Gene-based editing can potentially correct the genetic defect in methylmalonic acidemia (MMA). SUNRISE, a first-in-human phase 1/2 open-label study, evaluated the safety/tolerability (primary endpoints) of liver-targeted hLB-001 in four pediatric participants (ages 20-114 months) with mitochondrial methylmalonyl-CoA mutase (MMUT)-deficient MMA. We designed a single-infusion adeno-associated viral capsid (hLB-001) to nondisruptively integrate functional MMUT at the 3 ' end of the albumin (ALB) locus to produce both albumin and MMUT. All four participants experienced at least one treatment-emergent adverse event. Three participants had treatment-emergent serious adverse events of cytokine release syndrome (one participant) and thrombotic microangiopathy (two participants); all resolved during the trial. Biologic activity, clinical efficacy, and 1-year survival were secondary endpoints. MMUT expression (measured by 2A-tagged ALB biomarker expression) increased in two participants over two years, confirming homology-based integration and positive selection of transgenic cells. However, serum methylmalonic acid (sMMA), serum FGF21, serum methylcitric acid (sMCA), and propionate oxidation remained abnormal in all four participants. All participants were alive at 1 year and at database lock. SUNRISE was terminated due to lack of efficacy. These results provide proof-of-concept for use of liver-targeted gene editing without nucleases for MMA and other genetic metabolic disorders. ClinicalTrials.gov identifier: NCT04581785Target journal: Gene Therapy (Springer Nature)

tRNA-derived small RNAs (tsRNAs) are generated by specific cleavage of transfer RNAs that are essential for delivering amino acids during protein synthesis. They have emerged as important regulators of gene expression across diverse biological contexts and have been implicated in multiple diseases, including cancers and neurological disorders. Advances in next-generation sequencing (NGS) have enabled high-throughput profiling of tsRNA abundance; however, extensive base modifications and heterogeneous termini frequently lead to inaccurate outcomes in small RNA sequencing. These technical limitations highlight the need for reliable validation strategies. Here, we present two complementary methods for tsRNA validation: small RNA northern blotting, which allows direct visualization of RNA size and abundance, and dumbbell PCR, which enables isoform-specific quantification with single-nucleotide resolution. Together, these approaches provide robust tools to validate tsRNA expression and facilitate mechanistic studies of their biological functions.

Recombinant adeno-associated virus (rAAV) vectors and CRISPR-Cas9 are widely used in gene therapy. However, how endogenous DNA secondary structures may potentially affect genome editing outcomes is not fully understood. RNA/DNA hybrids (R-loops), which form mostly during transcription, are dynamically regulated in cells and have been implicated in influencing DNA repair pathway choice. Here, we investigated whether genomic R-loops are associated with differences in Cas9-mediated genome editing outcomes in vitro and in vivo. By targeting regions with relatively low or high R-loop levels within the murine albumin (Alb) and actin (Actb) loci, we observed comparable insertion/deletion (indel) frequencies across sites with different R-loop abundance. In contrast, homology-directed repair (HDR) efficiency appeared reduced at R-loop-enriched sites in proliferating hepatocyte-derived cells (HEPA1-6) but not in quiescent hepatocytes in vivo. Manipulations resulting in reducing R-loop levels, including RNaseH1 overexpression or pharmacological induction of G1 arrest were associated with increased HDR at these genomic sites. In addition, T cell activation correlated with elevated R-loop accumulation suggesting they might influence ex vivo genome editing. Together, these observations suggest that endogenous R-loop levels might influence HDR efficiency during Cas9-mediated editing and is a parameter to consider when designing genome editing strategies.

