STRAIGHT-IN Dual: a platform for dual, single-copy integrations of DNA payloads and gene circuits into human induced pluripotent stem cells.

Targeting DNA payloads into human (h)iPSCs involves multiple time-consuming, inefficient steps that must be repeated for each construct. Here, we present STRAIGHT-IN Dual, which enables simultaneous, allele-specific, single-copy integration of two DNA payloads with 100% efficiency within one week. Notably, STRAIGHT-IN Dual leverages the STRAIGHT-IN platform to allow near-scarless cargo integration, facilitating the recycling of components for subsequent cellular modifications. Using STRAIGHT-IN Dual, we investigated how promoter choice and gene syntax influences transgene silencing, and demonstrate the impact of these design features on forward programming of hiPSCs into neurons. Furthermore, we designed a grazoprevir-inducible synZiFTR system to complement the widely-used tetracycline-inducible system, providing independent, tunable, and temporally controlled expression of both transcription factors and functional reporters. The unprecedented efficiency and speed with which STRAIGHT-IN Dual generates homogenous genetically engineered hiPSC populations represents a major advancement for synthetic biology in stem cell applications and opens opportunities for precision cell engineering.

Two Accreditation Options for Biorepositories.

CONTEXT.­: Biomedical research relies on available biomaterials and associated data, and the quality of this starting material can have a significant impact on the quality of the experimental results. In the 2000s, best-practice documents and guidelines for biorepositories were published, followed in the 2010s by standards documents used to support accreditation. The College of American Pathologists Biorepository Accreditation Program and the International Standards Organization's standard 20387 were launched in 2012 and 2018, respectively. OBJECTIVE.­: To identify quantitative and qualitative differences between the two aforementioned biorepository accreditation standards for use by the larger biomedical research community; the results will empower biorepositories to select an accreditation program that best fits their goals. DESIGN.­: Individual requirements of both accreditation standards were identified and a bidirectional crosswalk was performed to identify gaps. Requirements were assigned to one of several standardized categories to enable comparison of the relative emphasis of different categories between the standards. RESULTS.­: Quantitatively, the College of American Pathologists program is comprehensive and stands alone, with 523 requirements, whereas the International Standards Organization program contains 167 requirements and is comprehensive through its incorporation and reference to numerous related standards documents. Qualitatively, both programs rely heavily on the implementation of an overarching quality management system and both programs can accommodate different types of biobanks (eg, human and animal). CONCLUSIONS.­: The standards differ in number of requirements, distribution of requirements across categories, and amount of reliance on separate standard documents. This information may aid in selection of an appropriate accreditation standard.

STRAIGHT-IN: a platform for rapidly generating panels of genetically modified human pluripotent stem cell lines.

Targeted integration of large DNA cargoes (>10 kb) or genomic replacements in mammalian cells, such as human pluripotent stem cells (hPS cells), remains challenging. Here we describe a platform termed serine and tyrosine recombinase-assisted integration of genes for high-throughput investigation (STRAIGHT-IN) to circumvent this. First, a landing pad cassette is precisely inserted or used to replace specific genomic regions. The site-specific integrase Bxb1 then enables DNA constructs, including those >50 kb, to be integrated into the genome, while Cre recombinase excises auxiliary DNA sequences to prevent postintegrative silencing. Using a strategy whereby the positive selection marker is only expressed if the donor plasmid carrying the payload is correctly targeted, we can obtain 100% enrichment for cells containing the DNA payload. Procedures for expressing Cre efficiently also mean that a clonal isolation step is no longer essential to derive the required genetically modified hPS cells containing the integrated DNA, potentially reducing clonal variability. Furthermore, STRAIGHT-IN facilitates rapid and multiplexed generation of genetically matched hPS cells when multiple donor plasmids are delivered simultaneously. STRAIGHT-IN has various applications, which include integrating complex genetic circuits for synthetic biology, as well as creating panels of hPS cells lines containing, as necessary, hundreds of disease-linked variants for disease modeling and drug discovery. After establishing the hPS cell line containing the landing pad, the entire procedure, including donor plasmid synthesis, takes 1.5-3 months, depending on whether single or multiple DNA payloads are integrated. This protocol only requires the researcher to be skilled in molecular biology and standard cell culture techniques.

Avian influenza viruses in wild birds in Canada following incursions of highly pathogenic H5N1 virus from Eurasia in 2021-2022.

