BackgroundPreviously, we identified three loci affecting HDL-cholesterol levels in a screen for ENU-induced mutations in mice and discovered two mutated genes. We sought to identify the third mutated gene and further characterize the mouse phenotype.MethodsWe engaged, DNA sequencing, gene expression profiling, western blotting, lipoprotein characterization, metabolomics assessment, histology and electron microscopy in mouse tissues.ResultsWe identify the third gene as Ampd2, a liver isoform of AMP Deaminase (Ampd), a central component of energy and purine metabolism pathways. The causative mutation was a guanine-to-thymine transversion resulting in an A341S conversion in Ampd2. Ampd2 homozygous mutant mice exhibit a labile hypercholesterolemia phenotype, peaking around 9 weeks of age (251 mg/dL vs. wildtype control at 138 mg/dL), and was evidenced by marked increases in HDL, VLDL and LDL. In an attempt to determine the molecular connection between Ampd2 dysfunction and hypercholesterolemia, we analyzed hepatic gene expression and found the downregulation of Ldlr, Hmgcs and Insig1 and upregulation of Cyp7A1 genes. Metabolomic analysis confirmed an increase in hepatic AMP levels and a decrease in allantoin levels consistent with Ampd2 deficiency, and increases in campesterol and β-sitosterol. Additionally, nephrotic syndrome was observed in the mutant mice, through proteinuria, kidney histology and effacement and blebbing of podocyte foot processes by electron microscopy.ConclusionIn summary we describe the discovery of a novel genetic mouse model of combined transient nephrotic syndrome and hypercholesterolemia, resembling the human disorder.Electronic supplementary materialThe online version of this article (doi:10.1186/1476-511X-13-167) contains supplementary material, which is available to authorized users.

Background: FLT3 (CD135) is a receptor tyrosine kinase expressed by hematopoietic progenitor cells, and when activated by ligand binding, FLT3 signaling can induce survival, proliferation and differentiation. Acute myeloid leukemia (AML), an indication with high unmet need, derives from hematopoietic progenitor lineage cells; consequently, many AML patient samples retain FLT3 expression on circulating leukemic cells. In comparison to the wide expression seen in AML patient samples, normal tissue expression of FLT3 is restricted to the hematopoietic compartment: a subset of hematopoietic stem cells, a subset of lineage committed progenitor cells and dendritic cells. Cell surface FLT3 protein was not detectable in those solid tissues that express quantifiable FLT3 mRNA (cerebellum, pancreas). As a result of this favorable expression profile, targeting AML cells using T cells expressing a chimeric antigen receptor (CAR) directed against FLT3, is expected to provide benefit to patients. Methods: A panel of fully-human, anti-human FLT3 antibodies were generated, screened for specific FLT3 binding, converted to single chain variable fragments (scFv) and re-evaluated for binding and stability. Three anti-FLT3 scFv sequences were selected for incorporation into CAR constructs. T cells transduced with these CAR constructs were evaluated in vitro for cytotoxicity, proliferation and cytokine secretion, and in vivo in a mouse xenograft model. The lead anti-FLT3 scFv was combined with an anti-CD3 scFv (bispecific T cell engager (BiTE®) format) for evaluation in non-human primates (NHP). Results: Human T cells engineered to express anti-FLT3 CAR constructs were evaluated for cytotoxicity, proliferation and cytokine secretion in the presence of three FLT3-positive cell lines, each with a different number of surface-expressed FLT3 receptors, and one FLT3-negative cell line. While all constructs were selectively active against FLT3-positive cells, one construct was more active than the others against cells expressing very low levels of FLT3; 90% depletion of target cells with ~1600 receptors/cell was observed after 40 hours of co-culture with CAR T cells in a 1:1 ratio. This lead construct was further evaluated in a mouse xenograft model, where FLT3 CAR-T cells provided a survival advantage. The anti-FLT3 scFv used in the lead CAR construct was evaluated in NHP in the form of a BiTE®. In this 16-day, multi-dose study, the FLT3 BiTE® induced elimination of FLT3+ cells as assessed by depletion of FLT3 mRNA-expressing cells (97% reduction in FLT3 mRNA) in the circulation and increased levels of soluble FLT3 ligand in serum. Conclusions: These results demonstrate the potential of using FLT3 CAR-T cell therapy for the treatment of AML. Citation Format: Tara L. Arvedson, Alice Bakker, Herve Lebrec, Gregor Adams, Armen Madiros, Priya Koppikar, Mercedesz Balazs, Mei Gong, Yan Zheng, Rebecca Goldstein, Tony Polverino, Lawren Wu, Angela Coxon. Generation and evaluation of an FLT3 CAR-T cell therapy for the treatment of acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2559.