Extreme elevations of donor-derived cell-free DNA increases the risk of chronic lung allograft dysfunction and death, even without clinical manifestations of disease.

BACKGROUND: Lung transplant recipients are traditionally monitored with pulmonary function testing (PFT) and lung biopsy to detect post-transplant complications and guide treatment. Plasma donor-derived cell free DNA (dd-cfDNA) is a novel molecular approach of assessing allograft injury, including subclinical allograft dysfunction. The aim of this study was to determine if episodes of extreme molecular injury (EMI) in lung transplant recipients increases the risk of CLAD or death. METHODS: This multicenter prospective cohort study included 238 lung transplant recipients. Serial plasma samples were collected for dd-cfDNA measurement by shotgun sequencing. EMI was defined as a dd-cfDNA above the third quartile of levels observed for acute rejection (dd-cfDNA level of ≥ 5% occurring after 45 days post-transplant). EMI was categorized as Secondary if associated with co-existing acute rejection, infection or PFT decline; or Primary if not associated to these conditions. RESULTS: EMI developed in 16% of patients at a median 343.5 (IQR: 177.3-535.5) days post-transplant. Over 50% of EMI episodes were classified as Primary. EMI was associated with an increased risk of severe CLAD or death (HR: 2.52, 95% CI: 1.10 - 3.82, p= 0.024). The risk remained consistent for Primary EMI (HR: 2.34, 95% CI 1.18-4.85, p=0.015). Time to first EMI episode was a significant predictor of the likelihood of developing CLAD or death (AUC=0.856, 95% CI =.805-908, p<.001). CONCLUSIONS: Episodes of EMI in lung transplant recipients are often isolated and not detectable with traditional clinical monitoring approaches. EMI is associated with an increased risk of severe CLAD or death, independent of concomitant transplant complications.

Antiviral cellular therapy for enhancing T-cell reconstitution before or after hematopoietic stem cell transplantation (ACES): a two-arm, open label phase II interventional trial of pediatric patients with risk factor assessment.

Viral infections remain a major risk in immunocompromised pediatric patients, and virus-specific T cell (VST) therapy has been successful for treatment of refractory viral infections in prior studies. We performed a phase II multicenter study (NCT03475212) for the treatment of pediatric patients with inborn errors of immunity and/or post allogeneic hematopoietic stem cell transplant with refractory viral infections using partially-HLA matched VSTs targeting cytomegalovirus, Epstein-Barr virus, or adenovirus. Primary endpoints were feasibility, safety, and clinical responses (>1 log reduction in viremia at 28 days). Secondary endpoints were reconstitution of antiviral immunity and persistence of the infused VSTs. Suitable VST products were identified for 75 of 77 clinical queries. Clinical responses were achieved in 29 of 47 (62%) of patients post-HSCT including 73% of patients evaluable at 1-month post-infusion, meeting the primary efficacy endpoint (>52%). Secondary graft rejection occurred in one child following VST infusion as described in a companion article. Corticosteroids, graft-versus-host disease, transplant-associated thrombotic microangiopathy, and eculizumab treatment correlated with poor response, while uptrending absolute lymphocyte and CD8 T cell counts correlated with good response. This study highlights key clinical factors that impact response to VSTs and demonstrates the feasibility and efficacy of this therapy in pediatric HSCT.

Third-party virus specific T cells for the treatment of double stranded DNA viral reactivation and PTLD after solid organ transplant.

Reactivation or primary infection with double stranded DNA viruses is common in recipients of solid organ transplants and is associated with significant morbidity and mortality. Treatment with conventional anti-viral medications is limited by toxicities, resistance, and lack of effective options for adenovirus and BKPyV. Virus-specific T-cells (VSTs) have been shown to be an effective treatment for infections with adenovirus, BKPyV, cytomegalovirus (CMV), and Epstein-Barr virus (EBV). Most of these studies have been conducted in stem cell recipients and no large studies have been published in the solid organ transplant population to date. In this study we report on the outcome of quadrivalent third party VST infusions in 98 recipients of solid organ transplants in the context of an open label phase 2 trial. The ninety-eight patients received a total of 181 infusions, with a median of 2 infusions per patient. The overall response rate was 45% for BKPyV, 65% for CMV, 68% for Adenovirus, and 61% for EBV. Twenty percent of patients with PTLD had a complete response and 40% a partial response. All the VST infusions were well tolerated. We conclude that VSTs are safe and effective in the treatment of viral infections in solid organ transplant recipients.

