HIV-1 establishes latency in resting memory CD4+ T cells, creating a long-lived reservoir that poses a barrier to HIV-1 cure. This reservoir is thought to form by initial infection of antigen-activated CD4+ T-cells that then transition into quiescent memory CD4+ T cells. However, naïve CD4+ T cells have been shown to harbor integrated HIV-1 in adults with non-perinatally acquired HIV-1, and more recently in young children. Given that perinatal HIV-1 transmission is established in an immune environment dominated by naïve CD4+ T cells, we hypothesized that naïve CD4+ T cells may be a substantial reservoir in young adults with longstanding perinatal infection. To investigate this, we studied a cohort of 10 adolescents and young adults (AYA) living with well-controlled, perinatally-acquired HIV-1. We quantified and characterized intact and defective HIV-1 proviruses and their location in sorted naïve and memory CD4+ T cells using an intact proviral DNA assay, near full-length single genome sequencing, and integration site profiling. We found that a median of 93.5% of HIV-1 proviruses resided in memory CD4+ T cells in AYA with perinatal HIV-1, with a few participants also having intact proviruses in naïve CD4+ T cells, but at substantially lower frequencies. HIV-1 integration site analysis, using the unbiased ligation-mediated PCR method, showed that 78.0% of proviruses were located within active transcription units in introns and 46.9% of integration sites were clonal, with detection in multiple cells. Overall, similar to adults with non-perinatally acquired HIV-1, integration sites were enriched in regions with epigenetic marks for active enhancers and transcription. Therapies to attain HIV-1 ART-free remission and cure for this population will need to consider reservoirs in non-canonical T cells subsets, as well as substantial HIV-1 infected cells arising from cellular proliferation.

Abstract Sickle cell disease (SCD) is characterized by chronic hemolysis, and painful vaso-occlusive episodes (VOE). High levels of fetal hemoglobin (HbF) attenuate the disease phenotype. We used a lentivirus vector (LVV) expressing a short hairpin RNA embedded in a microRNA (shmiR) that targets BCL11A in erythrocytes to induce HbF in a first-in-human study in SCD. The purpose of the study was to assess hematopoietic stem/progenitor cells collection, transduction parameters, safety, HbF induction, and durability. Eleven eligible patients with SCD underwent hematopoietic stem cell (HSC) collection. Plerixafor-mobilized peripheral blood HSCs required for manufacturing were obtained in 1 mobilization cycle for 10 of 11 participants, and 11 of 11 patient products were successfully manufactured with a median time to release of product of 39 days. Ten patients were infused with HSCs transduced with the shmiR vector. Engraftment occurred in all 10 patients. At a median follow-up of 58 months (range, 35-82) after infusion, there were no adverse events attributed to the vector. The transduction efficiency was 93.1%. One patient demonstrated low engraftment of the transduced cells and had suboptimal HbF induction. In the remaining 9 patients, at 2 years after treatment, the peripheral blood demonstrated 71% F cells with 11.9 pg HbF per F cells, and both parameters remained stable in 9 patients with ≥48 months follow-up. All patients who had VOEs before gene therapy demonstrated sustained mitigation of pain events. These data demonstrate excellent manufacturing efficiency, efficacy and safety of targeting BCL11A using a shmiR LVV, and long-term durability of the shmiR vector, leading to a pivotal, multisite, phase 2 trial that is currently underway (NCT05353647). This trial was registered at www.clinicaltrials.gov as NCT03282656.

ABSTRACTMetachromatic leukodystrophy (MLD) is a fatal lysosomal storage disease (LSD) characterized by the deficient enzymatic activity of arylsulfatase A (ARSA). Combined autologous hematopoietic stem cell transplant (HSCT) with lentiviral (LV) based gene therapy has great potential to treat MLD. However, if enzyme production is inadequate, this could result in continued loss of motor function, implying a high vector copy number (VCN) requirement for optimal enzymatic output. This may place children at increased risk for genomic toxicity due to higher VCN. We increased the expression of ARSA cDNA at single integration by generating novel LVs, optimizing ARSA expression, and enhancing safety. In addition, our vectors achieved optimal transduction in mouse and human HSC with minimal multiplicity of infection (MOI). Our top-performing vector (EA1) showed at least 4X more ARSA activity than the currently EU-approved vector and a superior ability to secrete vesicle-associated ARSA, a critical modality to transfer functional enzymes from microglia to oligodendrocytes. Three-month-oldArsa-KO MLD mice transplanted withArsa-KO BM cells transduced with 0.6 VCN of EA1 demonstrated behavior and CNS histology matching WT mice. Our novel vector boosts efficacy while improving safety as a robust approach for treating early symptomatic MLD patients.

