Keith R. Jerome’s research while affiliated with University of Washington and other places

Chimeric antigen receptor T (CAR-T) cell therapy has revolutionized treatment for B-cell malignancies, yet over 60% of patients relapse within one year, often due to insufficient CAR-T persistence. While mouse and primary cell models have been instrumental in advancing CAR-T therapy, they frequently fail to predict clinical outcomes, underscoring the need for more translationally relevant models. To address this limitation, we conducted the first systematic evaluation of CAR structure-function relationships in an immunocompetent nonhuman primate (NHP) model. We engineered an array of 20 CD20-targeted CARs with distinct combinations of hinge, transmembrane, and costimulatory domains. Following ex vivo characterization, we administered pooled autologous CAR-T arrays to three NHPs and tracked CAR abundance longitudinally using a novel digital droplet PCR assay. Ex vivo , CAR-T cells incorporating the MyD88-CD40 costimulatory domain exhibited markedly distinct functional profiles, including increased activation, unique cytokine secretion, tonic signaling, and resistance to exhaustion. In vivo , MyD88-CD40 CARs expanded dramatically, comprising up to 100% of peripheral T cells and significantly outperforming canonical CD28- and 4-1BB-based CARs. This expansion was associated with robust B-cell depletion across all animals. MyD88-CD40 CARs, particularly those with a CD28 hinge and transmembrane domain, demonstrated superior trafficking to secondary lymphoid tissues and persistence through study endpoint, unlike other CARs which waned by day 28. Our findings highlight the value of NHP models for screening CAR designs and identify MyD88-CD40 CARs as candidates with unmatched potency. The unique functional attributes conferred by this domain may provide key insights into features that drive enhanced CAR-T cell activity. Key points We developed the first pooled CAR-T screening platform in an immunocompetent nonhuman primate model to directly compare CAR designs. We identified MyD88-CD40 costimulatory domain as vastly superior to conventional domains in proliferation, trafficking and persistence.

Despite antiretroviral therapy (ART), people with HIV (PWH) on ART experience higher rates of morbidity and mortality vs. age-matched HIV negative controls, which may be driven by chronic inflammation due to persistent virus. We performed bulk RNA sequencing (RNA-seq) on peripheral CD4+ T cells, as well as quantified plasma immune marker levels from 154 PWH on ART to identify host immune signatures associated with immune recovery (CD4:CD8) and HIV persistence (cell-associated HIV DNA and RNA). Using a novel dimension reduction tool - Pairwise Controlled Manifold Approximation (PaCMAP), we defined three distinct participant transcriptomic clusters. We found that these three clusters were largely defined by differential expression of genes regulated by the transcription factor NF-κB. While clustering was not associated with HIV reservoir size, we observed an association with CD4:CD8 ratio, a marker of immune recovery and prognostic factor for mortality in PWH on ART. Furthermore, distinct patterns of plasma IL-1β, TNF-α and GCSF were also strongly associated with the clusters, suggesting that these immune markers play a key role in CD4+ T cell transcriptomic diversity and immune recovery in PWH on ART. These findings reveal novel subgroups of PWH on ART with distinct immunological characteristics, and define a transcriptional signature associated with clinically significant immune parameters for PWH. A deeper understanding of these subgroups could advance clinical strategies to treat HIV-associated immune dysfunction.

The treatment of monogenetic disorders, such as hemoglobinopathies and lysosomal storage diseases, has markedly improved with the advent of cell and gene therapies, particularly allogeneic or gene-modified autologous stem cell transplantations. However, therapeutic efficacy is reliant on maintaining engraftment above a critical threshold. To maintain such engraftment levels, we and others have pursued approaches to shield edited cells from antibody or CAR T-cell mediated selection. Here we focused on CD33, which is expressed early on hematopoietic stem and progenitor cells (HSPC) as well as on myeloid progenitors. Rhesus macaques were engrafted with HSPCs edited to ablate CD33 utilizing either CRISPR/Cas9 or adenine base editor. Both editing strategies showed similar post-transplant recovery kinetics and yielded equivalent levels of engraftment. We then created a V-set domain specific chimeric antigen receptor construct (CAR33), validated its functionality in vitro, and treated both animals with autologous CAR33 T cells. CAR33 T cells expanded after infusion and caused specific depletion of CD33WT but not CD33null progeny - leading to a transient enrichment for gene-edited cells in the blood. No depletion was seen in the bone marrow stem cell compartment with CD34+CD90+ HSCs expressing lower levels of CD33 in comparison to monocytes. Thus, we show proof of concept and safety of an epitope editing based enrichment/protection strategy in macaques.

