Use of HSC-targeted LNP to generate a mouse model of lethal α-thalassemia and treatment via lentiviral gene therapy.

ABSTRACT: α-Thalassemia (AT) is one of the most commonly occurring inherited hematological diseases. However, few treatments are available, and allogeneic bone marrow transplantation is the only available therapeutic option for patients with severe AT. Research into AT has remained limited because of a lack of adult mouse models, with severe AT typically resulting in in utero lethality. By using a lipid nanoparticle (LNP) targeting the receptor CD117 and delivering a Cre messenger RNA (mRNACreLNPCD117), we were able to delete floxed α-globin genes at high efficiency in hematopoietic stem cells (HSC) ex vivo. These cells were then engrafted in the absence or presence of a novel α-globin-expressing lentiviral vector (ALS20αI). Myeloablated mice infused with mRNACreLNPCD117-treated HSC showed a complete knock out (KO) of α-globin genes. They showed a phenotype characterized by the synthesis of hemoglobin H (HbH; also known as ß-tetramers or ß4), aberrant erythropoiesis, and abnormal organ morphology, culminating in lethality ∼8 weeks after engraftment. Mice infused with mRNACreLNPCD117-treated HSC with at least 1 copy of ALS20αI survived long term with normalization of erythropoiesis, decreased production of HbH, and amelioration of the abnormal organ morphology. Furthermore, we tested ALS20αI in erythroid progenitors derived from α-globin-KO CD34+ cells and cells isolated from patients with both deletional and nondeletional HbH disease, demonstrating improvement in α-globin/ß-globin mRNA ratio and reduction in the formation of HbH by high-performance liquid chromatography. Our results demonstrate the broad applicability of LNP for disease modeling, characterization of a novel mouse model of severe AT, and the efficacy of ALS20αI for treating AT.

Serum cytokine panels in pediatric clinical practice.

BACKGROUND: Cytokines are soluble signaling proteins that regulate inflammation and coordinate immune responses. Serum cytokine panels are increasingly used in medical practice, yet our understanding of cytokines as biomarkers for disease remains limited. OBJECTIVE: We sought to analyze real-world single-center use of a multiplexed cytokine panel, correlate its results with diagnosis and severity, and explore its use in pediatric practice. METHODS: A multiplexed cytokine panel, able to return same-day results, was implemented in April 2020 at the Children's Hospital of Philadelphia (Philadelphia, Pa) and its performance was validated for clinical use. Coded patient data were collected using the REDCap database, and correlations between cytokine levels and outcomes of interest were analyzed retrospectively. RESULTS: Cytokine levels correlate with acuity of care, with patients admitted to the pediatric intensive care unit having the highest cytokine values. Patients with familial hemophagocytic lymphohistiocytosis (fHLH) showed prominent peaks in IFN-γ, IL-10, and TNF, whereas patients with sepsis exhibited high IL-6 and IL-8 with relatively modest IFN-γ. Cytokine release syndrome (CRS) after chimeric antigen receptor T-cell therapy often demonstrated pan-panel positivity at peak levels, with a similar pattern as that of fHLH. A ratio of [IFN-γ] + [IL-10]/[IL-6] + [IL-8] levels was able to distinguish fHLH and CRS from severe sepsis. CONCLUSIONS: Cytokine levels correlate with severity of illness and can help differentiate between syndromes that present similarly, including fHLH and CRS compared with sepsis. Cytokine panels can be used as biomarkers to inform diagnosis and management decisions, but significant work remains to dissect complex clinical patterns of disease.

Gene therapy for hemophilia: Progress to date and challenges moving forward.

Over the past decades hemophilia has been transformed from a debilitating disease to a manageable condition. However, the current treatment options are expensive, complex, and inaccessible to a large portion of the global population. Moreover, the development of antibodies to replacement factors, termed inhibitors, is a common complication that not only renders conventional prophylaxis regimens ineffective but also increases the annual bleeding rate in affected patients. Fortunately, much progress has been made toward developing a curative gene therapy treatment for hemophilia and these efforts have led to a series of human trials with promising results. This review seeks to address some of the new issues raised by recent progress in the field, including the differences between available recombinant adeno-associated viral (rAAV) vectors, the etiology of transaminitis following vector administration, and techniques to induce long-term factor expression. We also address other unresolved questions, including strategies to overcome pre-existing neutralizing antibodies to AAV, approaches that can make vector re-administration possible, and whether gene therapy can be used to induce factor tolerance and treat inhibitors. Finally, we discuss logistical and ethical issues related to hemophilia gene therapy including how to accurately measure therapeutic outcomes, when to consider treatment of pediatric patients, and how to equitably price the medication to ensure fair compensation while maximizing accessibility. As the field marches forward from clinical trials towards clinical application, answers to these questions will determine the future of gene therapy for hemophilia.

Proliferative retinopathy and retinal detachment in pediatric atypical hemolytic uremic syndrome.

We report the case of a 14-year-old boy with history of microangiopathic hemolytic crises secondary to atypical hemolytic uremic syndrome presenting with new-onset decreased vision, flashes, and floaters in his left eye. The patient had a history of chronic retinal detachment in the right eye and retinal neovascularization in the left eye treated with panretinal photocoagulation at age 5. He was now found to have a new combined tractional-rhegmatogenous retinal detachment in the left eye. Despite surgical reattachment of the retina, he had progressive retinal and optic nerve ischemia, with resultant left eye visual acuity of light perception. To our knowledge, this is the first reported case of proliferative retinopathy and tractional and rhegmatogenous retinal detachments in a pediatric patient with atypical hemolytic uremic syndrome.

The human tyrosine hydroxylase gene promoter.

13.329 kilobases of the single copy human tyrosine hydroxylase (hTH) gene were isolated from a genomic library. The 5' flanking 11 kilobases fused to the reporter green fluorescent protein (GFP) drove high level expression in TH+ cells of the substantia nigra of embryonic and adult transgenic mice as determined by double label fluorescence microscopy. To provide a basis for future analysis of polymorphisms and structure-function studies, the previously unreported distal 10.5 kilobases of the hTH promoter were sequenced with an average coverage of 20-fold, the remainder with 4-fold coverage. Sequence features identified included four perfect matches to the bicoid binding element (BBE, consensus: BBTAATCYV) all of which exhibited specific binding by electrophoretic mobility shift assay (EMSA). Comparison to published sequences of mouse and rat TH promoters revealed five areas of exceptional homology shared by these species in the upstream TH promoter region -2 kb to -9 kb relative to the transcription start site. Within these conserved regions (CRs I-V), potential recognition sites for NR4A2 (Nurr1), HNF-3beta, HOXA4, and HOXA5 were shared across human, mouse, and rat TH promoters.