BACKGROUND & AIMS:Extensive research has shown that the microRNA-122 (miR-122) plays an important role in liver homeostasis, and its dysregulation has been implicated in various liver pathologies such as liver cancer and non-alcoholic fatty liver disease. The primary transcript for miR-122, long non-coding RNA 122 (lnc122) has traditionally only been considered an intermediate product for mature miR-122. METHODS:We used CRISPRi to inhibit lnc122 RNA expression in hepatoma cells and mouse livers with and without expression of mature miR-122 RNA to determine if a lnc122 RNA has a separate function. These models were used in combination of RNA-seq, Gene Set Enrichment Analysis, RNA pulldown-mass spectrometry, and a CRSIRPi/CRISPRa mediated liver tumor model to establish the specific role of lnc122 in hepatic homeostasis. RESULTS:We demonstrate that lnc122 RNA has a distinct tumor suppressive role that is separate from the antiproliferative function of miR-122. Specifically, lnc122 RNA promotes MYC protein degradation by stabilizing the MYC-UBR5 E3 ubiquitin ligase protein complex. Unlike other E3 ubiquitin ligases for MYC, UBR5 is co-amplified with MYC in >40% of human cancers because of their close location on the human genome and lnc122 is indispensable for efficient MYC degradation by UBR5. Paired patient liver and tumor samples showed decreased concentrations of lnc122 RNA in tumors. Furthermore, reducing the lnc122 RNA transcripts in mouse liver exacerbated MYC-driven liver tumorigenesis. Although lnc122 expression is restricted to the liver, exogenous expression of lnc122 in non-hepatic transformed cells also destabilized MYC protein. CONCLUSIONS:Our findings indicate that lnc122 RNA has a synergistic role with miR-122 in the surveillance and prevention of liver tumorigenesis and may represent a new target for treating MYC-driven human cancers. IMPACT AND IMPLICATIONS:The study demonstrates that lnc122 RNA not only is a precursor for miR-122, but also functions in forming a ubiquitination complex via UBR5, and responsible for limiting the half-life of MYC protein. Loss of lnc122 RNA expression promotes liver cancer perhaps categorizing it as tumor suppressor gene. Further studies to develop agents to enhance the activity of this complex may offer a new therapeutic approach to treat hepatocellular carcinoma.

Site-directed RNA editing is a promising and potentially safer alternative to genome editing. Previous methods have been developed that recruit the endogenously and ubiquitously expressed ADAR enzymes to initiate site-specific A-to-I edits, but often suffer from low efficacy or dependency on viral delivery. Chemically modified oligonucleotides may be a promising alternative, but the approach still lacks systematic in-depth studies. Furthermore, the best characterized platform uses stereo-pure backbone chemistry, which is not widely used, commercially unavailable and challenging to manufacture. Here, we report on single-stranded oligonucleotides of 30-60 nt length, which are fully chemically stabilized by applying commercially available, classical RNA drug modifications, like 2´-O-methyl, 2´-fluoro, and DNA on a stereo-random phosphate/phosphorothioate backbone. We demonstrate our so-called RESTORE 2.0 oligonucleotides to induce the correction of pathogenic point mutations, efficacy after GalNAc-mediated uptake into human primary hepatocytes, and proof of in-vivo efficacy in mice upon lipid nanoparticle-mediated delivery. The discovered design principles may increase the accessibility of site-directed RNA base editing to expand and support further research in this field.

INTRODUCTION:Gene editing therapies offer the possibility of substantial improvement in treatment and quality of life for people with haemophilia (PWH) in a landscape of dynamic therapeutic advancement. Developing a common and understandable language to discuss gene editing will be essential to ensure these treatments can be deployed in a safe and effective manner with fully informed and shared decision-making between healthcare professionals (HCPs) and PWH. A lexicon explaining and clarifying key concepts is one potential tool to address these aims. Here we evaluate how a gene editing lexicon could be deployed to maximise impact and improve patient outcomes. AIM:To operationalise the gene editing lexicon for successful adoption by the haemophilia community. METHODS:Through an innovative, iterative process, representatives from the haemophilia community, including multidisciplinary HCPs, PWH, and caregivers, with support from language strategy experts, developed a gene editing lexicon and evaluated operational aspects for real-world adoption of this resource. RESULTS:A gene editing lexicon was developed, including infographics illustrating key concepts. Infographics were adapted from the lexicon to further clarify and communicate these concepts. Infographics were found to be a potentially vital tool for enhancing the practical use of the lexicon to promote shared decision-making and attain informed consent for gene editing therapies. CONCLUSION:A gene editing lexicon shows promise for improving the understanding of gene editing for all stakeholders in the haemophilia community. Ensuring the lexicon remains up to date with current therapies and appropriate strategies for adoption such as infographics will enable this resource to have maximum impact.