Background Limited data exist on the association between specific symptoms and viral loads of common cold coronavirus (ccCOV). We investigated this potential association in allogeneic hematopoietic cell transplant (HCT) recipients.Figure 1.Proportion of sampling time points with each symptom Methods Four hundred seventy-one children and adults were prospectively followed with weekly symptom questionnaires through one year after allogeneic HCT at the Fred Hutchinson Cancer Center (12/2005-2/2010). Multiplex PCR for 11 respiratory viruses was performed on combined nasal wash and throat swab samples collected weekly through day 100 post-HCT, and then every three months or if symptoms were present through one-year post-HCT. Positive samples were evaluated for viral loads with quantitative real-time PCR (copies/ml). We included time points with the detection of ccCOV only and corresponding 15-point symptom survey data available post-HCT. To assess the association between specific symptoms and viral loads, the Mann-Whitney or Jonckheere-Terpstra tests were used to compare 2 or >2 groups for continuous variables, respectively.Figure 2.Symptoms associated with higher viral loads Results Among 6,276 samples tested, 251 samples (79 patients) were positive for ccCoV. One hundred thirty-six sampling time points (52 patients) met the inclusion criteria. The proportion of sample time points with each symptom present is shown in Figure 1. When we compared viral loads with the presence of each of the 15 symptoms, higher viral loads were associated with the presence of four respiratory tract symptoms (rhinorrhea, sneezing, sore throat, and coughing) and one systemic symptom (diarrhea) (p< 0.01 for each) (Figure 2). Viral loads appeared to increase with increasing number of respiratory tract symptoms (p< 0.001) (Figure 3).Figure 3.Viral loads compared with the number of respiratory tract symptoms Conclusion The presence of specific respiratory tract symptoms is associated with higher viral loads of ccCOV in the upper respiratory tract in allogeneic HCT recipients. The association with diarrhea suggests the possibility of fecal shedding, but this was not evaluated in our study. Patients with specific symptoms may be more likely to transmit virus given their higher viral loads. Studies of these associations for other respiratory viruses are warranted. Disclosures Chikara Ogimi, MD, PhD, AstraZeneca: Honoraria|bioMerieux Japan Ltd.: Honoraria|ELSEVIER: Honoraria|KYORIN: Honoraria|Miyarisan: Honoraria|MSD: Honoraria|NOVARTIS: Honoraria|Pfizer: Honoraria Alpana waghmare, MD, Allovir: Grant/Research Support|Ansun Biopharma: Grant/Research Support|GlaxoKlineSmith: Advisor/Consultant|GlaxoKlineSmith: Grant/Research Support|Pfizer: Grant/Research Support|Vir: Advisor/Consultant Janet A. Englund, MD, Abbvie: Advisor/Consultant|AstraZeneca: Advisor/Consultant|AstraZeneca: Grant/Research Support|GlaxoSmithKline: Advisor/Consultant|GlaxoSmithKline: Grant/Research Support|Meissa Vaccines: Advisor/Consultant|Merck: Advisor/Consultant|Pfizer: Board Member|Pfizer: Grant/Research Support|Pfizer: Speaker at meeting|SanofiPasteur: Advisor/Consultant|Shinogi: Advisor/Consultant Michael J. Boeckh, MD PhD, Allovir: Advisor/Consultant|Allovir: Grant/Research Support|AstraZeneca: Advisor/Consultant|AstraZeneca: Grant/Research Support|Merck: Advisor/Consultant|Merck: Grant/Research Support|Moderna: Advisor/Consultant|Moderna: Grant/Research Support|Symbio: Advisor/Consultant