Following the detection of novel highly pathogenic avian influenza virus (HPAIV) H5N1 clade 2.3.4.4b in Newfoundland, Canada, in late 2021, avian influenza virus (AIV) surveillance in wild birds was scaled up across Canada. Herein, we present the results of Canada's Interagency Surveillance Program for Avian Influenza in Wild Birds during the first year (November 2021-November 2022) following the incursions of HPAIV from Eurasia. The key objectives of the surveillance program were to (i) identify the presence, distribution, and spread of HPAIV and other AIVs; (ii) identify wild bird morbidity and mortality associated with HPAIV; (iii) identify the range of wild bird species infected by HPAIV; and (iv) genetically characterize detected AIV. A total of 6,246 sick and dead wild birds were tested, of which 27.4% were HPAIV positive across 12 taxonomic orders and 80 species. Geographically, HPAIV detections occurred in all Canadian provinces and territories, with the highest numbers in the Atlantic and Central Flyways. Temporally, peak detections differed across flyways, though the national peak occurred in April 2022. In an additional 11,295 asymptomatic harvested or live-captured wild birds, 5.2% were HPAIV positive across 3 taxonomic orders and 19 species. Whole-genome sequencing identified HPAIV of Eurasian origin as most prevalent in the Atlantic Flyway, along with multiple reassortants of mixed Eurasian and North American origins distributed across Canada, with moderate structuring at the flyway scale. Wild birds were victims and reservoirs of HPAIV H5N1 2.3.4.4b, underscoring the importance of surveillance encompassing samples from sick and dead, as well as live and harvested birds, to provide insights into the dynamics and potential impacts of the HPAIV H5N1 outbreak. This dramatic shift in the presence and distribution of HPAIV in wild birds in Canada highlights a need for sustained investment in wild bird surveillance and collaboration across interagency partners. IMPORTANCE: We present the results of Canada's Interagency Surveillance Program for Avian Influenza in Wild Birds in the year following the first detection of highly pathogenic avian influenza virus (HPAIV) H5N1 on the continent. The surveillance program tested over 17,000 wild birds, both sick and apparently healthy, which revealed spatiotemporal and taxonomic patterns in HPAIV prevalence and mortality across Canada. The significant shift in the presence and distribution of HPAIV in Canada's wild birds underscores the need for sustained investment in wild bird surveillance and collaboration across One Health partners.

End resection and telomere healing of DNA double-strand breaks during nematode programmed DNA elimination.

Most DNA double-strand breaks (DSBs) are harmful to genome integrity. However, some forms of DSBs are essential to biological processes, such as meiotic recombination and V(D)J recombination. DSBs are also required for programmed DNA elimination (PDE) in ciliates and nematodes. In nematodes, the DSBs are healed with telomere addition. While telomere addition sites have been well characterized, little is known regarding the DSBs that fragment nematode chromosomes. Here, we used embryos from the human and pig parasitic nematode Ascaris to characterize the DSBs. Using END-seq, we demonstrate that DSBs are introduced before mitosis, followed by extensive end resection. The resection profile is unique for each break site, and the resection generates 3'-overhangs before the addition of neotelomeres. Interestingly, telomere healing occurs much more frequently on retained DSB ends than on eliminated ends. This biased repair of the DSB ends may be due to the sequestration of the eliminated DNA into micronuclei, preventing neotelomere formation at their ends. Additional DNA breaks occur within the eliminated DNA in both Ascaris and Parascaris, ensuring chromosomal breakage and providing a fail-safe mechanism for PDE. Overall, our data indicate that telomere healing of DSBs is specific to the break sites responsible for nematode PDE.

Reduced Likelihood of Hospitalization with the JN.1 or HV.1 SARS-CoV-2 Variants Compared to the EG.5 Variant.

Within a multi-state viral genomic surveillance program, we evaluated whether proportions of SARS-CoV-2 infections attributed to the JN.1 variant and to XBB-lineage variants (including HV.1 and EG.5) differed between inpatient and outpatient care settings during periods of cocirculation. Both JN.1 and HV.1 were less likely than EG.5 to account for infections among inpatients versus outpatients (aOR=0.60 [95% CI: 0.43-0.84; p=0.003] and aOR=0.35 [95% CI: 0.21-0.58; p<0.001], respectively). JN.1 and HV.1 variants may be associated with a lower risk of severe illness. The severity of COVID-19 may have attenuated as predominant circulating SARS-CoV-2 lineages shifted from EG.5 to HV.1 to JN.1.

