced bleeding and stable factor IX expression.

Chromodomain helicase DNA-binding (CHD1-9) enzymes reposition nucleosomal DNA for transcription, recombination, and replication. They possess highly conserved ATPase domains flanked by poorly characterised N- and C-termini, which are enriched with intrinsically disordered regions (IDRs) and short aggregation-prone regions (APRs). The roles of IDRs and APRs in CHD function has remained elusive. Here, by integrating proteomics and AlphaFold Multimer analysis, we defined the protein-protein interaction (PPI) networks within the N- and C-termini of all CHDs. We generated a comprehensive map of CHD1-9-specific binding proteins, revealing dozens of novel interactions with transcription regulators. We identified APR regions that contribute to PPI formation and demonstrated that a highly conserved APR within the C-terminus of CHD4 is critical for its interaction with the nucleosome remodeling and deacetylase (NuRD), as well as the CHD, ADNP, and HP1 (ChAHP) complexes. Further analysis unravels a regulatory role for the CHD4 APR in gene transcription during erythrocyte formation. Our results emphasize that the N- and C-termini of CHD chromatin remodelers establish PPI networks that drive unique transcriptional programs. ### Competing Interest Statement The authors have declared no competing interest.