Background We previously identified novel risk factors for progression to lower respiratory tract disease (LRTD) among allogeneic hematopoietic cell transplant (HCT) recipients presenting with upper respiratory tract infection (URTI) with 12 viruses in the PCR era and proposed a simple prediction model by a number of risk factors (BMT Ogimi C, et al. Bone Marrow Transplant 2022; 57(4):649-657). We aimed to investigate whether the presence of these risk factors at the time of URTI diagnosis also predicts overall mortality and pulmonary death.Figure 1.Kaplan-Meier plot of overall death and cumulative incidence plot of pulmonary death by day 90 among patients with any viral URTI (N=947) Methods We retrospectively analyzed patients with the first respiratory virus detected by multiplex PCR after allogeneic HCT (4/2008-9/2018). We created Kaplan-Meier plots for overall mortality and cumulative incidence plots for pulmonary death within 90 days among patients presenting with URTI by a single virus. Candidate risk factors were age >=40 years, a history of multiple HCT, early timing post-HCT (< =30 days), albumin < =3 g/dL, monocytopenia (< =100 cells/µL), highest glucose value >200 mg/dl, and systemic steroid use within 14 days.Figure 2.Kaplan-Meier plot of overall death by day 90 among patients with viral URTI Results A total of 947 patients (199 children and 748 adults) presented with URTI only as follows: 407 human rhinoviruses (HRV), 174 parainfluenza viruses (PIV) 1–4, 140 common cold coronaviruses, 84 respiratory syncytial virus (RSV), 58 influenza A/B, 43 human metapneumovirus (MPV), and 41 adenoviruses. Among these, 81 (18%) died with 44 pulmonary deaths within 90 days from URTI. No patients with any viral URTI without risk factors died (Figure 1). Patients with 3 or more risk factors appear to have high overall mortality, and pulmonary death accounted for more than half of cases as a cause of death (Figure 1). These trends were seen across viruses evaluated (Figure 2). Conclusion Established risk factors for progression from viral URTI to LRTD also predict overall mortality and pulmonary death. Patients without any risk factors appeared to be completely protected from death, while patients with >3 risk factors were at the highest risk for death, suggesting an intervention opportunity and close monitoring. These trends were observed across several viruses and future studies are needed to validate our findings in different cohorts. Disclosures Chikara Ogimi, MD, PhD, AstraZeneca: Honoraria|bioMerieux Japan Ltd.: Honoraria|ELSEVIER: Honoraria|KYORIN: Honoraria|Miyarisan: Honoraria|MSD: Honoraria|NOVARTIS: Honoraria|Pfizer: Honoraria Alpana waghmare, MD, Allovir: Grant/Research Support|Ansun Biopharma: Grant/Research Support|GlaxoKlineSmith: Advisor/Consultant|GlaxoKlineSmith: Grant/Research Support|Pfizer: Grant/Research Support|Vir: Advisor/Consultant Janet A. Englund, MD, Abbvie: Advisor/Consultant|AstraZeneca: Advisor/Consultant|AstraZeneca: Grant/Research Support|GlaxoSmithKline: Advisor/Consultant|GlaxoSmithKline: Grant/Research Support|Meissa Vaccines: Advisor/Consultant|Merck: Advisor/Consultant|Pfizer: Board Member|Pfizer: Grant/Research Support|Pfizer: Speaker at meeting|SanofiPasteur: Advisor/Consultant|Shinogi: Advisor/Consultant Michael J. Boeckh, MD PhD, Allovir: Advisor/Consultant|Allovir: Grant/Research Support|AstraZeneca: Advisor/Consultant|AstraZeneca: Grant/Research Support|Merck: Advisor/Consultant|Merck: Grant/Research Support|Moderna: Advisor/Consultant|Moderna: Grant/Research Support|Symbio: Advisor/Consultant

Therapeutic in vivo gene editing with highly specific nucleases has the potential to revolutionize treatment for a wide range of human diseases, including genetic disorders and latent viral infections like herpes simplex virus (HSV). However, challenges regarding specificity, efficiency, delivery, and safety must be addressed before its clinical application. A key concern is the risk of off-target effects, which can cause unintended and potentially harmful genetic changes. We previously developed a curative in vivo gene editing approach to eliminate latent HSV using HSV-specific meganuclease delivered by an AAV vector. In this study, we investigate off-target effects of meganuclease by identifying potential off-target sites through GUIDE-tag analysis and assessing genetic alterations using amplicon deep sequencing in tissues from meganuclease treated mice. Our results show that meganuclease expression driven by a ubiquitous promoter leads to high off-target gene editing in the mouse liver, a non-relevant target tissue. However, restricting the meganuclease expression with a neuron-specific promoter and/or a liver-specific miRNA target sequence efficiently reduces off-target effects in both liver and trigeminal ganglia. These findings suggest that incorporation of regulatory DNA elements for tissue-specific expression in viral vectors can reduce off-target effects and improve the safety of therapeutic in vivo gene editing.

