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1. Using a Human Liver Tissue Equivalent (hLTE) Platform to Define the Functional Impact of Liver-Directed AAV Gene Therapy

   Ramamurthy, R ; Zheng, WT ; George, S ; Wan, M ; Zhou, Y ; Lu, B ; Bishop, C ; Atala, A ; Porada, C ; Almeida-Porada, G ( December 2021 , ASH)

   2938 Using a Human Liver Tissue Equivalent (hLTE) Platform to Define the Functional Impact of Liver-Directed AAV Gene Therapy 63rd ASH Annual Meeting and Exposition, December 11-14, 2021, Georgia World Congress Center, Atlanta, GA Program: Oral and Poster Abstracts Session: 801. Gene Therapies: Poster II Hematology Disease Topics & Pathways: Bleeding and Clotting, Biological, Translational Research, Hemophilia, Genetic Disorders, Clinically Relevant, Diseases, Gene Therapy, Therapies Sunday, December 12, 2021, 6:00 PM-8:00 PM Ritu M Ramamurthy1*, Wen Ting Zheng2*, Sunil George, PhD1*, Meimei Wan1*, Yu Zhou, PhD1*, Baisong Lu, PhD1*, Colin E Bishop, PhD1*, Anthony Atala, M.D.1*, Christopher D Porada, PhD1* and M. Graca Almeida-Porada, MD3 1Fetal Research and Therapy Program, Wake Forest Institute for Regenerative Medicine, Winston Salem, NC 2Massachusetts Institute of Technology, Cambridge, MA 3Fetal Research and Therapy Program, Wake Forest Institute For Regenerative Medicine, Winston-Salem, NC Clinical trials employing AAV vectors for hemophilia A have been hindered by unanticipated immunological and/or inflammatory responses in some of the patients. Also, these trials have often yielded lower levels of transgene expression than were expected based upon preclinical studies, highlighting the poor correlation between the transduction efficiency observed in traditional 2D cultures of primary cells in vitro, and that observed inmore »those same cell types in vivo. It has been also recognized that there are marked species-specific differences in AAV-vector tropism, raising the critical question of the accuracy with which various animal models will likely predict tropism/vector transduction efficiency, and eventual treatment success in humans. Human liver tissue equivalents (hLTEs) are comprised of major cell types in the liver in physiologically relevant frequencies and possess the ability to recapitulate the biology and function of native human liver. Here, we hypothesize that hLTEs can be used as a better model to predict the efficacy and safety of AAV gene therapy in humans. We fabricated hLTEs using 75% hepatocytes, 10% stellate cells, 10% Kupffer cells, and 5% liver sinusoid-derived endothelial cells in 96-well Elplasia plates with 79 microwells per well. hLTEs were transduced at an MOI of 105vg/cell, on the day of fabrication, with the clinically relevant serotypes AAV5 (hLTE-5) or AAV3b (hLTE-3b), both encoding a GFP reporter. After 4 days of self-aggregation, live/dead assay was performed to confirm viability. Non-transduced hLTEs served as negative controls (hLTE(-)), and hLTEs exposed to 20 mM acetaminophen were used as positive controls for liver inflammation/damage. Incucyte® Live-Cell Imaging system was used to track the aggregation and GFP expression of hLTEs. Over the course of the next 5 days, media was collected to determine hepatic functionality, RNA was isolated to assess dysregulation of genes involved in inflammation and fibrosis, DNA was isolated to determine whether AAV vectors integrate into the genome of human hepatocytes and, if so, to define the frequency at which this occurs and the genomic loci of integration, and hLTEs were fixed and processed at appropriate times for histological analyses and transmission electron microscopy (TEM). TEM analysis revealed that all groups exhibited microvilli and bile-canaliculus-like structures, demonstrating the formation of a rudimentary biliary system and, more importantly, proving that hLTEs resemble native liver structure. Incucyte® imaging showed that AAV5 and AAV3b transduction impaired formation of hLTEs (57.57 ± 2.42 and 24.57 ± 4.01 spheroids/well, respectively) in comparison with hLTE(-) (74.86 ± 3.8 spheroids/well). Quantification of GFP expression demonstrated that AAV5 yielded the most efficient transduction of hLTEs (fold change in GFP expression compared to control: 2.73 ± 0.09 and 1.19 ± 0.03 for hLTE-5 and hLTE-3b, respectively). Chromogenic assays showed decreased urea production in cell culture supernatants of AAV transduced groups compared to the non-transduced hLTEs on days 6 and 10 of culture, demonstrating decreased hepatocyte functionality. However, ALT and AST levels were similar in all groups. On day 10, hLTEs were either used for RNA isolation or fixed in 4% PFA and processed for histology. Masson’s Trichrome and Alcian Blue/Sirius Red staining was performed to detect fibrosis, which was then quantified using ImageJ. These analyses showed no significant increase in fibrosis in either hLTE-5 or hLTE-3b compared to hLTE(-). Nevertheless, RT2 PCR Array for Human Fibrosis detected dysregulation of several genes involved in fibrosis/inflammation in both hLTE-5 and hLTE-3b (16/84 and 26/84, respectively). In conclusion, data collected thus far show successful recapitulation of native liver biology and demonstrate that AAV5 transduces hLTEs more efficiently than AAV3b. However, impaired self-aggregation and decreased hepatocyte functionality was observed in both AAV-transduced groups. Studies to address the incidence and location(s) of AAV integration are ongoing. We have thus shown that the hLTE system can provide critical new knowledge regarding the efficacy and safety of AAV gene therapy in the human liver. Disclosures: No relevant conflicts of interest to declare.« less
2. Combined subsampling and analytical integration for efficient large-scale GW calculations for 2D systems