Analysis of EGR2 and type I IFN pathway regulation in CAR T-cells. (A-C) ATAC-seq tracks of genes associated with dysfunction, type I IFN signaling, and memory differentiation are shown, with differentially accessible regions indicated. D, Heatmap showing expression of exhaustion genes in tonically signaling GD2 and functional CD19 naïve CD8+ CAR-T cells (GSE136891). E, Comparison of EGR2 and type I IFN gene signature scores in central memory CD8+ T-cells expressing a tonically signaling GD2 CAR and control CD19 CAR. CAR T-cells were generated using T-cells from a healthy donor (GSE136891). F, Top transcription factor co-expression signatures overexpressed in CD19 CAR T-cell products of CLL complete responders. G, Comparison of EGR2 and type I IFN signature scores in unstimulated CD19 CAR T-cell products from CLL patients (CR: complete response, PRTD: very good partial response, PR: conventional partial response, NR: no response). H, Comparison of type I IFN and EGR2 module scores in PSMA CAR T-cells from lymphodepleted prostate cancer patients, and their association with in vivo CAR T-cell proliferation and PSA decline.

Alpha thalassemia major (ATM) is an inherited blood disorder caused by the absence of all four α-globin genes (HBA2/1), resulting in severe anemia and lifelong transfusion dependence. While allogeneic hematopoietic stem cell transplantation (HSCT) offers a potential cure, donor availability remains limited. We present a gene therapy approach for autologous HSCT using lentiviral vectors (LVs) to deliver HBA2 under the regulation of optimized β-globin locus control region (LCR) enhancers, restoring α-globin expression in red blood cells. The best-performing LVs, erythroid vector-alpha (EV-α) and EV-α-UV, achieved up to 100% transduction efficiency in human hematopoietic stem and progenitor cells (HSPCs), optimal vector copy numbers, and safe integration profiles. ATM-derived HSPCs from three donors treated with these LVs yielded α/β-globin mRNA and chain ratios within the therapeutic range (∼0.5+), and restored hemoglobin levels by 50%-100%. These findings establish the safety and clinical potential of EV-α and EV-α-UV as a promising autologous stem cell gene therapy for ATM.

Analysis of survival outcomes and EGR2 gene expression in CD19 CAR T-cell products. The figure presents the P values and hazard ratio of different EGR2 molecular marker stratification points in relation to A, overall survival and B, event-free survival The black arrows indicate the stratification points used in the study. C, EGR2-targeted gene expression scores in CD19 CAR T-cell products from responders and non-responders in pediatric ALL. D, Summary of how EGR2 regulates resistance to CAR T-cell therapy through the type I IFN pathway.

Marker gene expression in CAR T-cell clusters. A, Uniform manifold approximation and projection (UMAP) plot of AAVS1 and EGR2 knockout (KO) CAR T-cell samples is shown. B, UMAP plots showing expression levels of CD4 and CD8. C, Violin plot depicting expression of cluster-defining markers in CD4+ T-cells. D, Differentially expressed genes in IL7R+ versus CTLA4+ CD4+ T-cells. E, Violin plots showing expression levels of cluster-defining markers in CD8+ T-cells. F, Heatmap displaying differentially expressed genes between CD8+ cell clusters. G, Cell cycle scores mapped on UMAP plots.

Malignant T-cell transformation after chimeric antigen receptor (CAR) T-cell therapy has been described, but the contribution of CAR integration to oncogenesis is not clear. Here we report a case of a T-cell lymphoma harboring a lentiviral integration in a known tumor suppressor, TP53, which developed in a patient with multiple myeloma after B-cell maturation antigen (BCMA) CAR T-cell therapy.