Secondary bone marrow graft loss after third-party virus-specific T cell infusion: Case report of a rare complication.

Virus-specific T cells (VST) from partially-HLA matched donors have been effective for treatment of refractory viral infections in immunocompromised patients in prior studies with a good safety profile, but rare adverse events have been described. Here we describe a unique and severe adverse event of VST therapy in an infant with severe combined immunodeficiency, who receives, as part of a clinical trial (NCT03475212), third party VSTs for treating cytomegalovirus viremia following bone marrow transplantation. At one-month post-VST infusion, rejection of graft and reversal of chimerism is observed, as is an expansion of T cells exclusively from the VST donor. Single-cell gene expression and T cell receptor profiling demonstrate a narrow repertoire of predominantly activated CD4+ T cells in the recipient at the time of rejection, with the repertoire overlapping more with that of peripheral blood from VST donor than the infused VST product. This case thus demonstrates a rare but serious side effect of VST therapy.

Development of a Safety Surveillance Plan for the Academic Medicine Sponsor Performing First-in-Human Cellular Therapy Clinical Trials: A Report from the Consortium for Pediatric Cellular Immunotherapy.

Pharmacovigilance (PV), also known as drug safety, is the science of risk management involving the detection, assessment, understanding, and prevention of adverse effects related to a medication. This discipline has traditionally focused on the postmarketing period, with less attention to early-phase clinical trials. However, during the immunotherapy and cellular therapy investigational stage, regulatory agencies are increasingly emphasizing the need to identify and characterize safety signals earlier in clinical development as part of a comprehensive safety surveillance plan. Compliance with PV and safety regulations are further heightened as cell and gene therapy (CGT) trials grow in complexity and scope owing to ever-changing and increasingly rigorous regulatory mandates. Based on this changing landscape, a critical aspect of early-phase trials of cellular products where significant safety events are anticipated is to ensure that every effort is made to protect clinical trial participants by maximizing attention to the risk-versus-benefit profile. This includes the development of robust plans for safety surveillance that provide a continual assessment of safety signals to enable safety reporting to regulatory bodies and the Food and Drug Administration, a regular analysis of aggregate safety data, and a plan to communicate safety findings. This report focuses on PV in early-phase clinical trials of first-in-human investigational products sponsored by academic centers in which the availability of PV resources and subject matter experts is limited. To more fully understand the challenges of CGT PV oversight within pediatric academic medical centers conducting early-phase clinical trials, a working group from institutions participating in the Consortium for Pediatric Cellular Immunotherapy composed of faculty and regulatory professionals was convened to compare experiences, identify best practices, and review published literature to identify commonalities and opportunities for alignment. Here we present guidelines on PV planning in early-phase CGT clinical trials occurring in academic medical centers and offer strategies to mitigate risk to trial participants. Standards to address regulatory requirements and governance for safety signal identification and risk assessment are discussed.

Higher Molecular Injury at Diagnosis of Acute Cellular Rejection Increases the Risk of Lung Allograft Failure: A Clinical Trial.

