PurposeThe strongest genetic association with Fuchs' endothelial corneal dystrophy (FECD) is the presence of an intronic (CTG·CAG)n trinucleotide repeat (TNR) expansion in the transcription factor 4 (TCF4) gene. Repeat-associated non-ATG (RAN) translation, an unconventional protein translation mechanism that does not require an initiating ATG, has been described in many TNR expansion diseases, including myotonic dystrophy type 1 (DM1). Given the similarities between DM1 and FECD, we wished to determine whether RAN translation occurs in FECD.MethodsAntibodies against peptides in the C-terminus of putative RAN translation products from TCF4 were raised and validated by Western blotting and immunofluorescence (IF). CTG·CAG repeats of various lengths in the context of the TCF4 gene were cloned in frame with a 3× FLAG tag and transfected in human cells. IF with antipeptide and anti-FLAG antibodies, as well as cytotoxicity and cell proliferation assays, were performed in these transfected cells. Corneal endothelium derived from patients with FECD was probed with validated antibodies by IF.ResultsCTG·CAG repeats in the context of the TCF4 gene are transcribed and translated via non-ATG initiation in transfected cells and confer toxicity to an immortalized corneal endothelial cell line. An antipeptide antibody raised against the C-terminus of the TCF4 poly-cysteine frame recognized RAN translation products by IF in cells transfected with CTG·CAG repeats and in FECD corneal endothelium.ConclusionsExpanded CTG·CAG repeats in the context of the third intron of TCF4 are transcribed and translated via non-ATG initiation, providing evidence for RAN translation in corneal endothelium of patients with FECD.

Center (to the Baldwin lab). J. M. G. serves as a consultant to BioMarin Pharmaceutical for the development of histone deacetylase inhibitors as therapeutics and is an inventor on patents licensed by The Scripps Research Institute to BioMarin Pharmaceutical. E. S. is supported in part by BioMarin Pharmaceuticals. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This article contains Figs. S1-S3 and File S1-S2. The data discussed in this paper have been deposited to the Sequence Read Archive (SRA) under accession no. PRJNA495860.

TPS765 Background: PT886 is a novel bispecific antibody that targets Claudin 18.2 (CLDN18.2) and CD47. CLDN18.2 is overexpressed in a significant proportion of gastric and pancreatic adenocarcinomas and its restricted expression makes it a promising therapeutic target for the treatment of these carcinomas. Moreover, studies have found that immunoglobulin superfamily CD47 is overexpressed widely across tumor types, and CD47 plays an important role in suppressing phagocytes activity through binding to the transmembrane protein SIRPα in phagocytic cells. Hence, by targeting both pathways, one can direct macrophage-mediated phagocytotic activity to tumor cells by blocking the “don’t eat me” signal mediated by CD47/ SIRPα interaction, potentially offering a better safety profile than anti-CD47 monoclonal antibodies. Additionally, PT886 utilizes IgG1 to enhance antibody-dependent cellular cytotoxicity (ADCC) by NK cells and antibody-dependent cellular phagocytosis (ADCP) by macrophages and thus increases the antitumor activity. The combined cancer killing effects by the above mechanisms therefore represent a novel approach in treating CLDN18.2-positive malignancies. Methods: This is an open label, Phase I study to evaluate the safety, tolerability, pharmacokinetics (PK) and preliminary efficacy of PT886 in subjects with unresectable or metastatic gastric adenocarcinoma, gastroesophageal junction adenocarcinoma, and pancreatic ductal adenocarcinoma (PDAC) for which there is no available standard therapy. Approximately 34-58 patients will be enrolled. The study consists of 2 parts: Dose Escalation, and Dose Expansion. The dose escalation study of PT886 will be guided by a standard 3+3 dose escalation study design to determine the maximum tolerated dose (MTD) and/or the optimal biological doses for expansion. The MTD or the optimal biological dose and a lower dose level will be further evaluated in a dose expansion cohort to determine a recommended phase II dose (RP2D). Each enrolled patient will receive PT886 as a monotherapy (0.1, 0.3, 1, 3 or 6 mg/kg QW) as an intravenous infusion continuously (over 60 minutes) in 28-day cycles. The primary endpoints are Dose Limiting Toxicity (DLT) and MTD, if reached, and RP2D of PT886 as a single agent. PD markers of PT886 biological activity will be measured, including T-cell receptor sequencing on circulating T cells, T-cell activation studies, serum cytokines and assessing CLDN18.2 expression in tumor tissues. PT886, which was recently granted orphan drug designation by the FDA for the treatment of pancreatic cancer, has the potential to be a new treatment option for pancreatic and gastric cancer patients whose current standard of care is limited. Preliminary safety and efficacy data are anticipated in the second quarter of 2023. Clinical trial information: NCT02178241 .

Friedreich ataxia (FRDA) is a rare childhood neurodegenerative disorder with no effective treatment. FRDA is caused by transcriptional silencing of the FXN gene and consequent loss of the essential mitochondrial protein frataxin. Based on the knowledge that a GAA•TTC repeat expansion in the first intron of FXN leads to heterochromatin formation and gene silencing, we have shown that members of the 2-aminobenzamide family of histone deacetylase inhibitors (HDACi) reproducibly increase FXN mRNA levels in induced pluripotent stem cell (iPSC)derived FRDA neuronal cells and in peripheral blood mononuclear cells from patients treated with the drug in a phase I clinical trial. How the reduced expression of frataxin leads to neurological and other systemic symptoms in FRDA patients remains unclear. Similarly to other triplet repeat disorders, it is not known why only specific cells types are affected in the disease, primarily the large sensory neurons of the dorsal root ganglia and cardiomyocytes. The combination of iPSC technology and genome editing techniques offers the unique possibility of addressing these questions in a relevant cell model of the disease, without the confounding effect of different genetic backgrounds. We derived a set of isogenic iPSC lines that differ only in the length of the GAA•TTC repeats, using "scarless" gene-editing methods (helper-dependent adenovirus-mediated homologous recombination). To uncover the gene expression signature due to GAA•TTC repeat expansion in FRDA neuronal cells and the effect of HDACi on these changes, we performed transcriptomic analysis of iPSC-derived central nervous system (CNS)and isogenic sensory neurons by RNA sequencing. We find that multiple cellular pathways are commonly affected by the loss of frataxin in CNS and peripheral nervous system neurons and these changes are partially restored by HDACi treatment.