Chronic Mexiletine Administration Increases Sodium Current in Non-Diseased Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

A sodium current (INa) reduction occurs in the setting of many acquired and inherited conditions and is associated with cardiac conduction slowing and increased arrhythmia risks. The sodium channel blocker mexiletine has been shown to restore the trafficking of mutant sodium channels to the membrane. However, these studies were mostly performed in heterologous expression systems using high mexiletine concentrations. Moreover, the chronic effects on INa in a non-diseased cardiomyocyte environment remain unknown. In this paper, we investigated the chronic and acute effects of a therapeutic dose of mexiletine on INa and the action potential (AP) characteristics in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) of a healthy individual. Control hiPSC-CMs were incubated for 48 h with 10 µM mexiletine or vehicle. Following the wash-out of mexiletine, patch clamp analysis and immunocytochemistry experiments were performed. The incubation of hiPSC-CMs for 48 h with mexiletine (followed by wash-out) induced a significant increase in peak INa of ~75%, without any significant change in the voltage dependence of (in)activation. This was accompanied by a significant increase in AP upstroke velocity, without changes in other AP parameters. The immunocytochemistry experiments showed a significant increase in membrane Nav1.5 fluorescence following a 48 h incubation with mexiletine. The acute re-exposure of hiPSC-CMs to 10 µM mexiletine resulted in a small but significant increase in AP duration, without changes in AP upstroke velocity, peak INa density, or the INa voltage dependence of (in)activation. Importantly, the increase in the peak INa density and resulting AP upstroke velocity induced by chronic mexiletine incubation was not counteracted by the acute re-administration of the drug. In conclusion, the chronic administration of a clinically relevant concentration of mexiletine increases INa density in non-diseased hiPSC-CMs, likely by enhancing the membrane trafficking of sodium channels. Our findings identify mexiletine as a potential therapeutic strategy to enhance and/or restore INa and cardiac conduction.

Three-dimensional cardiac models: a pre-clinical testing platform.

Major advancements in human pluripotent stem cell (hPSC) technology over recent years have yielded valuable tools for cardiovascular research. Multi-cell type 3-dimensional (3D) cardiac models in particular, are providing complementary approaches to animal studies that are better representatives than simple 2-dimensional (2D) cultures of differentiated hPSCs. These human 3D cardiac models can be broadly divided into two categories; namely those generated through aggregating pre-differentiated cells and those that form self-organizing structures during their in vitro differentiation from hPSCs. These models can either replicate aspects of cardiac development or enable the examination of interactions among constituent cell types, with some of these models showing increased maturity compared with 2D systems. Both groups have already emerged as physiologically relevant pre-clinical platforms for studying heart disease mechanisms, exhibiting key functional attributes of the human heart. In this review, we describe the different cardiac organoid models derived from hPSCs, their generation methods, applications in cardiovascular disease research and use in drug screening. We also address their current limitations and challenges as pre-clinical testing platforms and propose potential improvements to enhance their efficacy in cardiac drug discovery.

Genome-wide Cas9-mediated screening of essential non-coding regulatory elements via libraries of paired single-guide RNAs.

The functions of non-coding regulatory elements (NCREs), which constitute a major fraction of the human genome, have not been systematically studied. Here we report a method involving libraries of paired single-guide RNAs targeting both ends of an NCRE as a screening system for the Cas9-mediated deletion of thousands of NCREs genome-wide to study their functions in distinct biological contexts. By using K562 and 293T cell lines and human embryonic stem cells, we show that NCREs can have redundant functions, and that many ultra-conserved elements have silencer activity and play essential roles in cell growth and in cellular responses to drugs (notably, the ultra-conserved element PAX6_Tarzan may be critical for heart development, as removing it from human embryonic stem cells led to defects in cardiomyocyte differentiation). The high-throughput screen, which is compatible with single-cell sequencing, may allow for the identification of druggable NCREs.

Validating human induced pluripotent stem cell-specific quality control tests for the release of an intermediate drug product in a Good Manufacturing Practice quality system.

