Biology subjects

Pfaff, S. L.

Publications and source records attributed to Pfaff, S. L..

4 recordsLinked to original sources

bioRxiv2026

RNA trans-splicing treatment for Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a fatal disorder caused by dystrophin mutations, leading to progressive muscle degeneration and cardiac failure. Although AAV-based DMD therapies with innovative designs to overcome the cargo limits of the virus are effective in animal models, these current systems may face clinical translation challenges related to efficiency, off-target effects, and immunogenicity. We developed a multi-vector RNA End Joining (REJ) system to split large genes into multiple co-delivered AAVs that engage the cell's intrinsic spliceosome to precisely reassemble RNA segments that encode very large scar-free proteins. We show the system is efficient and has negligible off-target interactions. In vivo, REJ vectors expressing the adenine base editor Abe8e, mini-dystrophin Dp253, or native full-length dystrophin Dp427 each prevented muscle degeneration. Machine-learning histopathology of more than 120,000 myofibers revealed robust reductions in nuclear infiltration, improved centronucleation, and normalized hypertrophy, supported by functional, transcriptomic, and behavioral analyses. These findings establish REJ as a clinically viable AAV-based strategy for DMD that enables expression of large therapeutic proteins while avoiding potentially antigenic bacterial protein- or DNA-recombinases.

molecular biology↗

bioRxiv2026

An intersectional expression platform for gene complementation using RNA-fragment end joining (REJ)

Gene complementation is a powerful tool for genetic selection and protein functional studies but typically requires extensive screening for complementary components. Here we engineered a platform for gene complementation based on RNA end-joining (REJ) that precisely and efficiently splices separate RNA units into functional coding mRNAs. REJ is mediated by short structured modular RNA segments of ~250bp that: (a) promote RNA::RNA interaction, (b) coopt the intrinsic cell splicing machinery to facilitate RNA trans-splicing, (c) minimizes translation of protein fragments from un-spliced RNA segments, and (d) encodes scar-free protein. We demonstrate that REJ is a broadly applicable system that can reliably split nearly any gene into complementary segments regardless of protein structure. To enable the use of this system we provide a toolbox of reporters and a web-based design tool to facilitate vector design. Demonstrated REJ applications include genetic complementation, intersectional labeling of cell types, and efficient expression of large proteins.

molecular biology↗

bioRxiv2023

A spinal synergy of excitatory and inhibitory neurons coordinates ipsilateral body movements

Innate and goal-directed movements require a high-degree of trunk and appendicular muscle coordination to preserve body stability while ensuring the correct execution of the motor action. The spinal neural circuits underlying motor execution and postural stability are finely modulated by propriospinal, sensory and descending feedback, yet how distinct spinal neuron populations cooperate to control body stability and limb coordination remains unclear. Here, we identified a spinal microcircuit composed of V2 lineage-derived excitatory (V2a) and inhibitory (V2b) neurons that together coordinate ipsilateral body movements during locomotion. Inactivation of the entire V2 neuron lineage does not impair intralimb coordination but destabilizes body balance and ipsilateral limb coupling, causing mice to adopt a compensatory festinating gait and be unable to execute skilled locomotor tasks. Taken together our data suggest that during locomotion the excitatory V2a and inhibitory V2b neurons act antagonistically to control intralimb coordination, and synergistically to coordinate forelimb and hindlimb movements. Thus, we suggest a new circuit architecture, by which neurons with distinct neurotransmitter identities employ a dual-mode of operation, exerting either synergistic or opposing functions to control different facets of the same motor behavior.

neuroscience↗

bioRxiv2022

Mitf is a Schwann Cell Sensor of Axonal Integrity that Drives Nerve Repair

Schwann cells respond to acute axon damage by transiently transdifferentiating into specialized repair cells that restore sensorimotor function. However, the molecular systems controlling repair cell formation and function are not well defined and consequently it is unclear whether this form of cellular plasticity has a role in peripheral neuropathies. Here we identify Mitf as a transcriptional sensor of axon damage under the control of Nrg-ErbB-PI3K-PI5K-mTorc2 signaling. Mitf regulates a core transcriptional program for generating functional repair Schwann cells following injury and during peripheral neuropathies caused by CMT4J and CMT4D. In the absence of Mitf, core genes for epithelial-to-mesenchymal transition, metabolism and dedifferentiation are misexpressed and nerve repair is disrupted. Taken together, our findings demonstrate that Schwann cells monitor axonal health using a phosphoinositide signaling system that controls Mitf, which is critical for activating cellular plasticity and counteracting neural disease. HighlightsO_LIMitf-induced Schwann cell plasticity is triggered by peripheral neuropathy. C_LIO_LINrg-ErbB signaling activates Mitf via cytoplasmic-to-nuclear translocation. C_LIO_LIMitf restores sensorimotor function following axonal breakdown. C_LIO_LIMitf regulates a core repair program across both injury and neurodegeneration. C_LI

neuroscience↗