Category Archives: Gene Medicine

Fabrication and endothelialization of spiral tubes in PDMS and collagen gels

We exploited subtractive molding techniques to fabricate spiral tubes in both polydimethylsiloxane (PDMS) and collagen hydrogels and tested the fabrication limit and fidelity. In PDMS, stainless steel springs of various dimensions were molded in liquid-phase PDMS (10:1, base:curing agent) and manually removed after cross-linking. Robust perfusable spiral tubes with constant curvature were generated in PDMS with a diameter larger than 200 m and a pitch greater than 1 mm per turn. The fabrication of smaller spiral tubes in PDMS is less consistent because of the distortion of the channel structure during spring removal. In collagen hydrogels (6 to 7.5 mg/ml), an automatic two-axis motion system was designed to retract the spring from the hydrogel after thermal gelation. Automatic retraction was critical to minimize distortion of the spiral pattern and maximize continuity of the luminal geometry in three dimensions in soft matrices (see Materials and Methods) (Fig. 1A). Spiral tubes of a wide range of wire diameter (dw = 120 to 400 m), spiral diameter (ds = 1 to 3 mm), and pitch (p 400 m) were formed in collagen hydrogels, corresponding to curvature in the range of 0.43 to 1.05 mm1 and torsion in the range of 0.32 to 0.72 mm1 (fig. S1A). Fluorescent beads were perfused to visualize the 3D structure of the spiral lumen [Fig. 1B (a); fig. S1, A and B; and movie S1], where loops of the spiral tubes were periodically spaced with distinct boundaries. Using off-the-shelf springs, we were able to achieve spacing between loops ( = p dw) as small as 210 m. We further modified the spiral mold by adding a cylinder in the center of the spring to generate a second independently perfusable lumen in the hydrogel structure [Fig. 1B (b)]. We fabricated constructs with a central tube concentrically wrapped with a spiral tube and separated by a wall as thin as 200 m.

(A) Schematic of spiral vessel fabrication strategy. Top: A hydrogel is cross-linked around an off-the-shelf spring (a), the pattern is retracted from the gel via a two-axis motion system (b), and vessels were seeded with cells by perfusion (c). Bottom: An independent rod was introduced at the center of the spring to form an additional lumen for independent access and cell seeding. (B) (a) Maximum intensity projection (MIP) of a confocal z-stack of a spiral vessel in collagen perfused with fluorescent beads. (b) Optical section of a collagen spiral vessel (magenta) with an independently perfused center channel (green). Scale bars, 500 m. (C to E) MIP of side (C) and top (D) views of an endothelialized spiral vessel in PDMS and top view of endothelialized collagen vessel (E). Scale bars, 750, 600, and 150 m. (F) Integrated fabrication of spiral vessel (z directional flow) and planar microvessel (x and y directional flow) showing MIP of side and top views with magnified views of regions near (a) and distant from the connection of spiral to planar microvessels (b and c). Magenta, CD31; green, von Willebrand factor; blue, nuclei. Scale bars, 200 m and 50 m (inset). (G) Engineered vascularized tumor model with ECs from the spiral vessel sprouting toward avascular tumor cells embedded in the center lumen of the spiral. Green, CD31; red, KG1a cancer cells; blue, nuclei. Scale bars, 200 m (left) and 100 m (right). (H) Vascularized cardiac chamber model. Green, CD31; red, cTnT; blue, nuclei. Scale bars, 500 m.

Next, we perfused human umbilical vein ECs (HUVECs) into the spiral tubes in either PDMS or collagen to allow cell attachment followed by culture under flow. Both materials supported the growth of a robust endothelium under steady flow for at least 1 week (Q = 1 l/min; Fig. 1, C to E). PDMS spiral vessels with a lumen diameter of less than 200 m often had sparse coverage of ECs on the vessel surface after seeding and were not used in experimental conditions. Collagen spiral vessels better supported endothelialization, and HUVECs were seeded and cultured under similar flow conditions for spiral vessels as small as 180 m with high reproducibility (fig. S1C). ECs in PDMS vessels (lumen diameter > 200 m) and all sized collagen vessels had robust junctions at cell-cell contacts and localized expression of CD31 to the plasma membrane (Fig. 1, C to E). Together, we successfully generated spiral microvessels with constant curvature and torsion at high fidelity and reproducibility and with robust endothelialization and perfusion.