One of the challenges in Good Manufacturing Practice (GMP)-compliant human induced pluripotent stem cell (hiPSC) production is the validation of quality control (QC) tests specific for hiPSCs, which are required for GMP batch release. This study presents a comprehensive description of the validation process for hiPSC-specific GMP-compliant QC assays; more specifically, the validation of assays to assess the potential presence of residual episomal vectors (REVs), the expression of markers of the undifferentiated state and the directed differentiation potential of hiPSCs. Critical aspects and specific acceptance criteria were formulated in a validation plan prior to assay validation. Assay specificity, sensitivity and reproducibility were tested, and the equipment used for each assay was subjected to performance qualification. A minimum input of 20 000 cells (120 ng of genomic DNA) was defined for accurate determination of the presence of REVs. Furthermore, since vector loss in hiPSC lines is a passage-dependent process, we advocate screening for REVs between passages eight and 10, as testing at earlier passages might lead to unnecessary rejection of hiPSC lines. The cutoff value for assessment of markers of the undifferentiated state was set to the expression of at least three individual markers on at least 75% of the cells. When multi-color flow cytometry panels are used, a fluorescence minus one control is advised to ensure the control for fluorescent spread. For the assay to assess the directed differentiation potential, the detection limit was set to two of three positive lineage-specific markers for each of the three individual germ layers. All of our assays proved to be reproducible and specific. Our data demonstrate that our implemented analytical procedures are suitable as QC assays for the batch release of GMP-compliant hiPSCs.

A machine learning algorithm improves the diagnostic accuracy of the histologic component of antibody mediated rejection (AMR-H) in cardiac transplant endomyocardial biopsies.

BACKGROUND: Pathologic antibody mediated rejection (pAMR) remains a major driver of graft failure in cardiac transplant patients. The endomyocardial biopsy remains the primary diagnostic tool but presents with challenges, particularly in distinguishing the histologic component (pAMR-H) defined by 1) intravascular macrophage accumulation in capillaries and 2) activated endothelial cells that expand the cytoplasm to narrow or occlude the vascular lumen. Frequently, pAMR-H is difficult to distinguish from acute cellular rejection (ACR) and healing injury. With the advent of digital slide scanning and advances in machine deep learning, artificial intelligence technology is widely under investigation in the areas of oncologic pathology, but in its infancy in transplant pathology. For the first time, we determined if a machine learning algorithm could distinguish pAMR-H from normal myocardium, healing injury and ACR. MATERIALS AND METHODS: A total of 4,212 annotations (1,053 regions of normal, 1,053 pAMR-H, 1,053 healing injury and 1,053 ACR) were completed from 300 hematoxylin and eosin slides scanned using a Leica Aperio GT450 digital whole slide scanner at 40X magnification. All regions of pAMR-H were annotated from patients confirmed with a previous diagnosis of pAMR2 (>50% positive C4d immunofluorescence and/or >10% CD68 positive intravascular macrophages). Annotations were imported into a Python 3.7 development environment using the OpenSlide™ package and a convolutional neural network approach utilizing transfer learning was performed. RESULTS: The machine learning algorithm showed 98% overall validation accuracy and pAMR-H was correctly distinguished from specific categories with the following accuracies: normal myocardium (99.2%), healing injury (99.5%) and ACR (99.5%). CONCLUSION: Our novel deep learning algorithm can reach acceptable, and possibly surpass, performance of current diagnostic standards of identifying pAMR-H. Such a tool may serve as an adjunct diagnostic aid for improving the pathologist's accuracy and reproducibility, especially in difficult cases with high inter-observer variability. This is one of the first studies that provides evidence that an artificial intelligence machine learning algorithm can be trained and validated to diagnose pAMR-H in cardiac transplant patients. Ongoing studies include multi-institutional verification testing to ensure generalizability.

End resection and telomere healing of DNA double-strand breaks during nematode programmed DNA elimination.

Most DNA double-strand breaks (DSBs) are harmful to genome integrity. However, some forms of DSBs are essential to biological processes, such as meiotic recombination and V(D)J recombination. DSBs are also required for programmed DNA elimination (PDE) in ciliates and nematodes. In nematodes, the DSBs are healed with telomere addition. While telomere addition sites have been well-characterized, little is known regarding the DSBs that fragment nematode chromosomes. Here, we used embryos from the nematode Ascaris to study the timing of PDE breaks and examine the DSBs and their end processing. Using END-seq, we characterize the DSB ends and demonstrate that DNA breaks are introduced before mitosis, followed by extensive end resection. The resection profile is unique for each break site, and the resection generates 3' overhangs before the addition of telomeres. Interestingly, telomere healing occurs much more frequently on retained DSB ends than on eliminated ends. This biased repair of the DSB ends in Ascaris may be due to the sequestration of the eliminated DNA into micronuclei, preventing their ends from telomere healing. Additional DNA breaks occur within the eliminated DNA in both Ascaris and Parascaris, ensuring chromosomal breakage and providing a fail-safe mechanism for nematode PDE.

