Nonclinical pharmacokinetics, pharmacodynamics and safety assessment of a FLT3L-Fc molecule for cancer immunotherapy.

FLT3L-Fc is a cytokine-Fc fusion agonizing receptor-type tyrosine-protein kinase FLT3 (fms-related tyrosine kinase 3; CD135). FLT3 is expressed on dendritic cells (DCs) as well as myeloid and lymphoid progenitors. Nonclinical pharmacokinetics, pharmacodynamics and safety of FLT3L-Fc were investigated in rats and cynomolgus monkeys. FLT3L-Fc induced robust pharmacodynamic responses, evidenced by marked expansion of peripheral blood cDC1s, cDC2s, and pDCs (up to 301-fold in rats and 378-fold in monkeys), peaking at 8-10 days after the first dose. FLT3L-Fc was well tolerated with no adverse findings at doses up to 10 mg/kg administered intravenously twice three weeks apart. In both species, major clinical pathology findings consisted of expansion of white blood cell (WBC) populations including lymphocytes, monocytes, neutrophils, basophils, and large unstained cells, which were pronounced after the first dose. The WBC findings were associated microscopically with histiocytic and mononuclear cell infiltrates in multiple organs. Tissue immunohistochemistry in monkeys showed that the leukocyte infiltrates consisted of hematopoietic progenitor cells and histiocytes with a reactive morphology and were associated with a slight stimulation of regional T and B cell populations. Additional FLT3L-Fc-associated changes included decreases in red blood cell (RBC) mass, increases in RBC distribution width, variable changes in reticulocytes, and transient alterations in platelet counts (rats only). The RBC and WBC findings were associated microscopically with increased hematopoietic cellularity of the bone marrow in both species and increased splenic megakaryocytic extramedullary hematopoiesis in rats. The totality of nonclinical safety data support the clinical development of FLT3L-Fc.

Ex Vivo Hepatocyte Reprograming Promotes Homology-Directed DNA Repair to Correct Metabolic Disease in Mice After Transplantation.

Ex vivo CRISPR/Cas9-mediated gene editing in hepatocytes using homology-directed repair (HDR) is a potential alternative curative therapy to organ transplantation for metabolic liver disease. However, a major limitation of this approach in quiescent adult primary hepatocytes is that nonhomologous end-joining is the predominant DNA repair pathway for double-strand breaks (DSBs). This study explored the hypothesis that ex vivo hepatocyte culture could reprogram hepatocytes to favor HDR after CRISPR/Cas9-mediated DNA DSBs. Quantitative PCR (qPCR), RNA sequencing, and flow cytometry demonstrated that within 24 hours, primary mouse hepatocytes in ex vivo monolayer culture decreased metabolic functions and increased expression of genes related to mitosis progression and HDR. Despite the down-regulation of hepatocyte function genes, hepatocytes cultured for up to 72 hours could robustly engraft in vivo. To assess functionality long-term, primary hepatocytes from a mouse model of hereditary tyrosinemia type 1 bearing a single-point mutation were transduced ex vivo with two adeno-associated viral vectors to deliver the Cas9 nuclease, target guide RNAs, and a 1.2-kb homology template. Adeno-associated viral Cas9 induced robust cutting at the target locus, and, after delivery of the repair template, precise correction of the point mutation occurred by HDR. Edited hepatocytes were transplanted into recipient fumarylacetoacetate hydrolase knockout mice, resulting in engraftment, robust proliferation, and prevention of liver failure. Weight gain and biochemical assessment revealed normalization of metabolic function. Conclusion: The results of this study demonstrate the potential therapeutic effect of ex vivo hepatocyte-directed gene editing after reprogramming to cure metabolic disease in a preclinical model of hereditary tyrosinemia type 1.