Applied Genetic Technologies (United States)

Human skeletal muscle comprises myofibers formed by fusion of thousands of myoblasts. This process depends on tightly regulated, muscle-specific fusogens, but its genetic control remains poorly understood. Here, we identify CHAMP1 (Chromosome Alignment Maintaining Phosphoprotein 1) as essential for human myoblast fusion in vitro and in vivo. Genomic and protein-interaction assays reveal a noncanonical role for CHAMP1 as a MyoD cofactor that directly activates expression of the key muscle fusogen Myomaker. As established in prior clinical reports, CHAMP1 mutations in patients cause developmental delay, hypotonia, and muscle weakness. Consistently, patient-derived cells show fusion defects that can be fully rescued by restoring Myomaker expression. Structure and function analyses identify C2H2-type zinc-finger motifs on CHAMP1 protein that are both necessary and sufficient for MyoD interaction and Myomaker expression. These findings highlight a cell-autonomous role for CHAMP1 in muscle development and disease and point to therapeutic avenues for treating CHAMP1-related muscle development defects.

SUMMARYThis study, the third in a three‐part series, investigates whether chromosomal instability persists in cultivated peanut. The allotetraploid peanut (Arachis hypogaea; genome type AABB) originated from the hybridization and polyploidization of A. duranensis (AA) and A. ipaënsis (BB). Our first study established that this was an extremely narrow genetic origin, likely from a single hybridization event. This raised a paradox: how did such narrow genetics give rise to the phenotypic diversity seen in cultivated peanut? The second study addressed this, showing that a single neoallotetraploid spontaneously generates striking diversity, and that homoeologous exchanges—abundant in early generations following polyploidy—are a key mechanism in creating this diversity. In contrast to this early‐generation instability, cultivated peanut is generally considered to be genetically stable, presumably due to selection. This third study tests whether residual instability still occurs in modern peanut. From a single plant of the highly selfed ‘genome stock’ of the cultivar ‘Tifrunner’, we advanced lineages through seven generations in a pollinator‐free greenhouse. Among 233 plants, we identified three new large‐scale chromosomal instability events: a large deletion on chromosome B01, associated with reduced pod width and seed weight, and two ABBB compositions involving chromosomes A02/B02 and A05/B05. With these observations in hand, we reinterpreted previously published data from two recombinant inbred populations. Together, these results indicate that at least 1% of pure pedigree A. hypogaea plants exhibit spontaneous large‐scale chromosomal changes—a surprising frequency of instability that likely contributes to peanut's long‐term adaptability and evolution.

Polycystic ovary syndrome (PCOS) is among the most common disorders affecting up to 15% of the menstruating population globally. It is the leading cause of anovulatory infertility and a major risk factor for type 2 diabetes. Elevated testosterone levels are a core endophenotype. Despite that prevalence, the underlying causes remain unknown. PCOS genome-wide association studies (GWAS) have reproducibly mapped a number of susceptibility loci, including one encompassing a gene regulating androgen biosynthesis, DENND1A. Identifying the causal variants within these loci will provide fundamental insight into the precise biological pathways that are disrupted in PCOS. Here, we report the discovery of gene regulatory mechanisms that help explain genetic association with PCOS in the GATA4, FSHB and DENND1A loci using a combination of high throughput reporter assays, CRISPR-based epigenome editing, and genetic association analysis from PCOS case and control populations. In addition, we find that increasing endogenous DENND1A expression causes elevated testosterone levels in an adrenal cell model, specifically by perturbing candidate regulatory elements. These results further highlight the potential for combining genetic variant analyses with experimental approaches to fine map genetic associations with disease risk.

Glycogen storage disease type IIIa (GSD IIIa) affects multiple tissues, including liver, heart, and skeletal muscles. We recently reported that an adeno-associated virus serotype 9 vector expressing pullulanase, a bacterial glycogen debranching enzyme, driven by an immunotolerizing dual promoter (AAV9-Dual-Pull), effectively decreased pullulanase-induced cytotoxic T lymphocyte response and corrected disease abnormalities in all major affected tissues in GSD IIIa mice. To reduce effective vector dosages for transgene delivery to skeletal muscles, we packaged the AAV-Dual-Pull vector into two muscle-tropic MyoAAV capsids, MyoAAV4A and MyoAAV4E. Six weeks after administration of the same dose vector (1 × 1013 vg/kg), both the MyoAAV vectors demonstrated remarkably greater transduction efficiency and glycogen clearance efficacy in the cardiac and skeletal muscles than the AAV9 vector, accompanied by the improvement of muscle function, reversal of liver abnormalities, and normalization of the disease biomarker, Glc4 in the urine. Furthermore, treatment with the MyoAAV4A-Dual-Pull vector at a 10-fold lower dose (1×1012 vg/kg) achieved significantly better therapeutic outcomes in the skeletal muscles than the AAV9-Dual-Pull vector at a high dose (1×1013 vg/kg). Validation in human liver chimeric mice revealed that the MyoAAV vectors and the AAV9 vectors had a similar efficiency in transducing human hepatocytes, indicating increased translatability for clinical applications.

