Citations

Abstract Chimeric Antigen Receptor (CAR) T cell therapy is a transformative treatment for hematologic malignancies. However, ex vivo autologous CAR T cell manufacturing presents substantial logistical and clinical challenges. Direct in vivo CAR-T cell engineering offers a new paradigm for CAR T delivery. We recently developed the CD3-targeted lentiviral vector LV-169 which can deliver a CAR transgene to resting niche-resident T cells in lymphoid tissues. LV169 is pseudotyped with a mix of chimeric and nonchimeric VSV-G proteins detargeted (blinded) from the natural LDL receptor and re-targeted to T cells through the display of a CD3-specific scFv. Here we demonstrate that LV169 can specifically deliver a functional aBCMA-CAR transgene to resting human T cells in PBMC cultures from the healthy volunteers and from multiple myeloma patients. For clinical translation, we selected an αBCMA-CAR construct comprising a fully human single heavy chain nanobody linked to 41BB and CD3z cytoplasmic signaling domains. Human T cells expressing this CAR construct have low tonic signaling and efficiently kill BCMA-positive myeloma target cells with similar or lower associated cytokine release compared to a clinically relevant benchmark BCMA-CAR. To further improve LV169 and to minimize CAR protein incorporation in the LV envelope which may lead undesirably to myeloma cell transduction, we placed the CAR transgene under a T-cell specific Lck promoter in reverse orientation relative to the 5' LTR. The transcriptional activity of the Lck promoter was comparable to that of a constitutive EF1a promoter in primary T cells and, in contrast to EF1a had minimal activity in a variety of other tested cell types including the HEK293T producer cell line and the THP-1 monocytic cell line. Western blotting confirmed the absence of CAR protein in LV particles (LVP) generated using the Lck promoter-driven reverse orientation construct. In contrast, high levels of CAR protein were detected in LVP generated using the parental EF1a promoter-driven construct. The LV-incorporated CAR protein mediated off-target gene delivery to BCMA-positive myeloma cells. The therapeutic efficacy of intravenously administered LV169 LV particles carrying BCMA-CAR constructs was tested in the OPM-2 human multiple myeloma xenograft model. NSG mice with knockout of MHC-I and MHC-II, and with advanced disseminated OPM-2-Luc tumors were engrafted with human PBMC one week prior to intravenous LV169-BCMA-CAR administration. Mice were treated with single intravenous injection of LV169 at different doses 3 weeks after tumor engraftment. OPM-2-Luc tumor burden was monitored by regular luciferase imaging. All treated mice cleared their tumors within 28-days following LV administration and the rate of clearance was correlated to the LV particle dose. Even at the 4e8 dose level (by p24) tumors were completely cleared. Eighty four days post-treatment the treated mice remained tumor-free and resisted re-challenge with OPM-2 tumor cells. Circulating CAR-T cells, monitored by flow cytometry, became detectable by 14 dpi and peaked between 21-35 dpi. Following tumor clearance, the number of circulating CAR-T cells declined dramatically. The treatment was well tolerated and analysis of cytokines associated with CAR-T cell cytotoxicity confirmed the absence of a severe cytokine storm. IFNγ was the only cytokine which was elevated during the expansion period of CAR-T cells, while IL-2, IL-6, TNFα and GM-CSF were barely detectable. In summary the characteristics of our new developed LV169-based platform make it suitable for further clinical translation which is underway.

