Context-Dependent Effects of Amplified MAPK Signaling during Lung Adenocarcinoma Initiation and Progression

Expression of oncogenic KrasG12D initiates lung adenomas in a mitogen-activated protein kinase (MAPK) signal-dependent manner from only a subset of cell types in the adult mouse lung. Amplification of MAPK signaling is associated with progression to malignant adenocarcinomas, but whether this is a cause or a consequence of disease progression is not known. To better understand the effects of MAPK signaling downstream of KrasG12D expression, we capitalized on the ability of Braf inhibition to selectively amplify MAPK pathway signaling in KrasG12D-expressing epithelial cells. MAPK signal amplification indeed promoted the rapid progression of established adenomas to malignant adenocarcinomas. However, we observed, surprisingly, a greater number of overall tumor-initiating events after MAPK signal amplification, due to induced proliferation of cell types that are normally refractory to KrasG12D-induced transformation. Thus, MAPK signaling in the lung is thresholded not only during malignant progression but also at the moment of tumor initiation

Toxicology Study of Intra-Cisterna Magna Adeno-Associated Virus 9 Expressing Human Alpha-L-Iduronidase in Rhesus Macaques

Mucopolysaccharidosis type I is a recessive genetic disease caused by deficiency of the lysosomal enzyme α-L-iduronidase, which leads to a neurodegenerative and systemic disease called Hurler syndrome in its most severe form. Several clinical trials are evaluating adeno-associated virus serotype 9 (AAV9) for the treatment of neurodegenerative diseases. Although these trials focus on systemic or lumbar administration, intrathecal administration via suboccipital puncture into the cisterna magna has demonstrated remarkable efficacy in large animals. We, therefore, conducted a good laboratory practice-compliant non-clinical study to investigate the safety of suboccipital AAV9 gene transfer of human α-L-iduronidase into nonhuman primates. We dosed 22 rhesus macaques, including three immunosuppressed animals, with vehicle or one of two doses of vector. We assessed in-life safety and immune responses. Animals were euthanized 14, 90, or 180 days post-vector administration and evaluated for histopathology and biodistribution. No procedure-related lesions or adverse events occurred. All vector-treated animals showed a dose-dependent mononuclear pleocytosis in the cerebrospinal fluid and minimal to moderate asymptomatic degeneration of dorsal root ganglia neurons and associated axons. These studies support the clinical development of suboccipital AAV delivery for Hurler syndrome and highlight a potential sensory neuron toxicity that warrants careful monitoring in first-in-human studies. Keywords: AAV9, intrathecal, MPS

Safe and Sustained Expression of Human Iduronidase After Intrathecal Administration of Adeno-Associated Virus Serotype 9 in Infant Rhesus Monkeys

Many neuropathic diseases cause early, irreversible neurologic deterioration, which warrants therapeutic intervention during the first months of life. In the case of mucopolysaccharidosis type I, a recessive lysosomal storage disorder that results from a deficiency of the lysosomal enzyme α-l-iduronidase (IDUA), one of the most promising treatment approaches is to restore enzyme expression through gene therapy. Specifically, administering pantropic adeno-associated virus (AAV) encoding IDUA into the cerebrospinal fluid (CSF) via suboccipital administration has demonstrated remarkable efficacy in large animals. Preclinical safety studies conducted in adult nonhuman primates supported a positive risk-benefit profile of the procedure while highlighting potential subclinical toxicity to primary sensory neurons located in the dorsal root ganglia (DRG). This study investigated the long-term performance of intrathecal cervical AAV serotype 9 gene transfer of human IDUA administered to 1-month-old rhesus monkeys (N = 4) with half of the animals tolerized to the human transgene at birth via systemic administration of an AAV serotype 8 vector expressing human IDUA from the liver. Sustained expression of the transgene for almost 4 years is reported in all animals. Transduced cells were primarily pyramidal neurons in the cortex and hippocampus, Purkinje cells in the cerebellum, lower motor neurons, and DRG neurons. Both tolerized and non-tolerized animals were robust and maintained transgene expression as measured by immunohistochemical analysis of brain tissue. However, the presence of antibodies in the non-tolerized animals led to a loss of measurable levels of secreted enzyme in the CSF. These results support the safety and efficiency of treating neonatal rhesus monkeys with AAV serotype 9 gene therapy delivered into the CSF

<i>Ahr</i>^<i>-/-</i> mice exhibit increased T cell activation following infection.