Transfer RNAs (tRNAs) are short non-coding RNA molecules that play a crucial role in protein synthesis by carrying amino acids to ribosomes during translation. tRNAs are highly conserved and abundant across species, with each type categorized based on its anticodon sequence. Although traditionally viewed as essential for protein synthesis, tRNAs have been found to have additional roles in cell proliferation, tumor metastasis, and neuronal homeostasis. In addition, tRNAs are cleaved by ribonucleases to produce smaller fragments. These fragments have previously been referred to as tRNA fragments (tRF RNAs) or tRNA-derived small RNAs (tsRNAs). More recently a nomenclature has been but forward for all tRNA derived RNAs referred to as tDRs. We will use tsRNA and tDR interterchangeably. The tDRs are processed at specific sites in tRNAs and can be differentially expressed in various tissues and diseases, indicating their potential as unique non-coding RNAs with specific functions. In a previous study, we identified a 3'tDR, which can regulate the translation of a target mRNA by altering its secondary structure. This chapter provides a detailed protocol to analyze the tDR-mediated translational regulation based on several molecular methods.

Gene therapy has revolutionized modern medicine by offering innovative treatments for genetic and acquired diseases. The liver has been and continues as a prime target for in vivo gene therapy due to its essential biological functions, vascular access to the major target cell (hepatocytes), and relatively immunotolerant environment. Adeno-associated virus (AAV) vectors have become the cornerstone of liver-directed therapies, demonstrating remarkable success in conditions such as hemophilia A and B, with US Food and Drug Administration (FDA)-approved therapies like etranacogene dezaparvovec, Beqvez, and Roctavian marking milestones in the field. Despite these advances, challenges persist, including vector immunogenicity, species-specific barriers, and high manufacturing costs. Innovative strategies, such as capsid engineering, immune modulation, and novel delivery systems, are continuing to address these issues in expanding the scope of therapeutic applications. Some of the challenges with many new therapies result in the discordance between preclinical success and translation into humans. The advent of various genome-editing tools to repair genomic mutations or insert therapeutic DNAs into precise locations in the genome further enhances the potential for a single-dose medicine that will offer durable life-long therapeutic treatments. As advancements accelerate, liver-targeted gene therapy is poised to continue to transform the treatment landscape for both genetic and acquired disorders, for which unmet challenges remain.

Background: Clustered regularly interspaced short palindromic repeats (CRISPR)associated protein 9 (Cas9)-based targeted gene editing platforms are being developed to treat genetic diseases like hemophilia. Such novel therapy involves complex concepts and terminology that require aligned language to engage key stakeholders in the hemophilia community. Thus, a globally aligned gene editing lexicon-a consistent language to communicate the fundamentals of gene editing in hemophilia, designed to be credible and accessible for people with hemophilia and caregivers while avoiding unnecessary complexity-is required to address this need. Objectives: To establish an aligned language and communications framework that facilitates informed consent and shared decision-making regarding gene editing and treatment considerations in hemophilia. Methods: Through an innovative partnership with global experts in hemophilia, gene editing, and biotechnology, initial insights were gathered via interviews, workshops, and analysis of existing language within the hemophilia community. Qualitative research involving lived experience experts (people with hemophilia and caregivers; n = 43) and hematologists (n = 24) informed the lexicon development, which was further validated by a steering committee of global experts in the hemophilia and gene editing fields. Finally, optimized language recommendations were developed for a clear, consistent gene editing lexicon. Results: Key themes included insights into audience mindsets, guiding language principles, and optimized terminology for key topics like gene editing concepts and post-treatment considerations. Audience mindsets revealed cautious optimism around gene therapy, with more skepticism around gene editing. Guiding language principles indicated a preference for plainspoken over technical language, definitions that link to patient benefits, and explanations that highlight the precise nature of gene editing. Conclusion: This collaborative approach ensures broad adoption of the lexicon within the hemophilia community and readiness for beta testing.

We investigated long-term human coagulation factor IX (huFIX) expression of a novel variant when delivered into mice and rhesus macaques and compared transduction ef fi ciencies using two different adeno-associated virus (AAV) capsids. In hemophilic mice injected with KP1-packaged recombinant AAV (rAAV) expressing the hyperactive FIX variant speci fi c activity plasma levels were 10-fold or 2-fold enhanced when compared with wild-type or Padua huFIX injected mice, respectively. In rhesus macaques AAV-LK03 capsid outperformed AAV-KP1 in terms of antigen expression and liver transduction. Two animals from each group showed sustained low-level huFIX expression at 3 months after administration, while one animal from each group lost huFIX mRNA and protein expression over time, despite comparable vector copies. We investigated whether epigenetic differences in the vector episomes could explain this loss of transcription. Cut&Tag analysis revealed lower levels of activating histone marks in the two animals that lost expression. When comparing rAAV genome associated histone modi fi cations in rhesus macaques with those in mice injected with the same vector, the activating histone marks were starkly decreased in macaque-derived episomes. Differential epigenetic marking of AAV genomes may explain different expression pro fi les in mice and rhesus macaques, as well as the wide dose response variation observed in primates in both preclinical and human clinical trials.