Rationale: The association of acute cellular rejection (ACR) with chronic lung allograft dysfunction (CLAD) in lung transplant recipients has primarily been described before consensus recommendations incorporating restrictive phenotypes. Furthermore, the association of the degree of molecular allograft injury during ACR with CLAD or death remains undefined. Objectives: To investigate the association of ACR with the risk of CLAD or death and to further investigate if this risk depends on the degree of molecular allograft injury. Methods: This multicenter, prospective cohort study included 188 lung transplant recipients. Subjects underwent serial plasma collections for donor-derived cell-free DNA (dd-cfDNA) at prespecified time points and bronchoscopy. Multivariable Cox proportional-hazards analysis was conducted to analyze the association of ACR with subsequent CLAD or death as well as the association of dd-cfDNA during ACR with risk of CLAD or death. Additional outcomes analyses were performed with episodes of ACR categorized as "high risk" (dd-cfDNA ⩾ 1%) and "low risk" (dd-cfDNA < 1%). Measurements and Main Results: In multivariable analysis, ACR was associated with the composite outcome of CLAD or death (hazard ratio [HR], 2.07 [95% confidence interval (CI), 1.05-4.10]; P = 0.036). Elevated dd-cfDNA ⩾ 1% at ACR diagnosis was independently associated with increased risk of CLAD or death (HR, 3.32; 95% CI, 1.31-8.40; P = 0.012). Patients with high-risk ACR were at increased risk of CLAD or death (HR, 3.13; 95% CI, 1.41-6.93; P = 0.005), whereas patients with low-risk status ACR were not. Conclusions: Patients with ACR are at higher risk of CLAD or death, but this may depend on the degree of underlying allograft injury at the molecular level. Clinical trial registered with www.clinicaltrials.gov (NCT02423070).

Implementation of a tier system for IVIG indications to address IVIG shortage at a tertiary care pediatric medical center.

BACKGROUND: Drug shortages are a common issue that healthcare systems face and can result in adverse health outcomes for patients requiring inferior alternate treatment. The United States recently experienced a national drug shortage of intravenous immunoglobulin (IVIG). Several reported strategies to address the IVIG and other drug shortages have been proposed; however, there is a lack of evidence-based methods for protocol development and implementation. OBJECTIVE: To evaluate the efficacy of introducing a multidisciplinary task force and tier system of indications and to minimize adverse effects during a shortage of IVIG. METHODS: Faculty members across disciplines with expertise in IVIG use were invited to participate in a task force to address the shortage and ensure adequate supply for emergent indications. A tier system of IVIG indications was established according to the severity of diagnosis, urgency of indication, and quality of supporting evidence. Based on inventory, indications in selected tiers were auto-approved. Orders that could not be automatically approved were escalated for task force review. RESULTS: Overall, there were 342 distinct requests for IVIG during the study period (August 1, 2019 to December 31, 2019). All Tier 1 indications were approved. Of all requests, only 2.6% (9) of requests were denied, none of which resulted in adverse effects based on retrospective chart review. Seven patients who regularly receive IVIG had possible adverse effects due to dose reduction or spacing of treatment; however, each complication was multifactorial and not attributed to the shortage or tier system implementation alone. CONCLUSION: Implementation of a multidisciplinary task force and tier system to appropriately triage high-priority indications for limited pharmaceutical agents should be considered in health institutions faced with a drug shortage.

Organizing pneumonia is associated with molecular allograft injury and the development of antibody-mediated rejection.

BACKGROUND: The association between organizing pneumonia (OP) after lung transplantation with the development of acute rejection (AR) remains undefined. In addition, molecular allograft injury, as measured by donor-derived cell-free DNA (dd-cfDNA), during episodes of OP and its relationship to episodes of AR, chronic lung allograft dysfunction (CLAD), or death is unknown. METHODS: This multicenter, prospective cohort study collected serial plasma samples from 188 lung transplant recipients for dd-cfDNA at the time of bronchoscopy with biopsy. Multivariable Cox regression was used to analyze the association between OP with the development of AR (antibody-mediated rejection (AMR) and acute cellular rejection (ACR)), CLAD, and death. Multivariable models were performed to test the association of dd-cfDNA at OP with the risk of AR, CLAD, or death. RESULTS: In multivariable analysis, OP was associated with increased risk of AMR (hazard ratio (HR) = 2.26, 95% confidence interval (CI) 1.04-4.92, p = 0.040) but not ACR (HR = 1.29, 95% CI: 0.66-2.5, p = 0.45) or the composite outcome of CLAD or death (HR = 0.88, 95% CI, 0.47-1.65, p = 0.69). Median levels of dd-cfDNA were higher in OP compared to stable controls (1.33% vs 0.43%, p = 0.0006). Multivariable analysis demonstrated that levels of dd-cfDNA at diagnosis of OP were associated with increased risk of both AMR (HR = 1.29, 95% CI 1.03-1.62, p = 0.030) and death (HR = 1.16, 95% CI, 1.02-1.31, p = 0.026). CONCLUSIONS: OP is independently associated with an increased risk of AMR but not CLAD or death. The degree of molecular allograft injury at the diagnosis of OP may further predict the risk of AMR and death.