<p>Wild type or <i>Ahr</i>^<i>-/-</i> mice were orally infected with <i>T</i>. <i>gondii</i> for 9 days. <b>(A)</b> Group 3 ILC frequency in the lamina propria of wild type or <i>Ahr</i>^<i>-/-</i> mice following infection. The plots on the left are gated on live CD90.2⁺CD11c^-B220^- cells. (<b>B)</b> Weight loss was monitored at various days post-infection. Data are pooled from 2 experiments with 6–8 mice per group. <b>(C)</b> H&E staining of small intestinal tissue sections. <b>(D)</b> H&E staining of small intestinal tissue from an infected <i>Ahr</i>^<i>-/-</i> mouse. The Peyer’s patch exhibits severe lymphocytolysis (*) and the lamina propria of adjacent villi is expanded by primarily lymphocytes and plasma cells (➜). <b>(E)</b> A higher magnification image of the section in Fig 2C shows that the Peyer’s patch exhibits severe lymphocytolysis characterized by pyknosis and other cellular debris. <b>(F)</b> H&E staining of small intestinal tissue from an infected <i>Ahr</i>^<i>-/-</i> mouse shows crypt loss (*) and multifocal necrotic enterocytes (➜). The lamina propria of the villi is expanded by lymphocytes and plasma cells (❋). <b>(G)</b> Expression of T-bet and Ki67 by FoxP3^- CD4⁺ T cells in the mesenteric lymph nodes of infected mice. Results are pooled from 3 separate experiments. <b>(H)</b> Cytokine production by CD4⁺ T cells following stimulation with PMA/ionomycin. Data are pooled from 2 experiments.</p

Increased T cell responses to <i>Toxoplasma</i> antigen and crude commensal antigen in <i>Ahr</i>^<i>-/-</i> mice following infection.

<p>Wild type or <i>Ahr</i>^<i>-/-</i> mice were orally infected with 100 <i>T</i>. <i>gondii</i> cysts for 7 days. <b>(A, B)</b> Splenocytes were stimulated with the indicated antigen preparations for 5 hours and then incubated overnight with brefeldin A. The cells were stained to assay IFN-γ expression by CD4⁺ T cells. The graph shows pooled data from 3 independent experiments. <b>(C)</b> Parasite burdens in the terminal ileum were assayed by RT-PCR. Results are pooled from 2 separate experiments.</p

Treg phenotype and IL-10 production in orally infected <i>Ahr</i>^<i>-/-</i> mice.

<p><i>Ahr</i>^<i>-/-</i> mice or wild type controls were infected orally with 20 Me49 cysts for 9 days. Results are pooled from 2–3 separate experiments. <b>(A)</b> Frequency of Tregs in the indicated tissues of wild type or <i>Ahr</i>^<i>-/-</i> mice. In the lamina propria, the plots shown are gated on live CD45⁺CD3⁺CD4⁺ cells, and Tregs were gated as live CD45⁺CD3⁺CD4⁺FoxP3⁺ cells. For the mesenteric lymph node and spleen, the plots shown are gated on CD3⁺CD4⁺ cells, and Tregs were gated as CD3⁺CD4⁺FoxP3⁺ cells. <b>(B)</b> Expression of T-bet, CXCR3, and Ki67 on Tregs in the spleens of wild type or <i>Ahr</i>^<i>-/-</i> mice. The plots are gated on CD3⁺CD4⁺FoxP3⁺ cells. <b>(C)</b> IL-10 secretion by cells isolated from the lamina propria or the spleen following restimulation with soluble <i>Toxoplasma</i> antigen. Results are pooled from 2 separate experiments with a total of 5–7 mice per group.</p

Spontaneous Pancreatitis Caused by Tissue-Specific Gene Ablation of Hhex in MiceSummary