Abstract Direct in vivo T cell engineering offers a new paradigm for CAR T delivery. We have evaluated several approaches to engineer lentiviral (LV) vectors capable of delivering CAR transgenes directly and selectively to T cells in vivo, within the lymphoid niche. Our goal was to develop an LV delivery platform that following intravenous administration can selectively bind and activate T cells, deliver and integrate the (CAR) transgene into genomic DNA, and drive CAR expression on the T cell surface. Further requirements were that the retargeted LV should be associated with minimal off-target transduction of tumor cells and normal host tissues and should be manufacturable at titers sufficient for human application. Our strategy for retargeting has been to modify the trimeric VSV-G protein, which is well established for efficient LV pseudotyping. Of the approaches evaluated, three retargeting platforms have proven to be effective: direct covalent display, indirect covalent display, and G-capping. Direct covalent display. This (LV-169) approach uses chimeric VSV-G protein that contains a CD3-targeting ligand displayed as a N-terminal extension of VSV-G protein that has been mutated to ablate (blind) its interaction with the LDL receptor. Chimeric and nonchimeric blinded G proteins are co- incorporated into LV particles within mixed envelopes which bind to CD3. Upon binding to cell surface CD3, the engineered particles activate T-cells and enter acidifying endosomes, whereupon they trigger membrane fusion and deliver their payloads. LV-169 displaying an anti-human CD3 scFv selectively transduced resting human T cells in PBMC cultures, in fresh human whole blood and following intravenous administration to humanized mice. In immunocompetent Cre reporter mice, surrogate vectors displaying anti-murine CD3 scFv selectively transduced lymph node resident T cells, with off-target transduction being limited to tissue-resident macrophages. To eliminate the incorporation of CAR protein into the LV-169 envelope, which can lead to highly undesirable transduction of tumor cells expressing the CAR target, we placed the CAR transgene in reverse to 5'LTR orientation, downstream of a T-cell specific promoter/enhancer. In vivo efficacy of LV-169-BCMA-CAR was demonstrated in humanized mice bearing disseminated OPM-2 myeloma tumors. Clinical translation of this construct is now underway. Indirect covalent display. In this G-protein engineering approach, a short peptide (SPY-tag) is displayed as an N-terminal extension of the LDLR-blinded VSV-G protein. The SPY-tagged LV vectors are then covalently modified to display a CD3-targeting ligand by simply mixing them at room temperature with a bifunctional fusion protein consisting of the SPY-catcher and the CD3 ligand. Under these conditions, the SPY-catcher and the SPY-tag spontaneously form a covalent isopeptide bond. The CD3 ligand-modified LV particles were shown to specifically activate and transduce T cells ex vivo within human PBMC cultures. G-capping. In the final approach, the VSV-G protein is not genetically modified. Instead, a “capping” protein crosslinker that binds directly to the unmodified G protein on preformed LV particles is used to mask their LDLR binding sites and redirect them to interact with CD3. Mixing of the CD3-targeted capping protein with traditional G-pseudotyped LV particles leads to efficient binding of the cap to the trimeric G proteins. The cap-modified LV are unable to interact with LDLR but interact efficiently with CD3. CD3-capped LV particles were highly specific and efficient in human T cell activation and transduction in PBMC cultures. The association between the G protein and the bound cap is highly stable during freeze-thaw cycles and at room temperature, a critical attribute of this platform to facilitate the manufacture of a clinically and commercially viable LV product. In summary, we have developed three translationally relevant platform technologies for the generation of LV particles capable of delivering CAR transgenes specifically and efficiently to resting niche-resident T cells after intravenous administration. Manufacturing studies are underway and, in the case of the LV-169 platform, clinical translation is already in progress.