A Quantitative Trait Locus on Maize Chromosome 5 Is Associated with Root-Knot Nematode Resistance.

This study provides the first report of a quantitative trait locus (QTL) in maize (Zea mays) for resistance to the southern root-knot nematode (SRKN) (Meloidogyne incognita). The SRKN can feed on the roots of maize in the U.S. Southern Coastal Plain region and can cause yield losses of 30% or more in heavily infested fields. Increases in SRKN density in the soil may reduce the yield for subsequently planted susceptible crops. The use of maize hybrids with resistance to SRKN could prevent an increase in SRKN density, yet no genetic regions have been identified that confer host resistance. In this study, a B73 (susceptible) × Ky21 (resistant) S5 recombinant inbred line (RIL) population was phenotyped for total number of eggs (TE) and root weight. This population had been genotyped using single-nucleotide polymorphisms (SNPs). By utilizing the SNP data with the phenotype data, a single QTL was identified on chromosome 5 that explained 15% of the phenotypic variation (PV) for the number of eggs and 11% of the PV for the number of eggs per gram of root (EGR). Plants that were homozygous for the Ky21 allele for the most associated marker PZA03172.3 had fewer eggs and fewer EGR than the plants that were homozygous or heterozygous for the B73 allele. Thus, the first QTL for SRKN resistance in maize has been identified and could be incorporated into maize hybrids.

A metagenomic investigation of phytoplasma diversity in Australian vegetable growing regions.

In this study, metagenomic sequence data was used to investigate the phytoplasma taxonomic diversity in vegetable-growing regions across Australia. Metagenomic sequencing was performed on 195 phytoplasma-positive samples, originating either from historic collections (n=46) or during collection efforts between January 2015 and June 2022 (n=149). The sampled hosts were classified as crop (n=155), weed (n=24), ornamental (n=7), native plant (n=6), and insect (n=3) species. Most samples came from Queensland (n=78), followed by Western Australia (n=46), the Northern Territory (n=32), New South Wales (n=17), and Victoria (n=10). Of the 195 draft phytoplasma genomes, 178 met our genome criteria for comparison using an average nucleotide identity approach. Ten distinct phytoplasma species were identified and could be classified within the 16SrII, 16SrXII (PCR only), 16SrXXV, and 16SrXXXVIII phytoplasma groups, which have all previously been recorded in Australia. The most commonly detected phytoplasma taxa in this study were species and subspecies classified within the 16SrII group (n=153), followed by strains within the 16SrXXXVIII group ('Ca. Phytoplasma stylosanthis'; n=6). Several geographic- and host-range expansions were reported, as well as mixed phytoplasma infections of 16SrII taxa and 'Ca. Phytoplasma stylosanthis'. Additionally, six previously unrecorded 16SrII taxa were identified, including five putative subspecies of 'Ca. Phytoplasma australasiaticum' and a new putative 16SrII species. PCR and sequencing of the 16S rRNA gene was a suitable triage tool for preliminary phytoplasma detection. Metagenomic sequencing, however, allowed for higher-resolution identification of the phytoplasmas, including mixed infections, than was afforded by only direct Sanger sequencing of the 16S rRNA gene. Since the metagenomic approach theoretically obtains sequences of all organisms in a sample, this approach was useful to confirm the host family, genus, and/or species. In addition to improving our understanding of the phytoplasma species that affect crop production in Australia, the study also significantly expands the genomic sequence data available in public sequence repositories to contribute to phytoplasma molecular epidemiology studies, revision of taxonomy, and improved diagnostics.

Septic Arthritis of the Sternoclavicular Joint.

Introduction: Sternoclavicular joint (SCJ) septic arthritis is a rare but rapidly fatal joint infection. Without proper medical or surgical management, it can progress to osteomyelitis, chest wall abscess, mediastinitis, or myositis. Case Report: A 57-year-old male with a past history of intravenous drug use presented to the emergency department (ED) with chest tenderness of one week duration. Vital signs were unremarkable, and exam was notable for tender, raised right SCJ without any fluctuance. On point-of-care ultrasound we noted fluid collection and capsular distention along the SCJ, which aided in rapidly diagnosing septic arthritis. The patient was immediately started on antibiotics and taken to the operating room for excision and debridement. Conclusion: While computed tomography is routinely used in the emergency department for diagnosing septic arthritis, ultrasound offers a rapid and safe alternative for diagnosis.