Human PLCG2 haploinsufficiency results in a novel natural killer cell immunodeficiency.

BACKGROUND: Although most individuals effectively control herpesvirus infections, some suffer from severe and/or recurrent infections. A subset of these patients possess defects in natural killer (NK) cells, lymphocytes that recognize and lyse herpesvirus-infected cells; however, the genetic etiology is rarely diagnosed. PLCG2 encodes a signaling protein in NK-cell and B-cell signaling. Dominant-negative or gain-of-function variants in PLCG2 cause cold urticaria, antibody deficiency, and autoinflammation. However, loss-of-function variants and haploinsufficiency have not been reported to date. OBJECTIVES: The investigators aimed to identify the genetic cause of NK-cell immunodeficiency in 2 families and herein describe the functional consequences of 2 novel loss-of-function variants in PLCG2. METHODS: The investigators employed whole-exome sequencing in conjunction with mass cytometry, microscopy, functional assays, and a mouse model of PLCG2 haploinsufficiency to investigate 2 families with NK-cell immunodeficiency. RESULTS: The investigators identified novel heterozygous variants in PLCG2 in 2 families with severe and/or recurrent herpesvirus infections. In vitro studies demonstrated that these variants were loss of function due to haploinsufficiency with impaired NK-cell calcium flux and cytotoxicity. In contrast to previous PLCG2 variants, B-cell function remained intact. Plcg2+/- mice also displayed impaired NK-cell function with preserved B-cell function, phenocopying human disease. CONCLUSIONS: PLCG2 haploinsufficiency represents a distinct syndrome from previous variants characterized by NK-cell immunodeficiency with herpesvirus susceptibility, expanding the spectrum of PLCG2-related disease.

Cell-Free DNA Maps Tissue Injury and Correlates with Disease Severity in Lung Transplant Candidates.

Rationale: Plasma cell-free DNA levels correlate with disease severity in many conditions. Pretransplant cell-free DNA may risk stratify lung transplant candidates for post-transplant complications. Objectives: To evaluate if pretransplant cell-free DNA levels and tissue sources identify patients at high risk of primary graft dysfunction and other pre- and post-transplant outcomes. Methods: This multicenter, prospective cohort study recruited 186 lung transplant candidates. Pretransplant plasma samples were collected to measure cell-free DNA. Bisulfite sequencing was performed to identify the tissue sources of cell-free DNA. Multivariable regression models determined the association between cell-free DNA levels and the primary outcome of primary graft dysfunction and other transplant outcomes, including Lung Allocation Score, chronic lung allograft dysfunction, and death. Measurements and Main Results: Transplant candidates had twofold greater cell-free DNA levels than healthy control patients (median [interquartile range], 23.7 ng/ml [15.1-35.6] vs. 12.9 ng/ml [9.9-18.4]; P < 0.0001), primarily originating from inflammatory innate immune cells. Cell-free DNA levels and tissue sources differed by native lung disease category and correlated with the Lung Allocation Score (P < 0.001). High pretransplant cell-free DNA increased the risk of primary graft dysfunction (odds ratio, 1.60; 95% confidence interval [CI], 1.09-2.46; P = 0.0220), and death (hazard ratio, 1.43; 95% CI, 1.07-1.92; P = 0.0171) but not chronic lung allograft dysfunction (hazard ratio, 1.37; 95% CI, 0.97-1.94; P = 0.0767). Conclusions: Lung transplant candidates demonstrate a heightened degree of tissue injury with elevated cell-free DNA, primarily originating from innate immune cells. Pretransplant plasma cell-free DNA levels predict post-transplant complications.