Recombinant adeno-associated viral vectors (rAAV) hold an intrinsic ability to stimulate homologous recombination (AAV-HR) and are the most used in clinical settings forin vivogene therapy. However, rAAVs also integrate throughout the genome. Here, we describe DNA-RNA immunoprecipitation sequencing (DRIP-seq) in murine HEPA1-6 hepatoma cells and whole murine liver to establish the similarities and differences in genomic R-loop formation in a transformed cell line and intact tissue. We show enhanced AAV-HR in mice upon genetic and pharmacological upregulation of R-loops. Selecting the highly expressedAlbumingene as a model locus for genome editing in bothin vitroandin vivoexperiments showed that the R-loop prone, 3’ end ofAlbuminwas efficiently edited by AAV-HR, whereas the upstream R-loop- deficient region did not result in detectable vector integration. In addition, we found a positive correlation between previously reported off-target rAAV integration sites and R-loop enriched genomic regions. Thus, we conclude that high levels of R-loops, present in highly transcribed genes, promote rAAV vector genome integration. These findings may shed light on potential mechanisms for improving the safety and efficacy of genome editing by modulating R-loops and may enhance our ability to predict regions most susceptible to off-target insertional mutagenesis by rAAV vectors.

INTRODUCTION:Despite the progress in gene editing platforms like CRISPR/Cas9 with the potential to transform the standard of care for haemophilia, the language used to explain and discuss gene editing is not aligned across the haemophilia community. Here, we present the objective and rationale for developing a clear, consistent, and globally aligned gene editing lexicon to address these communication gaps. METHODS:Effectively communicating complex gene editing concepts requires a clear and consistent vocabulary. Through collaboration with a diversity of haemophilia stakeholders, our main goal is to develop an accurate, informative lexicon which avoids overpromising or highly technical terminology. Using an innovative process, representatives from several patient and scientific haemophilia organizations and select biotechnology companies will develop and refine language concepts to be tested with approximately seventy participants across the United States of America, United Kingdom, and Germany. Participants will include lived experience experts (LEEs) and haematologists. The process will be overseen by the Lexicon Steering Committee of global experts from leading scientific and patient organizations in the haemophilia and gene editing fields. RESULTS:Initial feedback provided a robust foundation and rationale for building clear, consistent language around gene editing. This lexicon development framework will allow for increased understanding across the haemophilia community, including the development of valid informed consent and shared decision-making materials. CONCLUSION:Results provide important building blocks for stimuli development and highlight the need for a novel gene editing lexicon. In the next phase, language stimuli will be tested with LEEs and haematologists to better understand audience preferences and help shape the final lexicon.

Background: The experience in pediatric vascular diseases is limited in the United Kingdom and worldwide due to their rarity and variations in practice. We looked at types of cases presenting to a dedicated pediatric vascular clinic. Methods: Medical records of children seen in a dedicated pediatric vascular clinic at a tertiary referral service between 2016 and 2022 were reviewed. These patients were either seen for the first time in that clinic or had their appointments as a follow-up after inpatient review or intervention while being under the care of pediatric teams in local hospitals. Results: Fifty-five patients (34 males) were seen aged between 4 months and 17 years (mean 9.5 years). Common presentations were limb length discrepancy secondary to iatrogenic arterial occlusion, follow-up after bypass for trauma, lower limb swelling or discoloration, and varicose veins. Operative procedures included lower limb bypass, angioplasty, ligation of aneurysms, and varicose vein surgery. Conclusions: Pediatric vascular conditions are uncommon and therefore most vascular surgeons and trainees will have little exposure to such cases. Intervention is needed for arterial injury secondary to penetrating or iatrogenic trauma. A national registry is required for these rare cases to gain prospective data that can help build up more evidence for educational purposes and to establish guidelines.