The fabrication process for spiral vessels has the flexibility to integrate with existing vascularization approaches to further enhance tissue perfusion. By incorporating ECs into the bulk matrix, the endothelium in spiral tubes was readily anastomosed with self-assembled vessel networks and increased vascular density (fig. S1D). When combined with lithography and injection molding techniques (31), we successfully connected a spiral vessel with a microfabricated rectilinear vessel so that the spiral outflow was connected to the perfusion of microvessels in an orthogonal direction to the spiral. This integration allows the rotation of the spiral flow direction into another plane and mimics the architecture of the spiral artery to vascular bed connection found in vivo (Fig. 1F). We observed a continuous endothelium in the spiral microvessel connection [Fig. 1F (a)]. ECs in the planar microvessels near the spiral vessel outflow showed greater alignment with the direction of flow, likely due to higher flow stresses [Fig. 1F (a); average angle, 13.5 10.2] compared to cells in regions distant from the spiral microvessel interface [Fig. 1F (b and c); average angle, 39.3 23.7 and 58.13 29.25, respectively]. These findings illustrate the potential of spiral vessels as a new strategy for rapidly generating long and high surface area vascular structures that may enhance tissue vascularization.

Using the concentric spiral platform in collagen gel, we further demonstrated the potential of spiral vessels in supporting 3D tissue function. By dispensing tumor cells (KG1a, a leukemia cell line; Materials and Methods) in a collagen gel (6 mg/ml) into the spatially defined center cylinder (1.3 mm diameter), we formed an artificial tumor surrounded by spiral vessels and monitored the sprouting of vessels from the spiral. This cell-remodelable system mimics the physiological origins of some tumors, where malignancies begin as an avascular cellular mass surrounded by host vasculature that it must recruit for expansion (32). When cultured under flow (Q = 1 l/min) in normal growth medium, spiral vessels (dw = 400 m, ds = 3.0 mm, and p = 1 mm) maintained patency throughout 7 days of culture and sprouted consistently by day 7, but not at day 3 (N = 4 for each time point) (Fig. 1G). These sprouts extended exclusively toward the tumor, with sprouts reaching as far as 220 m from the vessel wall by day 7. No sprouts were observed when there were no tumor cells in the center.

We also created a thick cardiac chamber supported by a spiral vessel using the same concentric model (1.3-mm-diameter by 6-mm-long chamber surrounded by spiral vessel). GCaMP3-transduced human embryonic stem cellderived cardiomyocytes (hESC-CMs) and stromal cells (HS27a) were added into the bulk collagen matrix (33) and ECs (HUVECs) into both the bulk matrix and the spiral lumen, while the center of the tissue was kept open (Fig. 1H). By day 12 of culture, organized calcium waves were observed and appeared to propagate in three dimensions along the spiral vessel wall (movie S2). The conduction velocity in engineered cardiac tissues was 2.7 0.97 cm/s, as determined by analysis of the GCaMP3 signal (fig. S2). These proof-of-concept examples show that the spiral vessel platform can be used to support 3D vascularization and perfusion in large tissues, to study the vascular-tissue interaction in a spatially and temporally controlled manner, and to model complex tissue functions.

We next examined the flow characteristics in these spiral microvessels and compared them with straight vessels of the same caliber. We visualized the flow characteristics by perfusing fluorescent bead solutions in two parallel streams through straight and spiral PDMS vessels of the same diameter and length (dvessel = 400 m, dspiral = 3 mm, pspiral = 1 mm, spiral = 0.46 mm1, spiral = 0.31 mm1, and L = 6.5 cm) at three steady flow conditions (Q = 1, 50, and 100 l/min, corresponding to Re = 0.01, 0.76, and 1.52, respectively). The 3D flow images were taken under confocal fluorescence microscopy at set distances (Lv = 5, 30, and 55 mm in straight vessels, or loops 3/4, 3 3/4, and 6 3/4 in spiral vessels) from the vessel inlet. Straight tubes displayed a classical parallel flow profile where the two streams of beads traveled to the outlet and maintained their position over the whole vessel length at both flow rates (Fig. 2, A and C). In spiral tubes, the two bead streams remained distinct and parallel at low flow (Q = 1 l/min) but rotated over the vessel length without obvious mixing in the bulk [Fig. 2B (a to c)]. The orientation of the two parallel streams inverted after approximately four loops from the inlet [Fig. 2B (b)] and completed a full rotation at approximately loop 7 [Fig. 2B (c)]. At a higher flow rate (Q = 50 l/min) in the same spiral geometry (De = 2.77), the two bead streams developed obvious bulk mixing with the leading edge of flow rotating 270 after three loops [Fig. 2B (e)] and completed another full rotation by loop 7 [Fig. 2B (f)]. At even higher flow (Q = 100 l/min), a stronger mixing effect was observed in the same spiral geometry [Fig. 2B (g to i)], whereas the two streams remained parallel and unmixed in straight vessels under the same flow conditions.