Abstract Commercial potato in North America is a clonal autotetraploid crop, which complicates breeding. Efforts are underway to convert potato to a diploid inbred-hybrid crop that is amenable to additional breeding strategies and allows breeders to more quickly respond to demands for crop improvement. With the goal of preserving haplotypes developed over the last 200 years of selection, diploid potato breeding in the US started with the creation of diploids from tetraploid commercial varieties and advanced breeding lines through prickle pollination. This is an effective but slow method which presents a barrier to entry for individual breeding programs. Therefore, we developed 97 publicly available dihaploids (diploids from prickle pollination of tetraploids) as a resource for diploid breeding in the U.S. These clones contain the majority of alleles in the US breeding population for three market classes: chips, russets, and fresh market reds. To facilitate genomic informed breeding, all clones have been resequenced using short read sequencing technology, and we have developed de novo assemblies based on PacBio HiFi long reads for 20 individuals. As an illustration of how these data will be used in breeding, we explored the maturity locus (StCDF1) and identified 15 different alleles. The majority of dihaploids were heterozygous for early and late alleles, resulting in intermediate maturity. Beyond informing breeding, this data facilitates investigations into potato genomics. The dihaploid population is both highly heterozygous and incredibly diverse on a population level. In particular, there is extensive structural diversity, including copy number variation, segregating within the population. This contrasts with a relatively low genome-wide historical recombination rate (ρ). Taken together, these findings indicate that potato is highly diverse, with much of that diversity found within long linkage blocks.

SUMMARYSolanum verrucosum Schlechtendal (2x = 2n = 24) is unique among the clade 4 Solanum Sect Petota species. In addition to being one of the only fully self‐compatible diploid potato species, S. verrucosum is the only clade 4 species that lacks prezygotic interspecific reproductive barriers. This allows S. verrucosum to accept pollen from a broad range of Solanum species and thereby serving as a genetic “bridge” between the cultivated or primary potato gene pool and distantly related wild relatives in the tertiary gene pool. The genetic mechanisms underlying self‐compatibility in Solanum often underpin interspecific compatibility interactions, which in S. verrucosum, has been attributed to the lack of S‐RNase expression. Using an interspecific F2 mapping population (n = 150), we investigated the genetic mechanisms responsible for the lack of interspecific reproductive barriers in S. verrucosum. This F2 population was evaluated for the ability to accept pollen from two clade 1, 1 EBN species (S. pinnatisectum and S. tarnii); from which two QTL for interspecific compatibility were identified on chromosomes 1 and 11, explaining 56.6% of the phenotypic variation observed. To identify the genetic basis of interspecific compatibility, we generated a chromosome‐scale genome assembly of S. verrucosum MSII1813‐2 and performed gene expression profiling of reproductive organs. Differential gene expression of S‐RNase, located within the chromosome 1 QTL, confirmed the central role of the S‐locus and specifically, S‐RNase, in interspecific compatibility. Discovery of a non‐S‐locus QTL is consistent with previous findings that other non‐S‐locus factors are necessary for interspecific compatibility in S. verrucosum.

Monoterpene indole alkaloids (MIAs) are a large, structurally diverse class of bioactive natural products. These compounds are biosynthetically derived from a stereoselective Pictet-Spengler condensation that generates a tetrahydro-β-carboline scaffold characterized by a 3S stereocenter. However, a subset of MIAs contains a non-canonical 3R stereocenter. Herein, we report the basis for 3R-MIA biosynthesis in Mitragyna speciosa (Kratom). We discover the presence of the iminium species, 20S-3-dehydrocorynantheidine, which led us to hypothesize that isomerization of 3S to 3R occurs by oxidation and stereoselective reduction downstream of the initial Pictet-Spengler condensation. Isotopologue feeding experiments implicated young leaves and stems as the sites for pathway biosynthesis, facilitating the identification of an oxidase/reductase pair that catalyzes this epimerization. This enzyme pair has broad substrate specificity, suggesting that the oxidase and reductase may be responsible for the formation of many 3R-MIAs and downstream spirooxindole alkaloids in Kratom. The elucidation of this epimerization mechanism allows biocatalytic access to a range of previously inaccessible pharmacologically active spirooxindole alkaloid compounds.

Iridoids are specialized monoterpenes ancestral to asterid flowering plants1,2 that play key roles in defence and are also essential precursors for pharmacologically important alkaloids3,4. The biosynthesis of all iridoids involves the cyclization of the reactive biosynthetic intermediate 8-oxocitronellyl enol. Here, using a variety of approaches including single-nuclei sequencing, we report the discovery of iridoid cyclases from a phylogenetically broad sample of asterid species that synthesize iridoids. We show that these enzymes catalyse formation of 7S-cis-trans and 7R-cis-cis nepetalactol, the two major iridoid stereoisomers found in plants. Our work uncovers a key missing step in the otherwise well-characterized early iridoid biosynthesis pathway in asterids. This discovery unlocks the possibility to generate previously inaccessible iridoid stereoisomers, which will enable metabolic engineering for the sustainable production of valuable iridoid and iridoid-derived compounds.

Zusammenfassung Die Stargardt-Makuladystrophie (Morbus Stargardt, STGD1; OMIM 248200) ist die häufigste erbliche Makuladystrophie. STGD1 ist eine autosomal rezessive Erkrankung, die durch mehrere pathogene Sequenzvarianten im großen ABCA4-Gen (OMIM 601691) verursacht wird. Bedeutende Fortschritte im Verständnis der klinischen und molekularen Charakteristika sowie der zugrunde liegenden Pathophysiologie haben zu zahlreichen abgeschlossenen, laufenden und geplanten klinischen Studien mit neuartigen Therapien geführt. Ziel dieser kurzen Übersichtsarbeit ist es, 1) die detaillierten phänotypischen und genotypischen Merkmale der Erkrankung, die multimodalen bildgebenden Befunde, den Krankheitsverlauf und die Pathogenese zu beschreiben, 2) die vielfältigen Möglichkeiten der Forschung und therapeutischen Intervention, einschließlich pharmakologischer, zellulärer und anderer Arten von Therapien, zu beschreiben und 3) die neuen therapeutischen Ansätze am translationalen Horizont darzulegen, die darauf abzielen, STGD1 durch den Ersatz des gesamten, 6,8 kb großen, offenen Leserasters von ABCA4 zu behandeln.