TELO-SCOPE study: a randomised, double-blind, placebo-controlled, phase 2 trial of danazol for short telomere related pulmonary fibrosis

IntroductionRecent discoveries have identified shortened telomeres and related mutations in people with pulmonary fibrosis (PF). There is evidence to suggest that androgens, including danazol, may be effective in lengthening telomeres in peripheral blood cells. This study aims to assess the safety and efficacy of danazol in adults and children with PF associated with telomere shortening.Methods and analysisA multi-centre, double-blind, placebo-controlled, randomised trial of danazol will be conducted in subjects aged >5 years with PF associated with age-adjusted telomere length ≤10th centile measured by flow fluorescence in situ hybridisation; or in children, a diagnosis of dyskeratosis congenita. Adult participants will receive danazol 800 mg daily in two divided doses or identical placebo capsules orally for 12 months, in addition to standard of care (including pirfenidone or nintedanib). Paediatric participants will receive danazol 2 mg/kg/day orally in two divided doses or identical placebo for 6 months. If no side effects are encountered, the dose will be escalated to 4 mg/kg/day (maximum 800 mg daily) orally in two divided doses for a further 6 months. The primary outcome is change in absolute telomere length in base pairs, measured using the telomere shortest length assay (TeSLA), at 12 months in the intention to treat population.Ethics and disseminationEthics approval has been granted in Australia by the Metro South Human Research Ethics Committee (HREC/2020/QMS/66385). The study will be conducted and reported according to Standard Protocol Items: Recommendations for Interventional Trials guidelines. Results will be published in peer-reviewed journals and presented at international and national conferences.Trial registration numbersNCT04638517; Australian New Zealand Clinical Trials Registry (ACTRN12620001363976p).

Patient induced pluripotent stem cell-derived hepatostellate organoids establish a basis for liver pathologies in telomeropathies

Patients with dyskeratosis congenita (DC) and related telomeropathies resulting from premature telomere dysfunction suffer from multi-organ failure. In the liver, DC patients present with nodular hyperplasia, steatosis, inflammation, and cirrhosis. We model DC liver pathologies using isogenic human induced pluripotent stem (iPS) cells harboring a causal DC mutation in DKC1, or a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-corrected control allele. Differentiation of these iPS cells into hepatocytes or hepatic stellate cells followed by generation of genotype-admixed hepatostellate organoids revealed a dominant phenotype in the parenchyma, with DC hepatocytes eliciting a pathogenic hyperplastic response in stellate cells independent of stellate cell genotype. Pathogenic phenotypes could be rescued via suppression of AKT activity, a central regulator of MYC-driven hyperplasia downstream of DKC1 mutation. Thus, isogenic iPS-derived admixed hepatostellate organoids offer insight into the liver pathologies in telomeropathies and provide a framework for evaluating emerging therapies.

Genetic Dissection of Dyskeratosis Congenita-Causing Mutations in TINF2 Using Human Pluripotent and Hematopoietic Stem Cell Models

The length of telomeres, which cap the ends of linear chromosomes and provide genomic stability, is tightly regulated in adult stem cells. The telomere reserve in the stem cell population sets the replicative potential of its differentiated progeny. For this reason, abnormally short telomeres in stem cells restrict the number of cell divisions that their differentiated progenies can undergo, eventually resulting in stem cell depletion and tissue failure syndromes. Telomere biology disorders (TBDs) display a broad range of clinical features, age of onset, and severity, which are all correlated with the extent of abnormal telomere shortening. One such early-onset TBD is dyskeratosis congenita (DC), which is a bone marrow predisposition syndrome characterized by a mucocutaneous triad (oral leukoplakia, nail dystrophy, and abnormal skin pigmentation) as well as other conditions driven by premature tissue aging. The leading cause of death in DC patients is bone marrow failure and hematopoietic stem cell transplantation is the only definite intervention to restore hematopoiesis. TINF2, which encodes the TIN2 protein, is mutated in 12% of patients and thereby the second most frequently altered gene in DC cases. TIN2 is a member of the shelterin protein complex bridging the double-strand binding shelterin proteins TRF1/TRF2, and the TPP1/POT1 heterodimer. Such interactions implicate a complex role of TIN2 in telomere length regulation: First, TIN2 stabilizes TRF1, which is a negative regulator of telomere length. Secondly, TPP1, which recruits telomerase, strictly requires TIN2 for telomere elongation and maintenance. TINF2-DC mutations are uniformly heterozygous and localize to a 30 amino acid coding stretch in exon 6 called the 'DC cluster'. TINF2-DC mutations usually arise de novo and result in an earlier disease onset, shorter telomeres, and a more severe manifestation compared to other heterozygous DC-causing mutations in genes such as TR and TERT, which encode the components of the telomerase enzyme. How TINF2-DC mutations cause telomere shortening is unknown. Specifically, whether telomere shortening is caused by reduced telomerase action at telomeres or by degradation of telomeric DNA remains unresolved as studies using different model systems report contrasting results. The discrepancy could be attributed to differences in the model systems used in the studies, highlighting the need for a genetically trackable, primary preclinical human model system. Here, we report the development of two novel endogenous, isogenic model systems to study TINF2-DC mutations. First, we generated human embryonic stem cells (hESCs) engineered to express the TINF2-DC T284R mutation from the endogenous locus, which recapitulated the short telomere phenotype observed in DC patients. Using this model, we identified a gene editing strategy that elongates telomeres in the mutant stem cells and eventually restores replicative potential of the differentiated cells. Next, we used a xenotransplantation model of donor-derived human hematopoietic stem cells (hHSCs) to test the effects of target gene modifications on telomere length and proliferative capacity in vivo. We demonstrate that our models robustly complement each other and offer direct insights into the disease mechanism as well as avenues to potential therapeutic approaches. Figure 1 Figure 1. Disclosures Bertuch: Elixirgen Therapeutics: Consultancy; ImmunityBio: Current equity holder in publicly-traded company; NIH/NCI,: Research Funding; DOD: Research Funding; Hyundai Hope on Wheels: Research Funding.

