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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 in 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. 

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2. Three-dimensional microscale hanging drop arrays with geometric control for drug screening and live tissue imaging

   https://doi.org/10.1126/sciadv.abc1323  

   Ganguli, A. ; Mostafa, A. ; Saavedra, C. ; Kim, Y. ; Le, P. ; Faramarzi, V. ; Feathers, R. W. ; Berger, J. ; Ramos-Cruz, K. P. ; Adeniba, O. ; et al ( April 2021 , Science Advances)

   null (Ed.)

   Existing three-dimensional (3D) culture techniques are limited by trade-offs between throughput, capacity for high-resolution imaging in living state, and geometric control. Here, we introduce a modular microscale hanging drop culture where simple design elements allow high replicates for drug screening, direct on-chip real-time or high-resolution confocal microscopy, and geometric control in 3D. Thousands of spheroids can be formed on our microchip in a single step and without any selective pressure from specific matrices. Microchip cultures from human LN229 glioblastoma and patient-derived mouse xenograft cells retained genomic alterations of originating tumors based on mate pair sequencing. We measured response to drugs over time with real-time microscopy on-chip. Last, by engineering droplets to form predetermined geometric shapes, we were able to manipulate the geometry of cultured cell masses. These outcomes can enable broad applications in advancing personalized medicine for cancer and drug discovery, tissue engineering, and stem cell research. 

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3. Joint analysis of expression levels and histological images identifies genes associated with tissue morphology

   https://doi.org/10.1038/s41467-021-21727-x  

   Ash, Jordan T. ; Darnell, Gregory ; Munro, Daniel ; Engelhardt, Barbara E. ( March 2021 , Nature Communications)

   Histopathological images are used to characterize complex phenotypes such as tumor stage. Our goal is to associate features of stained tissue images with high-dimensional genomic markers. We use convolutional autoencoders and sparse canonical correlation analysis (CCA) on paired histological images and bulk gene expression to identify subsets of genes whose expression levels in a tissue sample correlate with subsets of morphological features from the corresponding sample image. We apply our approach, ImageCCA, to two TCGA data sets, and find gene sets associated with the structure of the extracellular matrix and cell wall infrastructure, implicating uncharacterized genes in extracellular processes. We find sets of genes associated with specific cell types, including neuronal cells and cells of the immune system. We apply ImageCCA to the GTEx v6 data, and find image features that capture population variation in thyroid and in colon tissues associated with genetic variants (image morphology QTLs, or imQTLs), suggesting that genetic variation regulates population variation in tissue morphological traits.

    

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4. Label-free histological imaging of tissues using Brillouin light scattering contrast

   https://doi.org/10.1364/BOE.414474  

   Ryu, Seungmi ; Martino, Nicola ; Kwok, Sheldon J. J. ; Bernstein, Liane ; Yun, Seok-Hyun ( February 2021 , Biomedical Optics Express)

   Brillouin light scattering offers a unique label-free approach to measure biomechanical properties non-invasively. While this technique is used in biomechanical analysis of cells and tissues, its potential for visualizing structural features of tissues based on the biomechanical contrast has not been much exploited. Here, we present high-resolution Brillouin microscopy images of four basic tissue types: muscular, connective, epithelial, and nervous tissues. The Brillouin contrast distinguishes between muscle fiber cells and endomysium in skeletal muscle and reveals chondrocytes along with spatially varying stiffness of the extracellular matrix in articular cartilage. The hydration-sensitive contrast can visualize the stratum corneum, epidermis, and dermis in the skin epithelium. In brain tissues, the Brillouin images show the mechanical heterogeneity across the cortex and deeper regions. This work demonstrates the versatility of using the Brillouin shift as histological contrast for examining intact tissue substructures via longitudinal modulus without the need for laborious tissue processing steps.

    

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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 by many inhibitory neuron subtypes that was accurately identifiable with a 3D STARmap approach. 

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