Doxycycline Sclerodesis-"Rhinodesis"-for Enhanced Soft Tissue Adhesion in Rhinoplasty: A Preliminary Study.

The use of doxycycline as a sclerosing agent is well-established. Given the clinical efficacy of doxycycline sclerosant therapy, we embarked upon a study to evaluate the efficacy of small-volume liquified doxycycline particularly in thick skinned rhinoplasty patients to promote re-adhesion of the nasal skin-soft tissue envelope (SSTE) thereby minimizing surgical dead space and enhancing surface contour, to improve the eventual outcome of surgery.We present two clinical case series using rhinodesis. All patients were treated with the same rhinodesis protocol that included conventional splinting and taping. The first series consisted of 102 consecutive primary rhinoplasties with medium to thick nasal skin treated via open rhinoplasty. Doxycycline solution at a concentration of 20 mg/mL was applied beneath the skin flap using a 14-gauge angiocath inserted through small gaps in the marginal suture line following closure, retained for 2 to 3 minutes, and then expressed from the dead space. Firm manual compression of the SSTE was maintained for at least 1 additional minute, and the splint was then applied. The second series consisted of 25 thick-skinned primary rhinoplasties that were also treated with open rhinoplasty using the same rhinodesis protocol. However, the second group was evaluated with serial postoperative ultrasonography to characterize the soft-tissue response to rhinodesis, particularly within the tip and supra-tip regions.Results revealed enhanced skin adherence in nearly all patients when compared to traditional taping and splinting alone. Ultrasonic examination demonstrated enhanced adherence of the subcutaneous tissue to the nasal framework and suggests that rhinodesis is effective at minimizing dead space in majority of thick-skinned rhinoplasty patients. No complications were observed. Doxycycline can be used easily and safely to seal the surgical dead space post-rhinoplasty and minimize degradation of nasal contour with excellent outcome.

Chimeric antigen receptor T cells to target CD79b in B-cell lymphomas.

BACKGROUND: Chimeric antigen receptor (CAR) T cells targeting CD19 mediate potent and durable effects in B-cell malignancies. However, antigen loss or downregulation is a frequent cause of resistance. Here, we report development of a novel CAR T-cell therapy product to target CD79b, a pan B-cell antigen, widely expressed in most B-cell lymphomas. METHODS: We generated a novel anti-CD79b monoclonal antibody by hybridoma method. The specificity of the antibody was determined by testing against isogenic cell lines with human CD79b knock-in or knock-out. A single-chain variable fragment derived from the monoclonal antibody was used to make a panel of CD79b-targeting CAR molecules containing various hinge, transmembrane, and co-stimulatory domains. These were lentivirally transduced into primary T cells and tested for antitumor activity in in vitro and in vivo B-cell lymphoma models. RESULTS: We found that the novel anti-CD79b monoclonal antibody was highly specific and bound only to human CD79b and no other cell surface protein. In testing the various CD79b-targeting CAR molecules, superior antitumor efficacy in vitro and in vivo was found for a CAR consisting CD8α hinge and transmembrane domains, an OX40 co-stimulatory domain, and a CD3ζ signaling domain. This CD79b CAR specifically recognized human CD79b-expressing lymphoma cell lines but not CD79b knock-out cell lines. CD79b CAR T cells, generated from T cells from either healthy donors or patients with lymphoma, proliferated, produced cytokines, degranulated, and exhibited robust cytotoxic activity in vitro against CD19+ and CD19- lymphoma cell lines and patient-derived lymphoma tumors relapsing after prior CD19 CAR T-cell therapy. Furthermore, CD79b CAR T cells were highly efficient at eradicating pre-established lymphoma tumors in vivo in three aggressive lymphoma xenograft models, including two cell line-derived xenografts and one patient-derived xenograft. Notably, these CAR T cells did not demonstrate any significant tonic signaling activity or markers of exhaustion. CONCLUSION: Our results indicated that this novel CD79b CAR T-cell therapy product has robust antitumor activity against B-cell lymphomas. These results supported initiation of a phase 1 clinical trial to evaluate this product in patients with relapsed or refractory B-cell lymphomas.