(A and B) Confocal cross sections of perfusion of two parallel streams of red and blue beads into a straight PDMS vessel (A) at a flow rate of Q = 50 l/min and a spiral PDMS vessel (B) at three flow rates (Q = 1, 50, and 100 l/min) at three distances from the vessel inlet (Lv,a 5 mm, Lv,b 30 mm, and Lv,c 55 mm), corresponding to the 3/4, 3 3/4, and 6 3/4 spiral loops (LL = linear length, Lv = vessel length, dv = vessel diameter, p = pitch). (C and D) Computational fluid dynamics plots of straight (C) and spiral (D) vessels at Q = 50 l/min for (a) streamlines (color expressed with primary velocity magnitude), (b) primary velocity magnitude, (c) secondary flow velocity orthogonal to cross-sectional plane, and (d) shear rate at the cross-sectional views.

Using numerical simulation with COMSOL, we confirmed these flow characteristics: (i) Idealized parallel streamlines were present in fully developed flow in straight vessels [Fig. 2C (a)]; (ii) parallel streamlines in spiral vessels slightly rotate along circumferential direction at low flow (Q = 1 l/min; fig. S3A); and (iii) streamline rotation was enhanced in spiral vessels and developed twists at higher flow [Q = 50 l/min; Fig. 2D (a)] and had clear twists at Q = 100 l/min (fig. S3B). The spiral geometry did not induce a significant change in the primary flow compared to straight vessels but did lead to the emergence of secondary flows with a peak magnitude of around 1% of the primary flow velocity [Q = 50 l/min; Fig. 2, C (b and c) and D (b and c)]. This also led to the development of a shear stress gradient in 3D space and a change in the wall shear stress (WSS), with a maximum (10% increase over the straight tube) on the surface of the inner curvature and minimum on the outer bend, unlike in a straight tube where the WSS was constant across the lumen cross section with zero gradients [Fig. 2, C (d) and D (d)]. These data demonstrated that spiral vessels induced bulk flow mixing and heterogeneous hemodynamic forces on the endothelium lining the wall due to 3D curvature and torsion.

To understand how the distinct hemodynamic features of flow in spiral vessels affected ECs, we cultured cells in both geometries under flow. In straight and spiral vessels, ECs formed robust junctions and a stable endothelium in low (Q = 1 l/min and WSS = 0.1 dyne/cm2 in straight vessels) and high (Q = 50 l/min and WSS = 4.6 dynes/cm2 in straight vessels) flow conditions. The increased flow appeared to change the EC morphology and enhance EC alignment in the direction of flow (Fig. 3A). Under low flow conditions (Q = 1 l/min; Fig. 3B), fewer Ki67+ proliferating cells were observed in spiral vessels than in straight vessels. When exposed to higher flow, however, more proliferating cells were observed in the spiral geometry than the straight geometry, suggesting distinct roles for geometry and flow on the ECs. Previous literature has highlighted that very low laminar flows activate ECs, whereas high laminar flow enhances EC quiescence (11). Our data were consistent with this in straight vessels with significantly reduced cell proliferation at higher flow. In spiral vessels, however, the flow rotation in low flow may alter transport and promote quiescence at low flow. Given that the magnitude of flow forces is very low in the low flow conditions, it is also likely that differences in substrate curvature between straight and spiral geometries contribute to these observed differences (34).