   https://doi.org/10.1038/s41524-020-00385-5  

   Xia, Weiyi ; Gao, Weiwei ; Lopez-Candales, Gabriel ; Wu, Yabei ; Ren, Wei ; Zhang, Wenqing ; Zhang, Peihong ( August 2020 , npj Computational Materials)

   Accurate and efficient predictions of the quasiparticle properties of complex materials remain a major challenge due to the convergence issue and the unfavorable scaling of the computational cost with respect to the system size. QuasiparticleGWcalculations for two-dimensional (2D) materials are especially difficult. The unusual analytical behaviors of the dielectric screening and the electron self-energy of 2D materials make the conventional Brillouin zone (BZ) integration approach rather inefficient and require an extremely densek-grid to properly converge the calculated quasiparticle energies. In this work, we present a combined nonuniform subsampling and analytical integration method that can drastically improve the efficiency of the BZ integration in 2DGWcalculations. Our work is distinguished from previous work in that, instead of focusing on the intricate dielectric matrix or the screened Coulomb interaction matrix, we exploit the analytical behavior of various terms of the convolved self-energy Σ(q) in the smallqlimit. This method, when combined with another acceleratedGWmethod that we developed recently, can drastically speed up (by over three orders of magnitude)GWcalculations for 2D materials. Our method allows fully convergedGWcalculations for complex 2D systems at a fraction of computational cost, facilitating future high throughput screening of the quasiparticle properties of 2D semiconductors for various applications. To demonstratemore »the capability and performance of our new method, we have carried out fully convergedGWcalculations for monolayer C₂N, a recently discovered 2D material with a large unit cell, and investigate its quasiparticle band structure in detail.

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3. Mixed-dimensional InAs nanowire on layered molybdenum disulfide heterostructures via selective-area van der Waals epitaxy

   https://doi.org/10.1039/D0NA00768D  

   Baboli, Mohadeseh A. ; Abrand, Alireza ; Burke, Robert A. ; Fedorenko, Anastasiia ; Wilhelm, Thomas S. ; Polly, Stephen J. ; Dubey, Madan ; Hubbard, Seth M. ; Mohseni, Parsian K. ( May 2021 , Nanoscale Advances)

   Self-assembly of vertically aligned III–V semiconductor nanowires (NWs) on two-dimensional (2D) van der Waals (vdW) nanomaterials allows for integration of novel mixed-dimensional nanosystems with unique properties for optoelectronic and nanoelectronic device applications. Here, selective-area vdW epitaxy (SA-vdWE) of InAs NWs on isolated 2D molybdenum disulfide (MoS 2 ) domains is reported for the first time. The MOCVD growth parameter space ( i.e. , V/III ratio, growth temperature, and total molar flow rates of metalorganic and hydride precursors) is explored to achieve pattern-free positioning of single NWs on isolated multi-layer MoS 2 micro-plates with one-to-one NW-to-MoS 2 domain placement. The introduction of a pre-growth poly- l -lysine surface treatment is highlighted as a necessary step for mitigation of InAs nucleation along the edges of triangular MoS 2 domains and for NW growth along the interior region of 2D micro-plates. Analysis of NW crystal structures formed under the optimal SA-vdWE condition revealed a disordered combination of wurtzite and zinc-blend phases. A transformation of the NW sidewall faceting structure is observed, resulting from simultaneous radial overgrowth during axial NW synthesis. A common lattice arrangement between axially-grown InAs NW core segments and MoS 2 domains is described as the epitaxial basis for vertical NWmore »growth. A model is proposed for a common InAs/MoS 2 sub-lattice structure, consisting of three multiples of the cubic InAs unit cell along the [21̄1̄] direction, commensurately aligned with a 14-fold multiple of the Mo–Mo (or S–S) spacing along the [101̄0] direction of MoS 2 hexagonal lattice. The SA-vdWE growth mode described here enables controlled hybrid integration of mixed-dimensional III–V-on-2D heterostructures as novel nanosystems for applications in optoelectronics, nanoelectronics, and quantum enabling technologies.« less
4. Full Tilt: Universal Constructors for General Shapes with Uniform External Forces