Introduction: Chimeric antigen receptor (CAR) T-cell therapy is standard care for relapsed or refractory hematologic malignancies. As clinical use expands, rare but serious toxicities beyond cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) are emerging. These include hyperleukocytosis and, specifically, following BCMA-directed CART (CART-BCMA) therapy, delayed neurotoxicity and enterocolitis. We named these post–CART-BCMA complications, distinct from CRS and ICANS, as CART immune-related adverse events (CirAE). CirAE are associated with elevated CART expansion in the first two weeks, suggesting that modulating CART-BCMA proliferation without affecting CART effector function could improve safety and expand therapeutic use. Method: We retrospectively analyzed a cohort of 198 multiple myeloma (MM) patients treated with CART-BCMA (idecabtagene vicleucel [ide-cel] and ciltacabtagene autoleucel [cilta-cel]) at the University of Pennsylvania (June 2021-December 2024) to identify predictors of CirAE and to investigate the mechanisms underlying the early onset of CirAE. Serial serum samples were analyzed using 32-plex proteomics (Luminex). Immune cell subsets were characterized serially by multiparametric flow cytometry. T-cell proliferation was assessed using CellTrace Violet. Cytotoxic CART function was evaluated via luciferase assays. Results: In this large cohort, both peak absolute lymphocyte count (ALC) ≥ 2.4 × 10³/μL and CD4:CD8 ratio >1 at apheresis independently predicted CirAE following CART-BCMA. To investigate mechanisms underlying elevated ALC, we analyzed a unique case (Cilta#1) marked by extreme, polyclonal CART expansion and three distinct post-infusion CirAE: facial palsy, delayed ICANS, and severe enterocolitis. Cilta#1 exhibited hyperleukocytosis (peak ALC: 197.5 × 10³/μL, Day 13) and profound CD4-skewed CART expansion (CD4:CD8 ratio: 12.6). Despite the magnitude, the CART population remained polyclonal, as confirmed by TCR Vβ sequencing and vector integration site analysis (>20,000 unique sites). Whole-exome sequencing of blood and marrow excluded clonal transformation or pathogenic mutations. Longitudinal flow cytometry from pre-lymphodepletion to month 15 showed persistent CD4 skewing, with CART displaying a highly activated, proliferative phenotype(HLA-DR+/Ki-67+). Serum proteomics at day 7, prior to peak expansion, revealed elevated lymphoproliferative cytokines (IL-2, IL-7, IL-15) and chemokines (CCL5, CXCL9, CXCL10). Given these findings, we assessed the dominant proliferative signal via cytokine-stimulated proliferation assays on Cilta#1 CART cells. IL-15 elicited the strongest proliferation and pSTAT5 activation. In vitro, IL-15 induced CCL5 secretion in both Cilta#1 and healthy donor CART (n=3); however, only Cilta#1 cells upregulated CCR5, a pattern absent in donor CART, where CCR5 was actually downregulated after cytokine exposure. These results suggest a potential IL-15–driven CCL5–CCR5 loop sustaining CART expansion in Cilta#1. We hypothesized that this axis contributes to the elevated ALC observed in patients with CirAE. To test this, we cultured healthy donor CART + 25% Cilta#1 day 7 serum, and observed significantly enhanced survival compared to serum from three other cilta-cel patients (p < 0.001). This effect was abrogated by 40 μM maraviroc,an FDA approved CCR5 antagonist, highlighting the key role of CCR5 signaling. Notably, maraviroc suppressed both IL-15–induced and antigen-dependent CART proliferation across multiple donors (n=4), suggesting broader applicability. Importantly, maraviroc did not impair CART viability or anti-myeloma cytotoxicity, as assessed by flow cytometry for CD107a, granzyme B, and MM.1S tumor killing. CCR5 blockade (20 µg/mL) inhibited IL-15–induced proliferation in Cilta#1 CART, further validating CCR5 as a key effector node. Finally, CCR5 knockout impaired antigen-driven proliferation and rendered CART insensitive to maraviroc, confirming on-target specificity.Conclusion: We identified the IL-15–CCL5–CCR5 circuit as a key driver of CART-BCMA proliferation in CirAE and demonstrated that CCR5 blockade safely restrains CART-BCMA expansion while preserving their anti-myeloma activity. These findings support CCR5-directed targeted strategies to selectively modulate CART expansion without compromising efficacy.