Gene drives are genetic modifications designed to propagate efficiently through a population. Most applications rely on homologous recombination during sexual reproduction in diploid organisms such as insects, but we recently developed a gene drive in herpesviruses that relies on co-infection of cells by wild-type and engineered viruses. Here, we report on a viral gene drive against human herpes simplex virus 1 (HSV-1) and show that it propagates efficiently in cell culture and during HSV-1 infection in mice. We describe high levels of co-infection and gene drive-mediated recombination in neuronal tissues during herpes encephalitis as the infection progresses from the site of inoculation to the peripheral and central nervous systems. In addition, we show evidence that a superinfecting gene drive virus could recombine with wild-type viruses during latent infection. These findings indicate that HSV-1 achieves high rates of co-infection and recombination during viral infection, a phenomenon that is currently underappreciated. Overall, this study shows that a viral gene drive could spread in vivo during HSV-1 infection, paving the way toward therapeutic applications.

Cytomegalovirus (CMV) disease occurs occasionally before allogeneic hematopoietic cell transplantation (HCT) and is associated with poor post-HCT outcomes; however, the impact of pre-HCT CMV reactivation is unknown. Pre-HCT CMV reactivation was assessed in HCT candidates from the preemptive antiviral therapy (2007-17) and letermovir prophylaxis (2018-21) eras. CMV DNA PCR surveillance was routinely performed during the pre-HCT work-up period, and antiviral therapy was recommended according to risk for progression to CMV disease. Risk factors for pre-HCT CMV reactivation were characterized and the associations of pre-HCT CMV reactivation with post-HCT outcomes were examined using logistic regression and Cox proportional hazard models, respectively. A total of 1694 patients were identified and 11% had pre-HCT CMV reactivation 14 days (median; IQR 6-23 days) before HCT. Lymphopenia (≤300 cells/uL) was the strongest risk factor for pre-HCT CMV reactivation at multiple PCR levels. In the preemptive therapy era, patients with pre-HCT CMV reactivation had a significantly increased risk of CMV reactivation by day 100 as well as CMV disease and death by 1 year post-HCT. Clearance of pre-HCT CMV reactivation was associated with a lower risk of post-HCT CMV reactivation. Similar associations with post-HCT CMV endpoints were observed in a cohort of patients receiving letermovir prophylaxis. Pre-HCT CMV reactivation can be routinely detected in high-risk HCT candidates and is a significant risk factor for post-HCT CMV reactivation and disease. Pre-HCT CMV DNA PCR surveillance is recommended in high-risk HCT candidates and antiviral therapy may be indicated to prevent post-HCT CMV reactivation.

Anti-HSV therapies are only suppressive because they do not eliminate latent HSV present in ganglionic neurons, the source of recurrent disease. We have developed a potentially curative approach against HSV infection, based on gene editing using HSV-specific meganucleases delivered by adeno-associated virus (AAV) vectors. Gene editing performed with two anti-HSV-1 meganucleases delivered by a combination of AAV9, AAV-Dj/8, and AAV-Rh10 can eliminate 90% or more of latent HSV DNA in mouse models of orofacial infection, and up to 97% of latent HSV DNA in mouse models of genital infection. Using a pharmacological approach to reactivate latent HSV-1, we demonstrate that ganglionic viral load reduction leads to a significant decrease of viral shedding in treated female mice. While therapy is well tolerated, in some instances, we observe hepatotoxicity at high doses and subtle histological evidence of neuronal injury without observable neurological signs or deficits. Simplification of the regimen through use of a single serotype (AAV9) delivering single meganuclease targeting a duplicated region of the HSV genome, dose reduction, and use of a neuron-specific promoter each results in improved tolerability while retaining efficacy. These results reinforce the curative potential of gene editing for HSV disease.