Mark A. Kay

MYC is among the most frequently amplified oncogenes in human cancer, yet its intrinsically disordered nature has rendered it refractory to direct pharmacological targeting. Here, we demonstrate that the long noncoding RNA NEAT1_1 functions as a molecular gatekeeper that controls MYC protein stability by regulating its access to the E3 ubiquitin ligase UBR5. We show that NEAT1_1 forms a ribonucleoprotein (RNP) complex with both MYC and UBR5, and that NEAT1_1 suppression disrupts this interaction, leading to decreased MYC polyubiquitination and enhanced protein stability. To systematically map the regulatory architecture of NEAT1_1 without altering its abundance, we screened 88 antisense oligonucleotide (ASO) mixmers tiling across the transcript. Strikingly, this phenotypic screen identified region-specific mixmers that elicit bidirectional control over MYC, either driving robust MYC destabilization or promoting its stabilization in a strictly expression-independent manner. Mechanistically, MYC-destabilizing mixmers selectively enhance the interaction between MYC and UBR5 to accelerate proteasomal degradation. A focused CRISPR screen targeting the ubiquitin–proteasome pathway independently validated UBR5 as the essential E3 ligase mediating this mixmer-induced turnover. In vivo, our lead mixmer ASO_#24 significantly suppressed tumor growth in MYC-high xenografts, whereas MYC-low tumors remained unaffected. Our findings reveal that the functional output of a scaffold lncRNA is encoded in its sub-domain architecture rather than bulk transcript levels, establishing domain-specific RNA targeting as a tractable strategy to rewire protein turnover pathways for otherwise undruggable oncogenes.

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: NCT04581785 Target journal : Gene Therapy (Springer Nature)

The variation in AAV vector mediated transduction between species makes it difficult to predict and translate from preclinical animal studies to clinical trials. We previously demonstrated that a 265G insertion (AAV-AM) into the AAV-LK03 primate selective capsid allows robust transduction between species. Here we demonstrate that the capsid species specificity cannot be complemented in trans and hence the transduction properties are cis mediated. We found that the 265G is in a surface-exposed region of AAV virion, facilitating its accessibility for binding to the universal AAVR receptor. We further demonstrated that human AAVR (hAAVR) supplementation rescued the low murine transduction of AAV-LK03 in vitro and in vivo. Sequence swap experiments demonstrated the four amino acid variation of mouse vs human PKD2 domain was important for the varied species tropism of AAV-LK03, with I426V having the greatest effect. Our findings imply that transduction efficiencies between various AAV capsids are substantially influenced by sequence variation in the AAVR protein observed between species. This may facilitate better approaches for translating preclinical to clinical application of AAV-based gene therapeutics.

Unbiased screening of patients seen in nephrology clinics indicate that ~ 10% of patients have a monogenetic cause of kidney disease. For these patients and possibly for the broader diaspora of patients living with chronic kidney disease, gene therapy may be efficacious and possibly curative. A major limitation of gene therapy to the kidney parenchyma thus far, has been seemingly poor delivery of cargo to cell types of interest, utilizing existing adeno-associated viral capsids (e.g. AAV9). Novel AAV capsid serotypes may enhance transduction efficiency, but compared to FDA-approved capsids for extrarenal diseases, safety and tolerability data in humans is unknown. We systematically varied promoter, cargo, genome configuration, enzymatic priming, capsid serotype, dose, and route of administration, and show that optimizing these parameters yields some of the highest kidney transduction efficiencies reported in mice—40 to 60% of kidney tubules transduced with systemic AAV9-Cbh-mCherry-WPRE—by multiple unbiased quantification methods, including a novel machine-learning-based image analysis tool. We also demonstrate AAV transduction in live human kidney using ex vivo normothermic perfusion. These data provide a roadmap for ongoing preclinical studies to enable translation of AAV-mediated kidney gene therapy into the clinic.

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.