Adeno-associated virus-based vector (AAV)-based gene therapy has been used to treat thousands of patients, but a limitation can be inefficient transgene expression from AAV vectors. AAV transduction can be affected by the small ubiquitin-like modifier (SUMO) system, in which SUMO proteins are attached to proteins after translation, thereby modulating their function and stability. However, to date, practical modulators of SUMOylation to increase AAV vector transgene expression have not been available. Here we demonstrate that small molecule inhibitors of SUMOylation can boost expression from AAV vectors. Treatment with the SUMOylation inhibitor TAK-981 sharply increased AAV transgene expression in transformed human cells, in primary human cells, and in mice. Increased transgene expression in vitro and in vivo was associated with increased mRNA levels per vector DNA template. Treatment of mice with TAK-981 during AAV delivery increased AAV transgene expression; in addition, TAK-981 could boost transgene expression when introduced at long times after initial AAV vector transduction, regardless of whether mice had been exposed to TAK-981 previously. Modulators of SUMOylation are currently in clinical trials in human patients and, thus, may soon represent a viable strategy for boosting AAV transgene expression to improve human gene therapy outcomes.

We conducted a first-in-human phase 1 dose-finding pilot and feasibility study testing the safety of gene therapy using a lentivirus vector (LVV) expressing a microRNA embedded shRNA (shmiR) targeting BCL11A in sickle cell disease (SCD) (NCT03282656). Eleven eligible subjects with SCD (HbSS or HbS/Hbβ0) had stem cell collection; one patient withdrew prior to infusion and 10 were infused with autologous hematopoietic stem and progenitor cells transduced with the shmiR vector. Engraftment occurred in all 10 patients. With a median follow-up of 58 months (range: 35-82) after infusion, no adverse events attributed to the gene vector have occurred. Plerixafor mobilized peripheral blood hematopoietic stem cells (HSC) required for manufacturing were obtained in 1 mobilization cycle (2 consecutive days of collection) for 10/11 subjects, with one patient requiring 2 cycles (mean of 2.3 apheresis days per patient for product manufacturing). Median time from first day of apheresis to final release of product was 39 days (range: 22–151; n=11), with only one product released after >50 days. The manufacturing efficiency, defined as the number of CD34+ cells in the final product divided by the number of CD34+ cells collected was 63% (mean, range 41-82%). Transduction efficiency was 93.1% (median, range: 62%-100%, n=11) with a vector copy number of 3.7+/-1.4 (mean+/-SD, n=11) and final product cell count was 5.1 (median, range: 3.1-8.7, n=11) CD34+ x 106cells/kg. BCL11A was effectively targeted with a median of 88% (range: 67-100%) of individual erythroid colonies derived from the manufactured product containing ≥30% fetal hemoglobin (HbF) by HPLC analysis. After busulfan conditioning and infusion, all subjects engrafted promptly (neutrophils 18-30d, platelets 26-62d) and at 6 months, the median vector copy number in whole blood (WB) was 0.7 copies/diploid genome (range: 0.2-1.5) which remained stable over the entire follow-up time. The subject with the lowest in vivo VCN (BCL-010) had the lowest HbF post-infusion of 14.1% at 6 months that remained low but stable over the entire follow-up time, defining the lower dosage of effective gene modfication. Of the remaining nine infused subjects there was a robust HbF induction with WB HbF 27.8% (range: 20.8%-38.2%), F cells 71% (range: 55.3%-80.3%) and F/Fcells 11.9 pg (range: 9.4-13.6 pg) at 2 years that remained stable at 48 months (N=8), with mRNA knock-down of BCL11A stable from 42-74% and no significant change in BCL11A expression in CD19+ B cells. These data defined the manufacturing parameters required for improved overall HbF induction and further increase in F cell numbers post-infusion that were implemented in the subsequent phase 2 GRASP trial (NCT05353647). Due to the cases of leukemia and myelodysplastic syndrome in two Group A subjects with SCD in the Lentiglobin HGB-206 gene addition trial, clonal hematopoiesis was monitored in all subjects by sequencing of defined clonal hematopoiesis-associated genes, in addition to tracking insertion site analysis (ISA). No subject had ISA-defined clones larger than 3% at any time during follow-up. One subject had a clonal pathogenic DNMT3A mutation noted after infusion that was retrospectively determined to be present prior to cell manufacturing below validated detection limits. The variant allele fraction of this mutation increased after infusion to a maximum of 9.3%, and has remained stable for over 4 years of follow-up with no evidence of dysplasia on the 2-year post-GT bone marrow. All patients demonstrated sustained mitigation of SCD phenotypes. These data demonstrate first-in-human efficiency, durability, safety and efficacy of targeting BCL11A using a shmiR LVV leading to a pivotal multi-site phase 2 trial currently underway (NCT05353647). Funding Funded by the National Institutes of Health; ClinicalTrials.gov number, NCT03282656