   https://doi.org/10.1137/1.9781611975482.167  

   Balanza-Martinez, Jose ; Luchsinger, Austin ; Caballero, David ; Reyes, Rene ; Cantu, Angel ; Schweller, Robert ; Garcia, Luis ; Wylie, Tim ( January 2019 , Proceedings of the annual ACM-SIAM Symposium on Discrete Algorithms)

   We investigate the problem of assembling general shapes and patterns in a model in which particles move based on uniform external forces until they encounter an obstacle. In this model, corresponding particles may bond when adjacent with one another. Succinctly, this model considers a 2D grid of “open” and “blocked” spaces, along with a set of slidable polyominoes placed at open locations on the board. The board may be tilted in any of the 4 cardinal directions, causing all slidable polyominoes to move maximally in the specified direction until blocked. By successively applying a sequence of such tilts, along with allowing different polyominoes to stick when adjacent, tilt sequences provide a method to reconfigure an initial board configuration so as to assemble a collection of previous separate polyominoes into a larger shape. While previous work within this model of assembly has focused on designing a specific board configuration for the assembly of a specific given shape, we propose the problem of designing universal configurations that are capable of constructing a large class of shapes and patterns. For these constructions, we present the notions of weak and strong universality which indicate the presence of “excess” polyominoes after the shape is constructed.more »In particular, for given integers h, w, we show that there exists a weakly universal configuration with O(hw) 1 × 1 slidable particles that can be reconfigured to build any h × w patterned rectangle. We then expand this result to show that there exists a weakly universal configuration that can build any h × w-bounded size connected shape. Following these results, which require an admittedly relaxed assembly definition, we go on to show the existence of a strongly universal configuration (no excess particles) which can assemble any shape within a previously studied “drop” class, while using quadratically less space than previous results. Finally, we include a study of the complexity of deciding if a particle within a configuration may be relocated to another position, and deciding if a given configuration may be transformed into a second given configuration. We show both problems to be PSPACE-complete even when no particles stick to one another and movable particles are restricted to 1 × 1 tiles and a single 2 × 2 polyomino.« less
5. Three-dimensional intact-tissue sequencing of single-cell transcriptional states.

   Wang, X. ( June 2018 , Science)

   The mammalian brain consists of an intricate tapestry of cell types, with diversity crucial for function that arises from both differential gene expression and circuit-specific anatomy. Yet, retrieving high-content gene-expression information while retaining 3D positional anatomy at cellular resolution has been difficult, limiting integrative understanding of brain structure and function. Here we introduce and apply a technology for 3D intact-tissue RNA sequencing, termed STARmap (Spatially-resolved Transcript Amplicon Readout Mapping), which integrates highly-specific signal amplification, novel hydrogel-tissue chemistry, and an error-reduction sequencing process. The capabilities of STARmap were tested by mapping from 160 to 1,020 distinct genes simultaneously in sections of mouse brain at single-cell resolution with unprecedented efficiency, accuracy and reproducibility. These experiments led to the discovery of multiple new neocortical cell types, with gene markers and spatial patterns of organization not previously described, by comparison of the molecularly-defined architectures of sensory versus cognitive neocortex, and by quantification of expression of activity-regulated genes as a function of stimulation condition, spatial position, and cell typology. By adapting STARmap to thick tissue blocks, we observed and confirmed a novel molecularly-defined gradient distribution of excitatory neuron subtypes across cubic millimeter-scale volumes (>30,000 cells), and discovered a short-range 3D pattern of self-clustering shared bymore »many inhibitory neuron subtypes that was accurately identifiable with a 3D STARmap approach.« less