(A) MIP of EC cultured under flow (Q = 50 l/min) for 24 hours. Blue, nuclei; green, CD31; red, Ki67. Scale bar, 50 m. (B) Quantification of the percentage of Ki67-positive nuclei by counting 100+ cells per vessel in N = 3 vessels at two flow conditions (Q = 1 and 50 l/min). Error bars represent 95% confidence interval of the mean. *P < 0.05 using a one-way analysis of variance (ANOVA) with Tukeys pairwise comparisons. (C) PCA of RNA-seq data from cells cultured at static and at two flow conditions in two vessel geometries (N = 3). (D) Venn diagram showing the overlap of genes significantly changed by increasing flow in straight and spiral geometries. (E) Heatmap of log counts per million (CPM) values of known flow-responsive genes. All genes are present in the overlapping region of (D) (green). (F) Heatmaps of the CPM values of significantly regulated transcripts belonging to the nonoverlapping regions of (D). Three hundred fifty-five genes uniquely regulated in straight high versus low (top, yellow) and 1261 genes uniquely regulated in spiral high versus low (bottom, blue). (G) Heatmaps of the CPM values of selected growth factors, transporters, and transcription factors. (H) IPA functional pathways identified by comparing spiral to straight vessels under high flow.

We next examined the transcriptional changes in ECs in these conditions via RNA sequencing (RNA-seq) for ECs cultured under both flow conditions in straight and spiral vessels and under static conditions. Principal components analysis (PCA) of gene expression data showed clustering of individual groups, with the largest variance between static and all flow conditions (Fig. 3C). Activation of classical flow-dependent genes was confirmed in all flow conditions compared to static culture (Fig. 3, D and E). Among these genes, KLF2 and KLF4 appeared to only change with the onset of flow but were not sensitive to a further increase in flow, whereas SMAD6, SMAD7, and NOS3 increased further at higher flow conditions. Among the genes differentially expressed in straight vessels due to the increase of flow, 52% (533 of 1012) overlap with genes differentially expressed in the onset of flow (static versus low flow condition) (fig. S4, A and B). The genes unique to the increase of flow include up-regulation of many genes previously reported to regulate vascular development and flow sensing (35), such as Notch ligands JAG1 and JAG2; Notch target HEY2 and other transcription factors such as SNAI2; transmembrane proteins IL21R and EFNB2; transporter GJA5; peptidases MMP10, MMP1, and MT1F; growth factors and cytokines NOG, DKK2, WNT4, CXCL12, and TGFB1; and other molecules such as VCAN and CYP1B1 (fig. S4C). Gene Ontology (GO)enriched terms for this group of genes showed up-regulation of cellular response to growth factors, vascular development, transmembrane receptor protein tyrosine kinase signaling pathway, blood vessel morphogenesis, cell migration and motility, and others (fig. S4D).

Approximately 66% (722 of 1136) of differentially expressed genes in straight vessels overlap with those in spiral vessels in response to increased flow (Fig. 3D). Almost all overlapping genes are changed in the same direction (99%), suggesting a conserved response to flow in both geometries (fig. S5A). MARC2, PTX3, and STX11 did not follow this trend and were up-regulated in spiral vessels with increased flow but down-regulated in straight vessels. PTX3 has been reported as a biomarker for endothelial dysfunction in preeclampsia, which is a disease caused by spiral artery dysfunction (36). Many genes down-regulated in straight vessels by the increase of flow did not show changes in spiral vessels, such as growth factors CTGF, FGF2, NRG1, and FGF16; transmembrane proteins CAV1, UNC5A, KIT, and SMAD4A; transcription regulators EGR1/2/3, MAF, MYRF, and MZF1; transporters such as LDLR; and cytokines TNFSF18, IL12A, CCL2, CCL16, and CCL28 (fig. S5B). This suggests that the EC response to flow in spiral vessels is a combination of both canonical flow pathways and a distinct response involving a wide range of other transcripts.

Increased flow also led to an additional 1294 genes significantly changed in spiral vessels that were not in straight ones (Fig. 3F). High flow in spiral vessels appeared to activate growth factors such as DKK1, ESM1, BMP2, PDGFA, OSGIN2, and VEGFC; many solute carrier (SLC) and adenosine triphosphate (ATP)binding cassette (ABC) superfamily transporters; transcriptional regulators such as GLI2; cytokine CXCL1; peptidases TLL1, ADAMTS1, ADAMTS9, and TASP1; and kinases PODXL, EPHA5, HK2, PRKCA, CCT2, and MAP2K1 (Fig. 3G and fig. S6A). In addition, high flow in spiral vessels repressed growth factors such as MST, NRG2, GDF3, GAS6, and IGF2; transmembrane receptors CHRNA1, SELP, LRP1, ITGB3, and ROBO3; transporters including MAL2, ATP2A3, RBP1, and APOL1 and several members of SLC and ABC superfamilies; transcriptional regulators such as NOTCH3, CITED4, CAND2, FOXO4, DACH1, and EBF3; cytokines DKK3, CSF1, and FLT3LG; GPCR (G proteincoupled receptor) group SIPR4, OPRL1, and HTR2B; and kinases PDGFRB, CKB, and SBK1 (Fig. 3F and fig. S6A). GO term analysis showed the up-regulation of primarily ribosome biogenesis, which would be critical for cellular growth and proliferation (fig. S6B). These expression profiles show that spiral vessels share a common set of flow-responsive elements with straight vessels but have an additional response that appeared to promote vascular growth.

