Rational Approach to Optimizing Conformation-Switching Aptamers for Biosensing Applications.

The utilization of structure-switching aptamers (SSAs) has enabled the development of novel sensing platforms for the sensitive and continuous detection of molecules. De novo development of SSAs, however, is complex and laborious. Here we describe a rational approach to SSA optimization that simultaneously improves aptamer binding affinity and introduces target-dependent conformation-switching for compatibility with real-world biosensor applications. Key structural features identified from NMR and computational modeling were used to optimize conformational switching in the presence of target, while large-scale, microarray-based mutation analysis was used to map regions of the aptamer permissive to mutation and identify combinations of mutations with stronger binding affinity. Optimizations were carried out in a relevant biofluid to ensure a seamless transition of the aptamer to a biosensing platform. Initial proof-of-concept for this approach is demonstrated with a cortisol binding aptamer but can easily be translated to other relevant aptamers. Cortisol is a hormone correlated with the stress response that has been associated with various medical conditions and is present at quantifiable levels in accessible biofluids. The ability to continuously track levels of stress in real-time via cortisol monitoring, which can be enabled by the aptamers reported here, is crucial for assessing human health and performance.

Enveloped viruses pseudotyped with mammalian myogenic cell fusogens target skeletal muscle for gene delivery.

Entry of enveloped viruses into cells is mediated by viral fusogenic proteins that drive membrane rearrangements needed for fusion between viral and target membranes. Skeletal muscle development also requires membrane fusion events between progenitor cells to form multinucleated myofibers. Myomaker and Myomerger are muscle-specific cell fusogens but do not structurally or functionally resemble classical viral fusogens. We asked whether the muscle fusogens could functionally substitute for viral fusogens, despite their structural distinctiveness, and fuse viruses to cells. We report that engineering of Myomaker and Myomerger on the membrane of enveloped viruses leads to specific transduction of skeletal muscle. We also demonstrate that locally and systemically injected virions pseudotyped with the muscle fusogens can deliver µDystrophin to skeletal muscle of a mouse model of Duchenne muscular dystrophy and alleviate pathology. Through harnessing the intrinsic properties of myogenic membranes, we establish a platform for delivery of therapeutic material to skeletal muscle.

Enveloped viruses pseudotyped with mammalian myogenic cell fusogens target skeletal muscle for gene delivery.

Entry of enveloped viruses into cells is mediated by fusogenic proteins that form a complex between membranes to drive rearrangements needed for fusion. Skeletal muscle development also requires membrane fusion events between progenitor cells to form multinucleated myofibers. Myomaker and Myomerger are muscle-specific cell fusogens, but do not structurally or functionally resemble classical viral fusogens. We asked if the muscle fusogens could functionally substitute for viral fusogens, despite their structural distinctiveness, and fuse viruses to cells. We report that engineering of Myomaker and Myomerger on the membrane of enveloped viruses leads to specific transduction of skeletal muscle. We also demonstrate that locally and systemically injected virions pseudotyped with the muscle fusogens can deliver micro-Dystrophin (µDys) to skeletal muscle of a mouse model of Duchenne muscular dystrophy. Through harnessing the intrinsic properties of myogenic membranes, we establish a platform for delivery of therapeutic material to skeletal muscle.

Nuclear numbers in syncytial muscle fibers promote size but limit the development of larger myonuclear domains.

Mammalian cells exhibit remarkable diversity in cell size, but the factors that regulate establishment and maintenance of these sizes remain poorly understood. This is especially true for skeletal muscle, comprised of syncytial myofibers that each accrue hundreds of nuclei during development. Here, we directly explore the assumed causal relationship between multinucleation and establishment of normal size through titration of myonuclear numbers during mouse neonatal development. Three independent mouse models, where myonuclear numbers were reduced by 75, 55, or 25%, led to the discovery that myonuclei possess a reserve capacity to support larger functional cytoplasmic volumes in developing myofibers. Surprisingly, the results revealed an inverse relationship between nuclei numbers and reserve capacity. We propose that as myonuclear numbers increase, the range of transcriptional return on a per nuclear basis in myofibers diminishes, which accounts for both the absolute reliance developing myofibers have on nuclear accrual to establish size, and the limits of adaptability in adult skeletal muscle.

Myonuclear content regulates cell size with similar scaling properties in mice and humans.

Muscle fibers are the largest cells in the body, and one of its few syncytia. Individual cell sizes are variable and adaptable, but what governs cell size has been unclear. We find that muscle fibers are DNA scarce compared to other cells, and that the nuclear number (N) adheres to the relationship N = aVb where V is the cytoplasmic volume. N invariably scales sublinearly to V (b < 1), making larger cells even more DNA scarce. N scales linearly to cell surface in adult humans, in adult and developing mice, and in mice with genetically reduced N, but in the latter the relationship eventually fails when they reach adulthood with extremely large myonuclear domains. Another exception is denervation-atrophy where nuclei are not eliminated. In conclusion, scaling exponents are remarkably similar across species, developmental stages and experimental conditions, suggesting an underlying scaling law where DNA-content functions as a limiter of muscle cell size.

Proteasome inhibition preserves longitudinal growth of denervated muscle and prevents neonatal neuromuscular contractures.

Muscle contractures are a prominent and disabling feature of many neuromuscular disorders, including the 2 most common forms of childhood neurologic dysfunction: neonatal brachial plexus injury (NBPI) and cerebral palsy. There are currently no treatment strategies to directly alter the contracture pathology, as the pathogenesis of these contractures is unknown. We previously showed in a mouse model of NBPI that contractures result from impaired longitudinal muscle growth. Current presumed explanations for growth impairment in contractures focus on the dysregulation of muscle stem cells, which differentiate and fuse to existing myofibers during growth, as this process has classically been thought to control muscle growth during the neonatal period. Here, we demonstrate in a mouse model of NBPI that denervation does not prevent myonuclear accretion and that reduction in myonuclear number has no effect on functional muscle length or contracture development, providing definitive evidence that altered myonuclear accretion is not a driver of neuromuscular contractures. In contrast, we observed elevated levels of protein degradation in NBPI muscle, and we demonstrate that contractures can be pharmacologically prevented with the proteasome inhibitor bortezomib. These studies provide what we believe is the first strategy to prevent neuromuscular contractures by correcting the underlying deficit in longitudinal muscle growth.

Myonuclear accretion is a determinant of exercise-induced remodeling in skeletal muscle.

Skeletal muscle adapts to external stimuli such as increased work. Muscle progenitors (MPs) control muscle repair due to severe damage, but the role of MP fusion and associated myonuclear accretion during exercise are unclear. While we previously demonstrated that MP fusion is required for growth using a supra-physiological model (Goh and Millay, 2017), questions remained about the need for myonuclear accrual during muscle adaptation in a physiological setting. Here, we developed an 8 week high-intensity interval training (HIIT) protocol and assessed the importance of MP fusion. In 8 month-old mice, HIIT led to progressive myonuclear accretion throughout the protocol, and functional muscle hypertrophy. Abrogation of MP fusion at the onset of HIIT resulted in exercise intolerance and fibrosis. In contrast, ablation of MP fusion 4 weeks into HIIT, preserved exercise tolerance but attenuated hypertrophy. We conclude that myonuclear accretion is required for different facets of exercise-induced adaptive responses, impacting both muscle repair and hypertrophic growth.