Abstract Introduction: The Duffy null variant (genotype or phenotype) is associated with lower circulating absolute neutrophil counts (ANC) without increased risk for infection. It is found in ~66% of people identifying as Black or African American vs <1% of people of Asian or European ancestry. Many ANC reference intervals (RI) are not representative of the Duffy null population. There are no adult Duffy null WBC RI and no pediatric Duffy null ANC or WBC RI. Thus, we aimed to assess Duffy null ANC and WBC distributions as well as develop WBC and ANC RI for adults and pediatrics. Methods: Twenty-three sites across the United States collected samples with all sites excluding participants with conditions or medications that could impact ANC or WBC. Only Duffy null participants were included. Sex, race, ethnicity, age, sample collection type, practice setting, Duffy testing method, ANC, and WBC were recorded. The non-parametric method for determining lower limit of normal (LLN), upper limit of normal (ULN), and 90% confidence intervals (CI) around the limits was performed according to the Clinical and Laboratory Standards Institute guidelines. Specific ULN and LLN are not reported as these are preliminary data. The Mann-Whitney U test was used to compare sex and prior data; differences by age groups were assessed with Kruskal-Wallis test. Results: 316 participants 18+ years-old (y/o) were assessed of whom 62.3% (n=197) are female, 97.5% (n=308) identified as Black or African American, and 94.9% (n=300) identified as non-Hispanic. Median age was 47 y/o (IQR: 32-60.5). Median ANC was 2600/uL (IQR: 1860-3530; range: 400-7400); 28.6% (n=90) of participants had ANC <2000/uL, 13.6% (n=43) had ANC <1500/uL, and 2.5% (n=8) had <1000/uL. The 90% CI around the LLN was 810-1070/uL and 5570-7130/uL around the ULN. Consistent with prior data, there was a difference in median ANC by sex (male: 2400/uL; female: 2710/uL; p=0.02). There was no significant difference between this ANC data and previously published Duffy null ANC data (p=0.28). Median WBC was 5.4x109/L (IQR: 4.4-6.5; range: 2.10-11.64) with 4.1% (n=13) of participants with WBC <3.0x109/L. The 90% CI around the LLN is 3.68-4.30x109/L and 11.02-12.97x109/L around the ULN. 515 participants between 0-17 y/o were assessed with 51.8% (n=267) females, 94.8% (n=488) identified as Black or African American, and 94.0% (n=484) identified as non-Hispanic. Among pediatric participants, 56.8% (n=291) had ANC <2000/uL, 38.7% (n=198) had ANC <1500/uL, and 14.4% (n=74) had ANC <1000/uL. For those <1 y/o (n=29), median ANC was 950/uL (IQR: 840-1360; range: 111-3910). There are currently too few samples to predict ULN and LLN. Median WBC was 8.85x109/L (IQR: 7.03-10.16; range: 4.17-13.53). For those 1-5 y/o (n=143), median ANC was 1735/uL (IQR: 1100-2550; range: 207-5810) with a 90% CI around the LLN of 207-820/uL and 4020-5810/uL around the ULN. Median WBC was 6.72x109/L (IQR: 5.64-8.25; range: 3.68-12.97). For those 6-11 y/o (n=128), median ANC was 1590/uL (IQR: 1090-2470; range: 420-5490) with a 90% CI around the LLN of 420-910/uL and 4340-5490/uL around the ULN. Median WBC was 5.42x109/L (IQR: 4.45-6.70; range: 2.80-10.50). For those 12-17 y/o (n=215), median ANC was 2190/uL (IQR: 1460-2930; range: 204-5850) with a 90% CI around the LLN of 540-930/uL and 4350-5510/uL around the ULN. Median WBC was 5.06x109/L (IQR: 4.26-6.20; range of 2.04-9.91). There is a significant difference in ANC by age category (p<0.0001). Conclusion: This is the first report of healthy pediatric Duffy null ANC and WBC data and the first large-scale verification of adult Duffy null ANC and WBC data. Final RI are expected in September 2025. Among adults with the Duffy null phenotype, ANC LLN are likely between 810-1070/uL. Pediatric ANC LLN are likely as low as 207-820/uL (1-5 y/o), 420-910/uL (6-11 y/o), and 540-930/uL (12-17 y/o). These findings redefine “normal” as a significant proportion of healthy participants have ANC or WBC below current RI or below thresholds typically defined as disease. Limitations include heterogeneous sample populations and collection methods. Future analyses will assess differences between collection sites or methods. Ultimately, this work lays the foundation for a more scientifically sound and equitable approach to medical care by ensuring that RI accurately reflects health for the entire community. Healthcare systems are strongly encouraged to adopt Duffy null-specific ANC and WBC RI.

Aerosol viral transmission monitoring enables early, noninvasive detection of infectious disease spread by identifying airborne viral particles in shared environments. Integrating sampling, sensing, and data analysis, this perspective outlines an integrated framework for transforming epidemic responses from reactive testing to proactive surveillance. It synthesizes current technological advances and deployment experiences to identify key challenges in detection efficiency, data management, and societal trust. This marks a shift from clinical diagnosis to environmental surveillance, transforming epidemic responses from testing individuals to monitoring shared air. By enabling earlier interventions, these systems help reduce transmission, protect vulnerable groups, and limit disruptions to daily life and the economy.

