Gene Therapy Laboratory

Excessive RNA damage activates cellular stress responses, triggering cell death. However, pathways that negatively regulate RNA damage responses are largely uncharacterized. Using genetic screens, we find that the ubiquitin ligase RNF25 provides tolerance to RNA damage caused by the nucleoside analogue azacytidine, a chemotherapeutic agent used to treat acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Mechanistically, we show that azacytidine is incorporated into mRNA, where it causes lesions that stall elongating ribosomes, leading to cytotoxic activation of the GCN2-dependent integrated stress response (ISR). Furthermore, we establish that RNF25 prevents ISR hyperactivation by ubiquitylation of ribosomal protein eS31, thereby suppressing cell death upon azacytidine treatment. Our study reveals an mRNA damage tolerance mechanism that determines cellular survival in response to azacytidine, highlighting RNA damage-induced stress response as a potentially critical component of chemosensitivity in AML and MDS.

Background Declining fertility rates are a globally documented problem, affecting even regions that have historically high fertility rates. In the Asia Pacific (APAC) region, most countries and territories are seeing birth rates drop far below the replacement rate of 2.1 children per woman. Policymakers in these countries have switched from a historic focus on population control policies to investing in pronatalist policies that aim to boost the national fertility rate by tackling the socioeconomic drivers of falling fertility. In this paper, we examine policies affecting the often-overlooked medical aspect of this challenge: infertility. Objective To review challenges in fertility policy and propose solutions to improve equitable access to assisted reproductive technologies (ART) and associated services. Methods A review of literature was conducted on fertility policy challenges and developments in the APAC region, covering fertility recognition and awareness; egg freezing; and access to ART treatment, psychosocial care, and supplementary care. Experts from nine different countries and territories were invited to validate secondary research findings and provide their perspectives on policy implications. The experts participated in individual 60-minute interviews, then a half-day Policy Forum discussion was held at the Asia-Pacific Assisted Conception Congress 2024, held in Kuala Lumpur, Malaysia. Results Whilst several countries in the region have long recognised falling total fertility rates (TFR) as a cause for concern, many have been slow to recognise infertility as a disease, resulting in limited policy development to support patients experiencing infertility. Commonly this presents as lack of funding allocated towards fertility services, limited or no reimbursement of fertility treatment or egg freezing, and poor societal awareness of infertility and hence delays in seeking care. Various policy changes can be adopted to improve care for patients across the APAC region. Conclusion Our review identified multiple gaps in policy development for supporting individuals experiencing infertility. Looking to countries in which the TFR dropped below replacement level many years ago, the importance of timely policy intervention before negative demographic consequences occur is critical.

The Deep Maniots, an isolated population at the southernmost tip of mainland Greece, have drawn scholarly interest for their unique dialect, culture, and patrilineal clan structure. Geographically shielded by the Mani Peninsula, they are thought to have been minimally affected by 6th-century CE migrations that transformed Balkan demography. To investigate their genetic origins, we analysed Y-DNA and mtDNA from 102 Deep Maniots using next-generation sequencing. Paternally, Deep Maniots exhibit an exceptional prevalence (~80%) of West Asian haplogroup J-M172 (J2a), with subclade J-L930 accounting for ~50% of lineages. We identify Bronze Age Greek ancestry in Y-haplogroups nearly absent elsewhere, highlighting their longstanding genetic isolation. The absence of northeast European-related paternal lineages, common in other mainlandGreeks, suggests preservation of southern Greece's pre-Medieval genetic landscape. Y-haplogroup phylogeny reveals strong founder effects dated to ~380-670 CE, while the emergence of clan-based social structure is estimated around 1350 CE, centuries earlier than previously thought. In contrast, maternal lineages display greater heterogeneity, primarily originating from ancient Balkan, Levantine, and West Eurasian sources. These results align with historical and anthropological accounts, showcasing Deep Maniots as a genetic snapshot of pre-Medieval southern Greece, offering new perspectives on population continuity and mobility in the Late Antique eastern Mediterranean.

Small interfering RNA (siRNA)-loaded lipid nanoparticles (LNPs) are a promising modality for gene silencing therapies. Pulmonary delivery offers an attractive, non-invasive route to target respiratory diseases. However, the development of stable dry powder formulations suitable for inhalation remains a key challenge. In this study, we investigated the impact of spray drying on the physicochemical integrity and biological performance of siRNA-LNPs. Four LNP formulations differing in PEG-lipid helper lipid content were subjected to spray drying in the presence of a lactose matrix. The impact of formulation parameters on physicochemical integrity, colloidal stability, structural preservation, and biological behaviour was systematically evaluated before and after spray drying and predicted by molecular dynamic simulations. Choosing this holistic approach demonstrates that LNP composition critically influences suitability for spray drying and provides key insights for the development of stable pulmonary siRNA therapies.