Zebrafish Models Provide Novel Insights into the Disease Biology of Parn-Mutant Dyskeratosis Congenita and DNAJC21-Mutant Shwachman-Diamond Syndrome

Inherited bone marrow failure syndromes (IBMFS) are a group of rare inherited genetic disorders defined by impaired hematopoiesis and multi- or unilineage cytopenias. Dyskeratosis Congenita (DC) is characterized by pancytopenia, abnormal skin pigmentation and leukoplakia and is caused by mutations in genes involved in telomere biogenesis and/or RNA processing such as TERT, TERC, DKC1 and PARN. Shwachman-Diamond syndrome (SDS) is characterized by neutropenia and exocrine pancreatic insufficiency and occurs as a result of mutations in genes required for ribosome subunit maturation such as SBDS, DNAJC21 and EFL1. How the disruption of such ubiquitous cellular processes leads to distinct cytopenic phenotypes is not fully understood. Further, IBMFS patients have a high risk of developing myelodysplastic syndrome and/or acute myeloid leukemia with a cumulative incidence of 36% by 30 years of age for SDS patients and 13% for DC patients who do not undergo a stem cell transplant. As IBMFS are rare and large numbers of primary human samples are not readily available for mechanistic studies, we employed zebrafish (Danio rerio) given their highly conserved hematopoietic program and ease of genetic manipulation. Using CRISPR-Cas9 genomic editing, we generated deletion mutations predicted to introduce premature stop codons in the zebrafish orthologs, parn and dnajc21, two more recently identified causes of DC and SDS, respectively. Homozygous zygotic parn mutants had normal morphology and were viable to adulthood. However, fertile homozygous adult females were not recoverable in subsequent generations, implicating parn as an essential factor in oocyte specification. In contrast, homozygous dnajc21 mutants showed normal development to adulthood. Using whole mount in situ hybridization (WISH), we found increased expression of hematopoietic precursor markers (erythroid - gata1 and myeloid - lcp1) and concurrently decreased expression of mature hematopoietic markers (erythroid - hbbe3, myeloid - mpx and thrombocyte - CD41) in parn mutants at 24- and 48-hours post-fertilization (hpf). Further, o-dianisidine staining showed reduced hemoglobinized erythrocytes at 48 hpf. These data indicate that multilineage embryonic hematopoiesis is compromised in parn mutants, recapitulating the pancytopenia observed in patients with DC. WISH for mpx in dnajc21 mutants revealed reduced expression at 24 and 48 hpf, recapitulating the neutropenia seen in SDS. Activation of the TP53 tumor suppressor pathway has been suggested to mediate marrow failure and leukemic progression in some types of IBMFS. Using quantitative PCR to measure the expression of tp53 and its downstream effector p21 at 48 hpf, we observed no significant changes in parn mutants but significantly upregulated tp53 expression in dnajc21 mutants. To further study the role of tp53 in dnajc21-mutant SDS, we crossed dnajc21 mutants with a zebrafish line carrying a tp53 R217H point mutation that confers anti-apoptotic phenotypes. Heterozygous tp53 loss in a dnajc21-/- or +/- background resulted in reduced overall larval growth and abnormal yolk sac development, suggesting defective lipid metabolism. Using the lipophilic dye, Oil Red O, we observed reduced lipid distribution in the vasculature and caudal hematopoietic tissue region (equivalent to mammalian fetal liver) of dnajc21 mutants at 48 hpf. In summary, these findings support a role for PARN in hematopoietic lineage specification through mechanisms that are predominantly TP53-independent. By contrast, DNAJC21 is required for neutrophil specification and normal lipid metabolism and may function in a TP53-dependent manner. These zebrafish models provide new insights into the unique biology underlying these IBMFS and can serve as an in vivo platform for identifying therapeutic compounds that restore normal hematopoiesis and prevent leukemic transformation. Disclosures Dror: Alexion Canada: Other: Received funding for a Marrow Failure and Myelodysplasia conference that I organized April 2021; RepaetDiagnostic Laboratory: Other: Received funding for a Marrow Failure and Myelodysplasia conference that I organized April 2021. Berman: Oxford Immune Algorithmics: Membership on an entity's Board of Directors or advisory committees.

Dyskeratosis congenita: rare case report of Syria

ABSTRACT Dyskeratosis congenita (DC) is an inherited disease characterized by the triad of abnormal skin pigmentation, nail dystrophy and mucosal leukoplakia. Non-cutaneous abnormalities (dental, gastrointestinal, genitourinary, neurological, ophthalmic, pulmonary and skeletal) have also been reported. Bone marrow failure (BMF) is the main cause of early mortality, with an additional predisposition to malignancy. DC results from an anomalous progressive shortening of telomeres resulting in DNA replication problems inducing replicative senescence. Men are more affected than women are and X-linked recessive, autosomal dominant and autosomal recessive forms of the disease are recognized. There are no targeted therapies for DC. Patients treated with androgens had a hematological response. We herein describe case of a 32-year-old man, presented with several characteristic systemic features of this condition, including the classic triad of lesions, dysplastic bone marrow, epiphora and liver cirrhosis with grade I esophageal varices. Therefore, a prophylactic propranolol was started in additional to danazol. Three-week later, the patient had subsequent increases in his platelet, red cell and white cell counts.