PCA analysis showed that the separation of straight and spiral geometries was enhanced under higher flow conditions (Fig. 3E). Under low flow conditions (Re << 1; inertia effect is negligible), ECs in the two geometries were largely similar, with only a handful of significantly regulated transcripts (fig. S7A). These included CYTL1, which is known to up-regulate proangiogenic function, but not proinflammatory pathways (37). HES2, a downstream Notch pathway gene, STK32B (serine/threonine kinase 32B), and CCND1, a cell cycle regulation gene, were also up-regulated in low flow spiral vessels. The up-regulation of these genes was further enhanced in high flow conditions. In addition, many genes that regulate vascular development were up-regulated when comparing spiral to straight vessels at high flow, for example, growth factors HGF, DKK1, ESM1, PGF, PDGFA, GDF6, PDGFB, CTGF, VEGFC, BMP2, and PDGFC; peptidases ADAMTS1, ADAMTS9, MME, and CTSS; kinases EPHA5, MPP4, PODXL, SPRY2, CDK7, and MAP2K1; transmembrane receptors KIT, SELE, ULBP2, PLXNA2, and LRP8; transcriptional regulators GLI2 (a hedgehog pathway mediator), ATF3 (required for endothelial regeneration) (38), and FOSL1 (required for vascular formation) (39); and many SLC and ATP family transporters (Fig. 3G). Ingenuity Pathway Analysis (IPA) showed that ECs in spiral vessels have activated upstream regulators including prosurvival factors HGF, PGF, EGF, VEGF, and HIF-1a. Up-regulated functional pathways included vascular development, angiogenesis, vasculogenesis, cell invasion, and cell survival, whereas cell death and necrosis were decreased compared to the straight vessel in high flow conditions (Fig. 3H and fig. S7B). Spiral vessels also showed the activation of antiapoptotic and proliferative pathways marked by cell cycle and mitotic genes. PDGF (platelet-derived growth factor) family members were relatively more abundant, as were molecules associated with IL-8 (interleukin-8) and HGF (hepatocyte growth factor) signaling (fig. S7B).

Together, the bulk RNA-seq showed that spiral vessels maintain a normal flow response to a certain extent, but curvature and torsion modified the response by up-regulating markers for transporters, cycling, and survival and down-regulating markers of cell death. These data suggest that flow in spiral vessels promoted vascular growth or development rather than inducing a common inflammatory response to disturbed flow.

We hypothesized that ECs exposed to flow within spiral vessels experienced a spatial variation in hemodynamic forces not present in straight vessels that would result in a heterogeneous transcriptional response to flow. To understand this heterogeneity at the single-cell level, we sequenced the transcriptomes of more than 2000 individual ECs pooled from three to four devices of each geometry cultured at high flow (Q = 50 l/min). Dimensionality reduction by Uniform Manifold Approximation and Projection (UMAP) and cluster analysis was performed using Monocle (4042). Projection in the top two UMAP dimensions shows overlapping contributions of ECs from spiral and straight vessels that form mostly contiguous clusters with nearly uniform expression of pan-endothelial markers such as CDH5 (VE-cadherin) (Fig. 4, A and B). Expression of classical flow-dependent genes, including KLF4 and NOS3, is distributed throughout the major clusters of ECs in this projection (Fig. 4C). We identified variation in gene expression across the first UMAP dimension driven largely by cell cycle genes that have been shown to be regulated, in part, by flow. Specifically, a large cluster of cells to the right in UMAP space (cluster 3) express genes such as MKI67, consistent with active cell cycle status, whereas cells clustered to the opposite pole (cluster 1) express genes implicated in cell cycle arrest and arterial phenotype shown to be regulated by the Notch pathway downstream of laminar shear stress (including CDKN1C, EFNB2, HEY1, GJA4, and IL33; Fig. 4C) (4345).