Abstract Background Perianal Crohn’s disease (pCD) is a challenging form of Crohn’s disease, which includes both fistulising and non-fistulising features, such as anorectal stricture, skin tags and erosive disease (1). Whilst non-fistulising pCD (nfpCD) can result in a significant disease burden for patients, these features are under-recognised in clinical practice, current guidelines and clinical research relating to pCD (2,3). Informed by a review of existing classification systems, this study aimed to develop a novel classification system to guide identification and management of non-fistulising pCD. Methods Following a systematic review to identify existing classification systems, a modified nominal group technique expert consensus process was used to develop a novel classification system for non-fistulising pCD. This process involved several phases including ideas generation, clarification of ideas, several rounds of voting and patient and public involvement (PPI). Results Twenty-two existing classification systems for non-fistulising pCD were identified, the majority (18/22, 88%) of which focused on categorising non-fistulising pCD. Participating panellists included multidisciplinary experts from the TOpClass consortium, with representation from 10 countries. The initial round of voting established a need for a novel classification system (n = 18 89% [A+], 11% [A]), and passed a number of statements relating to the content of the classification. The final classification structure was passed with 100% consensus (n = 27, 93%,[A+], 7% [A]) (Figure 1). The final statements to accompany the classification were passed with a mean 98% consensus (n = 28, 80% [A+], 18% [A]) (Table 1). The novel structure classifies nfpCD according to the presence of stricturing disease, erosive disease and skin tags with severity ranging from minimal symptoms (Class 1) to severe symptoms requiring defunctioning ostomy (Class 2c), proctectomy (Class 3) or perineal symptoms after proctectomy (Class 4). The structure complements the existing TOpClass classification for fistulising pCD, to enable classification of patients with both fistulising and non-fistulising features of pCD. The classification system incorporates patient goals and symptom burden, and introduces a number of novel concepts, such as velocity and destruction to provide a clinically useful framework. Conclusion This expert consensus has developed a dynamic, comprehensive and patient-focused system for optimising the classification of patients with non-fistulising pCD for clinical and research use. Further validation in prospective cohorts is planned to ensure clinical applicability.

Background: Adeno-associated viruses (AAVs) are widely used vectors for in vivo gene therapy, but their standard storage at −80 °C limits deployment in regions lacking ultracold infrastructure. Strategies enabling stable AAV storage at higher temperatures are needed to support global distribution. Methods: Nine AAV serotypes were lyophilized in simple sucrose-based buffers. Post-lyophilization vector integrity was assessed by measuring in vitro transduction efficiency using a luciferase reporter in cell-based assays. Stability of selected serotypes (AAV2.5 and AAV6) was further evaluated over 8 weeks under varying storage temperatures. Results: Lyophilization in sucrose preserved transduction activity for all tested serotypes (AAV1, AAV2, AAV2.5, AAV3, AAV4, AAV5, AAV6, AAV8, AAV9, and AAVrh10). Notably, AAV2, AAV2.5, and AAV6 exhibited 3- to 6-fold increases in transduction, an effect attributable to the sucrose excipient rather than the lyophilization process itself. Long-term stability of lyophilized vectors varied by serotype, temperature, and vial-seal integrity. AAV6 retained full activity for at least 8 weeks when stored at 4 °C or −20 °C. Conclusions: AAV vectors can be effectively lyophilized in simple sucrose solutions, enabling storage at standard −20 °C freezer temperatures while maintaining functional activity. Optimization of lyophilization buffers and excipients may further extend AAV stability at higher temperatures, improving feasibility for global gene therapy deployment.

The endoplasmic-reticulum (ER) transmembrane protein IRE1 mitigates ER stress through kinase-endoribonuclease and scaffolding activities. Cancer cells often co-opt IRE1 to facilitate growth. An IRE1-RNase inhibitor has entered clinical trials; however, recent work uncovered a significant nonenzymatic IRE1 dependency in cancer. To fully disrupt IRE1, we describe a proteolysis-targeting chimera (G6374) that couples an IRE1-kinase ligand to a compound that binds the ubiquitin Cullin-RING Ligase (CRL) substrate receptor, VHL. G6374 induces a stable, cooperative interaction between IRE1 and VHL, driving K48-linked ubiquitination on two principal lysine residues in the IRE1-kinase domain and inducing proteasomal IRE1 degradation. Cryogenic electron microscopy and mutagenesis studies reveal a 2:2 IRE1:VHL ternary-complex topology and critical interactional features, informing future designs. G6374 blocks growth of IRE1-dependent cancer cells irrespective of their dependency mode, while sparing IRE1-independent cells. We provide a proof-of-concept for VHL-based degradation of an ER-transmembrane protein, advancing strategies to fully disrupt IRE1.