Abstract Introduction Heterozygous germline pathogenic variants (PVs) in the 5' untranslated region of ANKRD26 cause inherited thrombocytopenia and predisposition to hematologic malignancy (HM). The estimated lifetime HM risk is ~10% based on families ascertained from an inherited thrombocytopenia cohort. However, the malignancy spectrum, molecular features, precursor states, and overall clinical outcomes remain poorly defined. Methods We established an international, multicenter cohort of individuals with ANKRD26-related thrombocytopenia diagnosed after undergoing genetic evaluation due to a personal and/or family history of thrombocytopenia and/or HM. Clinical, pathologic, and molecular data were collected for 27 individuals with germline ANKRD26 PVs from 21 unrelated families treated at 11 institutions across 5 countries. Statistical analyses were performed using R (v4.4.2). Results Patients were predominantly male (18/27) and white (25/27). Thrombocytopenia was typically identified in childhood (median age 10 years, range 0-48) with a genetic diagnosis made later (median age 53 years, range 2-80). The mean (+/- SD) pre-malignancy platelet count was 45 +/- 24 K/uL (range 5-87). ANKRD26 PVs observed in each family included: c.-126T>C (n=9), c.-128G>A (n=3), c.-134G>A (n=3), c.-116C>T (n=3), c.-118C>T (n=2), and c.-118C>A (n=1). Thirteen HMs were diagnosed in 11 of 27 (41%) patients, including myelodysplastic neoplasm/syndrome (MDS, n=5), acute myeloid leukemia (AML, n=3), chronic myeloid leukemia (CML, n=2), MDS/myeloproliferative neoplasm (MPN) overlap (n=2), and Langerhans cell histiocytosis (n=1). The median age at first HM diagnosis was 63 years (range 49-80). Recurrent somatic features in myeloid malignancies included complex karyotype (4/11, 36%) and TP53 mutations (5/10, 50%). Of the 3 patients with AML, 1 achieved a partial remission (PR) with venetoclax combined with cladribine, idarubicin, and cytarabine but relapsed, 1 achieved a PR with azacitidine but relapsed <100 days after haploidentical allogeneic stem cell transplantation (alloSCT), and 1 had primary refractory disease. Among 5 patients with MDS, 1 was refractory to decitabine, 1 underwent alloSCT but relapsed 2 years post-transplant, 1 remains in remission post-alloSCT with 1 year of follow-up, 1 is undergoing alloSCT evaluation, and 1 was lost to follow-up. Both patients with CML achieved durable major molecular remissions with imatinib. To better estimate HM risk, we assumed 100% penetrance of thrombocytopenia for ANKRD26 PV carriers and included family members with chronic thrombocytopenia who had not had germline genetic testing. In this expanded series (n=62), 16 individuals developed a HM (26%). Additional diagnoses included MDS with progression to AML (n=1), AML (n=1), CML (n=1), and leukemia of unknown type (n=2). Cumulative incidence of HM was 0% by age 40 years, 17% by age 60 years, and 60% by age 80 years. Somatic sequencing of peripheral blood was performed in 5 HM-unaffected ANKRD26 PV carriers. One individual was found to have clonal hematopoiesis driven by TET2 (c.1648C>T, p.Arg550*) with a variant allele frequency (VAF) of 9% at age 55. Bone marrow biopsy showed mild megakaryocytic atypia and 4% blasts without overt evidence of malignancy. To assess the prevalence of ANKRD26-related HM, we assessed the frequency of ANKRD26 PVs in 2 large MDS/AML cohorts. In the ARUP database (9662 AML, 6397 MDS), 2 individuals (0.01%) had an ANKRD26 PV with a VAF consistent with germline origin. In the MSK-IMPACT Heme Germline MDS/AML cohort (n = 284), no ANKRD26 P/LP variants were identified. Conclusions Germline ANKRD26 PVs underlie <1% of MDS/AML diagnoses but may represent a high-risk subtype characterized by complex karyotype, TP53 mutations, and high risk of relapse. Among our series, the incidence of HM was low early in life but increased substantially with age, reaching 60% by 80 years. This exceeds prior estimates, which were derived from individuals ascertained from thrombocytopenia-based registries. Together, our findings suggest that ANKRD26-associated HM may represent a high-risk clinical entity, and that age and family history should inform surveillance strategies. More studies are needed to define genetic and environmental modifiers of HM risk as well as precursor states to enable early detection and disease interception.