Impact of Surge Strain and Pandemic Progression on Prognostication by an Established COVID-19-Specific Severity Score.

IMPORTANCE: Many U.S. State crisis standards of care (CSC) guidelines incorporated Sequential Organ Failure Assessment (SOFA), a sepsis-related severity score, in pandemic triage algorithms. However, SOFA performed poorly in COVID-19. Although disease-specific scores may perform better, their prognostic utility over time and in overcrowded care settings remains unclear. OBJECTIVES: We evaluated prognostication by the modified 4C (m4C) score, a COVID-19-specific prognosticator that demonstrated good predictive capacity early in the pandemic, as a potential tool to standardize triage across time and hospital-surge environments. DESIGN: Retrospective observational cohort study. SETTING: Two hundred eighty-one U.S. hospitals in an administrative healthcare dataset. PARTICIPANTS: A total of 298,379 hospitalized adults with COVID-19 were identified from March 1, 2020, to January 31, 2022. m4C scores were calculated from admission diagnosis codes, vital signs, and laboratory values. MAIN OUTCOMES AND MEASURES: Hospital-surge index, a severity-weighted measure of COVID-19 caseload, was calculated for each hospital-month. Discrimination of in-hospital mortality by m4C and surge index-adjusted models was measured by area under the receiver operating characteristic curves (AUC). Calibration was assessed by training models on early pandemic waves and measuring fit (deviation from bisector) in subsequent waves. RESULTS: From March 2020 to January 2022, 298,379 adults with COVID-19 were admitted across 281 U.S. hospitals. m4C adequately discriminated mortality in wave 1 (AUC 0.779 [95% CI, 0.769-0.789]); discrimination was lower in subsequent waves (wave 2: 0.772 [95% CI, 0.765-0.779]; wave 3: 0.746 [95% CI, 0.743-0.750]; delta: 0.707 [95% CI, 0.702-0.712]; omicron: 0.729 [95% CI, 0.721-0.738]). m4C demonstrated reduced calibration in contemporaneous waves that persisted despite periodic recalibration. Performance characteristics were similar with and without adjustment for surge. CONCLUSIONS AND RELEVANCE: Mortality prediction by the m4C score remained robust to surge strain, making it attractive for when triage is most needed. However, score performance has deteriorated in recent waves. CSC guidelines relying on defined prognosticators, especially for dynamic disease processes like COVID-19, warrant frequent reappraisal to ensure appropriate resource allocation.

Investigations into mRNA Lipid Nanoparticles Shelf-Life Stability under Nonfrozen Conditions.

mRNA LNPs can experience a decline in activity over short periods (ranging from weeks to months). As a result, they require frozen storage and transportation conditions to maintain their full functionality when utilized. Currently approved commercially available mRNA LNP vaccines also necessitate frozen storage and supply chain management. Overcoming this significant inconvenience in the future is crucial to reducing unnecessary costs and challenges associated with storage and transport. In this study, our objective was to illuminate the potential time frame for nonfrozen storage and transportation conditions of mRNA LNPs without compromising their activity. To achieve this goal, we conducted a stability assessment and an in vitro cell culture delivery study involving five mRNA LNPs. These LNPs were constructed by using a standard formulation similar to that employed in the three commercially available LNP formulations. Among these formulations, we selected five structurally diverse ionizable lipids─C12-200, CKK-E12, MC3, SM-102, and lipid 23─from the existing literature. We incorporated these lipids into a standard LNP formulation, keeping all other components identical. The LNPs, carrying mRNA payloads, were synthesized by using microfluidic mixing technology. We evaluated the shelf life stability of these LNPs over a span of 9 weeks at temperatures of 2-8, 25, and 40 °C, utilizing an array of analytical techniques. Our findings indicated minimal impact on the hydrodynamic diameter, zeta potential, encapsulation efficiency, and polydispersity of all LNPs across the various temperatures over the studied period. The RiboGreen assay analysis of LNPs showed consistent mRNA contents over several weeks at various nonfrozen storage temperatures, leading to the incorrect assumption of intact and functional LNPs. This misunderstanding was rectified by the significant differences observed in EGFP protein expression in an in vitro cell culture (using HEK293 cells) across the five LNPs. Specifically, only LNP 1 (C12-200) and LNP 4 (SM-102) exhibited high levels of EGFP expression at the start (T0), with over 90% of HEK293 cells transfected and mean fluorescence intensity (MFI) levels exceeding 1. Interestingly, LNP 1 (C12-200) maintained largely unchanged levels of in vitro activity over 11 weeks when stored at both 2-8 and 25 °C. In contrast, LNP 4 (SM-102) retained its functionality when stored at 2-8 °C over 11 weeks but experienced a gradual decline of in vitro activity when stored at room temperature over the same period. Importantly, we observed distinct LNP architectures for the five formulations through cryo-EM imaging. This highlights the necessity for a deeper comprehension of structure-activity relationships within these complex nanoparticle structures. Enhancing our understanding in this regard is vital for overcoming storage and stability limitations, ultimately facilitating the broader application of this technology beyond vaccines.

