A minimally modified AAV9 variant (AAV9fyhr) for enhanced CNS gene delivery by intra-CSF administration.

Garza, I. T.; Ramesh, V.; Hu, Y.; Rioux, M.; Takar, M.; Higgs, H.; Lykken, E. A.; Adams, R.; Kunach, P.; Kieffer, D.; Trygg, C. B.; Fisher-Perkins, J. M.; Kumar, A.; Joachimiak, L. A.; Bailey, R. M.; Xing, C.; Braun, S. E. A.; Gray, S. J.

doi:10.64898/2026.10.07.756773

bioRxiv · 10.64898/2026.10.07.756773

A minimally modified AAV9 variant (AAV9fyhr) for enhanced CNS gene delivery by intra-CSF administration.

Garza, I. T.·Ramesh, V.·Hu, Y.·Rioux, M.·Takar, M.·Higgs, H.·Lykken, E. A.·Adams, R.·Kunach, P.·Kieffer, D.·Trygg, C. B.·Fisher-Perkins, J. M.·Kumar, A.·Joachimiak, L. A.·Bailey, R. M.·Xing, C.·Braun, S. E. A.·Gray, S. J.

View original source ↗Export metadata ↓

Abstract

Adeno-associated virus (AAV) mediated gene therapies continue to show promise for the treatment of genetic diseases. However, delivery and safety still represent significant prohibitive barriers restricting translation of these delivery modalities to humans. We describe a novel AAV9 variant, AAV9fyhr or FYHR, identified from screening a controlled randomization point mutation AAV9 library in mice and nonhuman primates followed by directed evolution of in vivo CNS-derived libraries screened in human iPSC differentiated cortical neurons. Capsid variants of interest were identified by coupling long read sequencing and computational structure modeling. Structure-function analysis of our lead candidate reveal that two targeted amino acid changes are sufficient to result in beneficial pleiotropic effects across viral stability, cell surface attachment, and gene delivery. We developed a novel multiplexed barcoding reporter system, enabling a time and cost-effective option to gather spatially resolved single cell capsid validation data in mice and nonhuman primates. When administered intrathecally, AAV9fyhr exhibits 2-to-6-fold higher gene delivery across several tissues in the CNS while minimally altering the tropism to peripheral tissues in comparison to AAV9. These findings demonstrate the potential of whole capsid engineering for the rational development of translational next generation AAV capsids.

Explore related subjects

bioengineering Preprint

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Garza, I. T., Ramesh, V., Hu, Y., Rioux, M., Takar, M., Higgs, H., Lykken, E. A., Adams, R., Kunach, P., Kieffer, D., Trygg, C. B., Fisher-Perkins, J. M., Kumar, A., Joachimiak, L. A., Bailey, R. M., Xing, C., Braun, S. E. A., Gray, S. J.. 2026-10-09. A minimally modified AAV9 variant (AAV9fyhr) for enhanced CNS gene delivery by intra-CSF administration.. https://doi.org/10.64898/2026.10.07.756773

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Development of a Low-cost Extrusion Multi-nozzle 3D Bioprinter for Complex Constructs

Extrusion-based multi-material bioprinting is a promising approach for fabricating complex tissue constructs, but current systems often require expensive hardware or support only one or a few numbers of bioinks. In this study, a low-cost, compact, and versatile multi-nozzle bioprinter (MNB) system was developed for pneumatic extrusion of up to 10 different inks using a rotational head. The system was integrated with a commercial 3D printer platform and controlled using custom G-code commands. Backlash compensation and nozzle-specific calibrations were experimentally determined to minimize positioning errors and airflow misalignment. The developed system showed stable repeatability in both clockwise and counterclockwise movements. Lines and various structures were successfully printed with different nozzle-switching directions and printing sequences. Representative structures including a diamond pattern, a multilayer scaffold, a pyramid structure, and a kidney-like construct were successfully fabricated. These results demonstrate that the proposed MNB system can produce complex 2D and 3D multi-material structures with good positional accuracy and repeatability.

bioengineering↗

bioRxiv2026

FluOpti: an open-source system for programmable whole-plate optogenetics and multi-fluorescence imaging

Bacteria are able to self-organize into complex spatial communities, such as colonies, biofilms and swarms. Probing cells and gene networks with dynamic stimuli while preserving their spatial organization is therefore essential for understanding how bacterial communities organize across spatial and temporal scales. Optogenetics offers a powerful approach for such a task given the precise control of stimulus delivery without the perturbations normally associated with the addition of liquid inducers. However, most optogenetics devices have been developed to work with liquid cell cultures or to deliver a spatial pattern without the concurrent recording of gene responses and colony growth. Here, we describe FluOpti, an open source, low-cost device for controlling gene expression, through programmable light inputs, and recording multi-fluorescence responses and colony growth across whole petri dishes. White light LEDs are used for brightfield imaging while green and red LEDs are used as optogenetic inputs to activate and inactivate target genes via the CcaS/R two component system in E. coli. Low cost blue and orange acrylic are used as excitation and emission filters for fluorescence imaging, respectively. We developed custom software to analyze colony growth and optogenetic responses from 72-hour time-lapse experiments. A mathematical model was developed to predict gene expression levels in response to green-to-red light ratio stimuli. We use FluOpti to reveal a spatially structured response to optogenetic stimuli, with gene activation rates differing in cells located at the center and edge of colonies. to validate its use at a whole petri dish scale, FluOpti was used to depict how Voronoi territories can act as predictors of colony size, in congruence with previous studies. Together, these results demonstrate the utility of the device for studying gene networks at multiple spatio-temporal scales, from individual colonies to the whole petri dish. Performing optogenetics studies in whole petri dishes while preserving spatial arrangements could offer valuable insights into the spatial biology of gene networks in microbial populations.

bioengineering↗

bioRxiv2026

Denoising broadband CARS hyperspectral images on variance-stabilized data: a unified benchmark

Broadband coherent anti-Stokes Raman scattering (BCARS) enables label-free hyperspectral chemical imaging, but weak signal components are often obscured by heteroscedastic noise. Here, we develop a physics-based variance stabilization transform and benchmark six hyperspectral denoising methods to identify effective strategies for BCARS noise reduction. The transform yields nearly intensity-independent noise variance while preserving the dispersive Raman response. Within this framework, we compare singular value decomposition, BM4D, Noise2Noise, deep image prior, self-supervised diffusion, and a cross-covariance-based singular value decomposition method (CCV) introduced here, using a shared phase-retrieval pipeline. Evaluation includes five simulated hyperspectral cubes with exact ground truth and experimental measurements of glycerol, polystyrene beads, and Caenorhabditis elegans. In simulations, variance stabilization reduces the ratio of maximum to minimum channel noise variance from approximately 72 to 1.2. Deep image prior and self-supervised diffusion achieve the highest mean foreground peak signal-to-noise ratios. CCV matches the mean foreground structural similarity of deep image prior with substantially shorter runtime and no per-cube rank tuning. Experimental comparisons reveal sample-dependent performance. These results establish physics-based variance stabilization as a common framework for adapting and evaluating hyperspectral denoisers for BCARS, with CCV offering a practical balance of reconstruction fidelity, computational speed, and automated operation.

bioengineering↗