Background & Aims: Perturbations in pancreatic ductal bicarbonate secretion cause chronic pancreatitis. The physiologic mechanism of ductal secretion is known, but its transcriptional control is not. We determine the role of the transcription factor hematopoietically expressed homeobox protein (Hhex) in ductal secretion and pancreatitis. Methods: We derived mice with pancreas-specific, Cre-mediated Hhex gene ablation to determine the requirement of Hhex in the pancreatic duct in early life and in adult stages. Histologic and immunostaining analyses were used to detect the presence of pathology. Pancreatic primary ductal cells were isolated to discover differentially expressed transcripts upon acute Hhex ablation on a cell autonomous level. Results: Hhex protein was detected throughout the embryonic and adult ductal trees. Ablation of Hhex in pancreatic progenitors resulted in postnatal ductal ectasia associated with acinar-to-ductal metaplasia, a progressive phenotype that ultimately resulted in chronic pancreatitis. Hhex ablation in adult mice, however, did not cause any detectable pathology. Ductal ectasia in young mice did not result from perturbation of expression of Hnf6, Hnf1β, or the primary cilia genes. RNA-seq analysis of Hhex-ablated pancreatic primary ductal cells showed mRNA levels of the G-protein coupled receptor natriuretic peptide receptor 3 (Npr3), implicated in paracrine signaling, up-regulated by 4.70-fold. Conclusions: Although Hhex is dispensable for ductal cell function in the adult, ablation of Hhex in pancreatic progenitors results in pancreatitis. Our data highlight the critical role of Hhex in maintaining ductal homeostasis in early life and support ductal hypersecretion as a novel etiology of pediatric chronic pancreatitis. Keywords: Npr3, Pancreatic Ducts, Primary Cili

Lipid nanoparticle-encapsulated mRNA therapy corrects serum total bilirubin level in Crigler-Najjar syndrome mouse model

Crigler-Najjar syndrome is a rare disorder of bilirubin metabolism caused by uridine diphosphate glucuronosyl transferase 1A1 (UGT1A1) mutations characterized by hyperbilirubinemia and jaundice. No cure currently exists; treatment options are limited to phototherapy, whose effectiveness diminishes over time, and liver transplantation. Here, we evaluated the therapeutic potential of systemically administered, lipid nanoparticle-encapsulated human UGT1A1 (hUGT1A1) mRNA therapy in a Crigler-Najjar mouse model. Ugt1 knockout mice were rescued from lethal post-natal hyperbilirubinemia by phototherapy. These adult Ugt1 knockout mice were then administered a single lipid nanoparticle-encapsulated hUGT1A1 mRNA dose. Within 24 h, serum total bilirubin levels decreased from 15 mg/dL (256 μmol/L) to <0.5 mg/dL (9 μmol/L), i.e., slightly above wild-type levels. This reduction was sustained for 2 weeks before bilirubin levels rose and returned to pre-treatment levels by day 42 post-administration. Sustained reductions in total bilirubin levels were achieved by repeated administration of the mRNA product in a frequency-dependent manner. We were also able to rescue the neonatal lethality phenotype seen in Ugt1 knockout mice with a single lipid nanoparticle dose, which suggests that this may be a treatment modality appropriate for metabolic crisis situations. Therefore, lipid nanoparticle-encapsulated hUGT1A1 mRNA may represent a potential treatment for Crigler-Najjar syndrome

FLT3 mutations confer enhanced proliferation and survival properties to multipotent progenitors in a murine model of chronic myelomonocytic leukemia

Despite their known transforming properties, the effects of leukemogenic FLT3-ITD mutations on hematopoietic stem and multipotent progenitor cells and on hematopoietic differentiation are not well understood. We report a mouse model harboring an ITD in the murine Flt3 locus that develops myeloproliferative disease resembling CMML and further identified FLT3-ITD mutations in a subset of human CMML. These findings correlated with an increase in number, cell cycling, and survival of multipotent stem and progenitor cells in an ITD dose-dependent manner in animals that exhibited alterations within their myeloid progenitor compartments and a block in normal B cell development. This model provides insights into the consequences of constitutive signaling by an oncogenic tyrosine kinase on hematopoietic progenitor quiescence, function, and cell fate