A biomass map of the Brazilian Amazon from multisource remote sensing.

The Amazon Forest, the largest contiguous tropical forest in the world, stores a significant fraction of the carbon on land. Changes in climate and land use affect total carbon stocks, making it critical to continuously update and revise the best estimates for the region, particularly considering changes in forest dynamics. Forest inventory data cover only a tiny fraction of the Amazon region, and the coverage is not sufficient to ensure reliable data interpolation and validation. This paper presents a new forest above-ground biomass map for the Brazilian Amazon and the associated uncertainty both with a resolution of 250 meters and baseline for the satellite dataset the year of 2016 (i.e., the year of the satellite observation). A significant increase in data availability from forest inventories and remote sensing has enabled progress towards high-resolution biomass estimates. This work uses the largest airborne LiDAR database ever collected in the Amazon, mapping 360,000 km2 through transects distributed in all vegetation categories in the region. The map uses airborne laser scanning (ALS) data calibrated by field forest inventories that are extrapolated to the region using a machine learning approach with inputs from Synthetic Aperture Radar (PALSAR), vegetation indices obtained from the Moderate-Resolution Imaging Spectroradiometer (MODIS) satellite, and precipitation information from the Tropical Rainfall Measuring Mission (TRMM). A total of 174 field inventories geolocated using a Differential Global Positioning System (DGPS) were used to validate the biomass estimations. The experimental design allowed for a comprehensive representation of several vegetation types, producing an above-ground biomass map varying from a maximum value of 518 Mg ha-1, a mean of 174 Mg ha-1, and a standard deviation of 102 Mg ha-1. This unique dataset enabled a better representation of the regional distribution of the forest biomass and structure, providing further studies and critical information for decision-making concerning forest conservation, planning, carbon emissions estimate, and mechanisms for supporting carbon emissions reductions.

Myelin oligodendrocyte glycoprotein antibody-associated disease as a novel presentation of central nervous system autoimmunity in a pediatric patient with Wiskott-Aldrich syndrome.

BACKGROUND: Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency caused by mutations in the WAS gene that leads to increased susceptibility to infections, thrombocytopenia, eczema, malignancies, and autoimmunity. Central nervous system (CNS) autoimmune manifestations are uncommon. CASE PRESENTATION: We describe the case of a five-year-old boy with refractory thrombocytopenia and iron deficiency anemia who developed relapsing bilateral optic neuritis. Myelin oligodendrocyte glycoprotein antibody (MOG-IgG) via serum fluorescence-activated cell sorting assay was positive (titer 1:100), confirming a diagnosis of myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD). At age six, molecular panel testing for genes associated with primary immunodeficiency identified a missense WAS gene variant. He was subsequently found to have decreased WAS protein expression, consistent with a diagnosis of WAS. CONCLUSIONS: This case expands the reported spectrum of CNS autoimmunity associated with WAS and may help to inform long-term therapeutic options.