ABSTRACT Background Adolescents with youth‐onset type 2 diabetes (YO‐T2D) have an increased risk for cardiometabolic complications. The impact of vertical sleeve gastrectomy (VSG) on fat distribution and cardiac morphology/function in YO‐T2D is unknown. Objectives To evaluate changes in body composition, abdominal and cardiac fat depots, and cardiometabolic health in adolescents with YO‐T2D undergoing VSG. Methods Anthropometrics, labs and imaging were used to assess participants pre‐surgery and 3–12 months post‐surgery. MRI quantified pericardial (PAT), epicardial (EAT), subcutaneous (SAT) and visceral adipose tissue (VAT), hepatic fat fraction (HFF) and cardiac morphology/function. Mixed‐effects models assessed longitudinal changes. Results By 12 months, weight decreased from 134.3 ± 5.1 to 103.3 ± 5.2 kg, VAT 1943 ± 148 to 1248 ± 150 cm 3 , HFF 23.2% ± 2.3% to 5.3% ± 2.3% and PAT and EAT by 27% and 33% (all p < 0.01). Homeostatic model assessment of insulin resistance (HOMA‐IR) improved (7.8 ± 1.2 to 1.7 ± 1.2 [ p < 0.01]). BMI, SAT, resting heart rate (RHR), left ventricular (LV) mass and cardiac output (CO) also decreased (all p < 0.05). VAT decreases correlated with decreases in HFF ( r = 0.70, p = 0.01), HOMA‐IR ( r = 0.60, p = 0.04) and CO ( r = 0.70, p = 0.03). HFF decreases correlated with decreases in BMI ( r = 0.70, p = 0.03), HOMA‐IR ( r = 0.90, p < 0.001), RHR ( r = 0.90, p = 0.002), CO ( r = 0.80, p = 0.007) and LV mass ( r = 0.70, p = 0.02). Conclusions VSG reduces ectopic and regional fat and improves associated insulin sensitivity and cardiac health in adolescents with YO‐T2D.

Galactic cosmic ray (GCR) radiation is a major barrier to human space exploration beyond Earth’s magnetic field protection. Mesenchymal stem cells (MSCs) are found in all organs and play a critical role in repair and regeneration of tissue. We engineered bone marrow-derived MSCs and evaluated their response to ionizing radiation exposure. Epidermal growth factor receptor (EGFR) expression by certain types of cancers has been shown to induce radioresistance. In this study, we tested the feasibility of transfecting MSCs to overexpress EGFR (eMSC-EGFR) and their capacity to tolerate and recover from X-ray exposure. Quantitative real-time PCR (qRT-PCR) and immunoblotting results confirmed the efficient transfection of EGFR into MSCs and EGFR protein production. eMSC-EGFR maintained characteristics of human MSCs as outlined by the International Society for Cell & Gene Therapy. Then, engineered MSCs were exposed to various dose rates of X-ray (1–20 Gy) to assess the potential radioprotective role of EGFR overexpression in MSCs. Post-irradiation analysis included evaluation of morphology, cell proliferation, viability, tumorigenic potential, and DNA damage. eMSC-EGFR showed signs of radioresistance compared to naïve MSCs when assessing relative proliferation one week following exposure to 1–8 Gy X-rays, and significantly lower DNA damage content 24 h after exposure to 4 Gy. We establish for the first time the efficient generation of EGFR overexpressing MSCs as a model for enhancing the human body to tolerate and recover from moderate dose radiation injury in long-term manned space travel.