Epigenetic remodeling of CAR T-cells by EGR2 knockout and effect of type I IFN signaling on the development of memory and exhaustion. A, Volcano plots showing differentially accessible chromatin regions within genes between KLF2+ and MKI67+ CD8+ T-cells. B, Volcano plots depicting differentially accessible chromatin regions within genes between EGR2 and AAVS1 knockout (KO) CD8+ CAR T-cells. C, Representative contour plots showing frequencies of TIM3- and LAG3-expressing CD8+ CAR-T cells after exposure to IFNβ (1ng/mL) following chronic CAR stimulation. D, Proportions of CD27+ (left) or CD62L+ (right) CD8+ CAR-T cells after exposure to IFNβ. E, Representative contour plots showing frequencies of CD45RO+CD27+ CD8+ CAR-T cells after IFNAR blockade (Anifrolumab, 1µg/mL) during chronic antigen stimulation. F, Frequencies of TIM3+LAG3+ CD8+ CAR-T cells after IFNAR blockade. G, Cytolytic capacity of CAR T-cells as measured by normalized cell index kinetics using the xCELLigence real-time cytotoxicity assay following chronic stimulation with target cancer cells in the setting of either IFNβ or IFNAR blockade. H, Normalized cell index at 75 hours after challenge with target cancer cells. All experiments were conducted using healthy donor T-cells from independent donors (Mann-Whitney test, n = 4). *P < 0.05, *P < 0.01, ***P < 0.001, ns.: not significant.

ABSTRACT:Chimeric antigen receptor T-cell (CAR-T) therapy has revolutionized the treatment of B-cell malignancies; however, >60% of patients relapse within 1 year, often due to insufficient CAR-T persistence. Although 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, to our knowledge, 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. After ex vivo characterization, we administered pooled autologous CAR-T arrays to 3 NHPs and tracked CAR abundance longitudinally using a novel digital droplet polymerase chain reaction 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 CAR-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 end point, 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 activity.

EGR2 knockout enhances the long-term potency and memory function of CAR T-cells in vivo. A, Schematic of the tumor rechallenge experiments. B, Tumor growth after the first (left) and second (right) NALM6 challenges (Mann-Whitney test, n = 10). C, Mouse survival after high-dose second tumor challenge (Log-rank test). D, Tumor growth inhibition in a prostate cancer model using PSMA-targeting CAR T-cells with EGR2 or AAVS1 knockout (KO) compared to control CD19 CAR T-cells (19BBz). The tumor growth was monitored over time using caliper-based measurements in NSG mice (n = 8).

Genes comprising the TCF7 regulon, the type I IFN signature, and the EGR2 Co-expression signatures. Gene names and symbols are listed.

Pathways regulated by EGR2 in CD8+ CAR T-cells. (A-C) Top pathways differentially expressed in EGR2 knockout CD8+ CAR-T cells compared to AAVS1 knockout CAR T-cells. Libraries used in this enrichment analysis: A, Reactome 2016. B, NCI-Nature Pathway Interaction Database 2015. C, ARCHS4 transcription factor (TF) co-expression.

Long-term risks of gene therapy are not fully understood. In this study, we evaluated safety outcomes in 783 patients over more than 2,200 total patient-years of observation from 38 T cell therapy trials. The trials employed integrating gammaretroviral or lentiviral vectors to deliver engineered receptors to target HIV-1 infection or cancer. Eighteen patients (2.3