Measuring the effect of newborn screening on survival after haematopoietic cell transplantation for severe combined immunodeficiency: a 36-year longitudinal study from the Primary Immune Deficiency Treatment Consortium.

BACKGROUND: Severe combined immunodeficiency (SCID) is fatal unless durable adaptive immunity is established, most commonly through allogeneic haematopoietic cell transplantation (HCT). The Primary Immune Deficiency Treatment Consortium (PIDTC) explored factors affecting the survival of individuals with SCID over almost four decades, focusing on the effects of population-based newborn screening for SCID that was initiated in 2008 and expanded during 2010-18. METHODS: We analysed transplantation-related data from children with SCID treated at 34 PIDTC sites in the USA and Canada, using the calendar time intervals 1982-89, 1990-99, 2000-09, and 2010-18. Categorical variables were compared by χ2 test and continuous outcomes by the Kruskal-Wallis test. Overall survival was estimated by the Kaplan-Meier method. A multivariable analysis using Cox proportional hazards regression models examined risk factors for HCT outcomes, including the variables of time interval of HCT, infection status and age at HCT, trigger for diagnosis, SCID type and genotype, race and ethnicity of the patient, non-HLA-matched sibling donor type, graft type, GVHD prophylaxis, and conditioning intensity. FINDINGS: For 902 children with confirmed SCID, 5-year overall survival remained unchanged at 72%-73% for 28 years until 2010-18, when it increased to 87% (95% CI 82·1-90·6; n=268; p=0·0005). For children identified as having SCID by newborn screening since 2010, 5-year overall survival was 92·5% (95% CI 85·8-96·1), better than that of children identified by clinical illness or family history in the same interval (79·9% [69·5-87·0] and 85·4% [71·8-92·8], respectively [p=0·043]). Multivariable analysis demonstrated that the factors of active infection (hazard ratio [HR] 2·41, 95% CI 1·56-3·72; p<0·0001), age 3·5 months or older at HCT (2·12, 1·38-3·24; p=0·001), Black or African-American race (2·33, 1·56-3·46; p<0·0001), and certain SCID genotypes to be associated with lower overall survival during all time intervals. Moreover, after adjusting for several factors in this multivariable analysis, HCT after 2010 no longer conveyed a survival advantage over earlier time intervals studied (HR 0·73, 95% CI 0·43-1·26; p=0·097). This indicated that younger age and freedom from infections at HCT, both directly driven by newborn screening, were the main drivers for recent improvement in overall survival. INTERPRETATION: Population-based newborn screening has facilitated the identification of infants with SCID early in life, in turn leading to prompt HCT while avoiding infections. Public health programmes worldwide can benefit from this definitive demonstration of the value of newborn screening for SCID. FUNDING: National Institute of Allergy and Infectious Diseases, Office of Rare Diseases Research, and National Center for Advancing Translational Sciences.

Propensities of Fatty Acid-Modified ASOs: Self-Assembly vs Albumin Binding.

We conducted a biophysical study to investigate the self-assembling and albumin-binding propensities of a series of fatty acid-modified locked nucleic acid (LNA) antisense oligonucleotide (ASO) gapmers specific to the MALAT1 gene. To this end, a series of biophysical techniques were applied using label-free ASOs that were covalently modified with saturated fatty acids (FAs) of varying length, branching, and 5'/3' attachment. Using analytical ultracentrifugation (AUC), we demonstrate that ASOs conjugated with fatty acids longer than C16 exhibit an increasing tendency to form self-assembled vesicular structures. The C16 to C24 conjugates interacted via the fatty acid chains with mouse and human serum albumin (MSA/HSA) to form stable adducts with near-linear correlation between FA-ASO hydrophobicity and binding strength to mouse albumin. This was not observed for the longer fatty acid chain ASO conjugates (>C24) under the experimental conditions applied. The longer FA-ASO however adopted self-assembled structures with increasing intrinsic stabilities proportional to the fatty acid chain length. For instance, FA chain lengths smaller than C24 readily formed self-assembled structures containing 2 (C16), 6 (C22, bis-C12), and 12 (C24) monomers, as measured by analytical ultracentrifugation (AUC). Incubation with albumin disrupted these supramolecular architectures to form FA-ASO/albumin complexes mostly with 2:1 stoichiometry and binding affinities in the low micromolar range, as determined by isothermal titration calorimetry (ITC) and analytical ultracentrifugation (AUC). Binding of FA-ASOs underwent a biphasic pattern for medium-length FA chain lengths (>C16) with an initial endothermic phase of particulate disruption, followed by an exothermic binding event to the albumin. Conversely, ASO modified with di-palmitic acid (C32) formed a strong, hexameric complex. This structure was not disrupted when incubated with albumin under conditions above the critical nanoparticle concentration (CNC; <0.4 µM). It is noteworthy that the interaction of parent, fatty acid-free malat1 ASO to albumin was below detectability by ITC (KD ≫150 µM). This work demonstrates that the nature of mono- vs multimeric structures of hydrophobically modified ASOs is governed by the hydrophobic effect. Consequently, supramolecular assembly to form particulate structures is a direct consequence of the fatty acid chain length. This provides opportunities to exploit the concept of hydrophobic modification to influence pharmacokinetics (PK) and biodistribution for ASOs in two ways: (1) binding of the FA-ASO to albumin as a carrier vehicle and (2) self-assembly resulting in albumin-inert, supramolecular architectures. Both concepts create opportunities to influence biodistribution, receptor interaction, uptake mechanism, and pharmacokinetics/pharmacodynamics (PK/PD) properties in vivo, potentially enabling access to extrahepatic tissues in sufficient concentration to treat disease.

Chromosomal microarray analysis supplements exome sequencing to diagnose children with suspected inborn errors of immunity.

Purpose: Though copy number variants (CNVs) have been suggested to play a significant role in inborn errors of immunity (IEI), the precise nature of this role remains largely unexplored. We sought to determine the diagnostic contribution of CNVs using genome-wide chromosomal microarray analysis (CMA) in children with IEI. Methods: We performed exome sequencing (ES) and CMA for 332 unrelated pediatric probands referred for evaluation of IEI. The analysis included primary, secondary, and incidental findings. Results: Of the 332 probands, 134 (40.4%) received molecular diagnoses. Of these, 116/134 (86.6%) were diagnosed by ES alone. An additional 15/134 (11.2%) were diagnosed by CMA alone, including two likely de novo changes. Three (2.2%) participants had diagnostic molecular findings from both ES and CMA, including two compound heterozygotes and one participant with two distinct diagnoses. Half of the participants with CMA contribution to diagnosis had CNVs in at least one non-immune gene, highlighting the clinical complexity of these cases. Overall, CMA contributed to 18/134 diagnoses (13.4%), increasing the overall diagnostic yield by 15.5% beyond ES alone. Conclusion: Pairing ES and CMA can provide a comprehensive evaluation to clarify the complex factors that contribute to both immune and non-immune phenotypes. Such a combined approach to genetic testing helps untangle complex phenotypes, not only by clarifying the differential diagnosis, but in some cases by identifying multiple diagnoses contributing to the overall clinical presentation.

Applications of virus-specific T cell therapies post-BMT.

Hematopoietic stem cell transplantation (HSCT) has been used as a curative standard of care for moderate to severe primary immunodeficiency disorders as well as relapsed hematologic malignancies for over 50 years [1,2]. However, chronic and refractory viral infections remain a leading cause of morbidity and mortality in the immune deficient period following HSCT, where use of available antiviral pharmacotherapies is limited by toxicity and emerging resistance [3]. Adoptive immunotherapy using virus-specific T cells (VSTs) has been explored for over 2 decades [4,5] in patients post-HSCT and has been shown prior phase I-II studies to be safe and effective for treatment or preventions of viral infections including cytomegalovirus, Epstein-Barr virus, BK virus, and adenovirus with minimal toxicity and low risk of graft vs host disease [6-9]. This review summarizes methodologies to generate VSTs the clinical results utilizing VST therapeutics and the challenges and future directions for the field.