(A) UMAP plots of spiral high flow and straight high flow cells, computationally derived clusters (B), and the distribution of endothelial and cell cycling genes across cells (C). (D) UMAP plots of the first and third UMAP dimensions, the corresponding location of clusters in this dimensional space (E) with examples of cluster specific genes (F), as well as a selection of genes identified as significantly differentially expressed (G).

To evaluate heterogeneity in the transcriptional response of ECs resulting from vessel geometry, we next identified differentially expressed genes on the basis of single-cell RNA-seq (scRNA-seq) data of EC from straight versus spiral vessels. Examination of the third UMAP dimension revealed separation in transcriptional space between EC from straight versus spiral vessels, with many of the identified differentially expressed genes polarized in this dimension (Fig. 4, D to F). Among the genes up-regulated in EC from spiral vessels are many that were also identified as differentially expressed in bulk RNA-seq analysis, including ATF3, SPRY2, IL8, JUN, AKAP12, ANGPTL4, FOSL1, ADAMTS1, and ADAMTS9 (Fig. 4G and fig. S8A). Most of these genes are expressed in a common pattern, with increased expression in cells localized in UMAP space to the lower (spiral) portion of cluster 2. This suggests a distinct transcriptional program among the primarily spiral ECs in this region that may correspond with their transcriptional response to specific hemodynamic conditions unique to spiral vessels under high flow. In further support of this hypothesis, analysis of the scRNA-seq data also identified differentially expressed genes not detected in bulk RNA-seq, including DCN, SLC6A9, GEM, NRG1, RSPO3, BAMBI, TGFBI, and PRRX2, that were highly specific to EC from spiral vessels localized in cluster 2 (Fig. 4G and fig. S8B). These data suggest that flow in spiral vessels induced a population of ECs with unique gene expression profiles that are not present in straight vessels, with potential roles in processes such as angiogenesis, vascular growth, and inflammatory and stress responses.

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3D curvature-instructed endothelial flow response and tissue vascularization - Science Advances

Depression is a dark horse.

The disease often goes unnoticed, but affects work performance, social interaction and the ability to take pleasure in everyday life. According to theNational Center for Biotechnology Information, antidepressants only help around 50 percent of those who struggle with depression and anxiety and, even when they are effective, scientists have yet to understand how they work in the brain.

MSU associate professor of physiology A.J. Robison and his lab used new CRISPR-based technology to uncover pathways of depression-like behavior in the mouse brain. Credit: College of Natural Science

But groundbreaking research in the lab of Michigan State University scientistA.J. Robison, associate professor in theDepartment of Physiologyand MSUsNeuroscience Program, is directing some new rays of light onto the molecular, cellular and circuit-level mechanisms underlying depression-like diseases.

Theresultswere recently published inNature Communications.

In this paper, we perform the first ever CRISPR-based gene editing [a genetic engineering technique in molecular biology by which the genomes of living organisms may be modified] in a single circuit between two areas of the mouse brain, explained Robison about the culmination of five years of research funded by the National Institutes of Mental Health. We can reach into the mouse brain and manipulate specific genes in a circuit involved in depression and anxiety-like behaviors a critical advance on the road to genetic medicine for psychiatric diseases.

Scientists estimate there are roughly 80-100 billion neurons connecting regions of the brain. To accomplish the feat of locating and manipulating a single gene in a single circuit required new and sophisticated technology. With the expertise of co-author Rachael Neve, director of theGene Transfer Core at Massachusetts General Hospital, they developed it.

The key advance is that we designed a dual-vector system to manipulate a specific gene in the connections between two brain areas, and that has never been done before, Robison said.

Cross section of a mouse brain. The projections of the cells between the vHPC and NAc, shown here in neon green, are manipulated by the new CRISPR viral vector-based technology developed by Rachael Neve and the Robison Lab. Credit: Andrew Eagle

The neurons that Robison and his team zeroed in on originate in the ventral hippocampus (vHPC), a deep-seated structure that projects to regions in the brain important in stress susceptibility, mood and social avoidance. Neurons rooted in the vHPC reach out with branch-like structures called axons to connect with the nucleus accumbens, or NAc. The completed circuit is regulated by the star of the pioneering paper, the transcription factor known as DFosB.

Using the viral vector technology specifically designed and packaged by Neve, the team split the CRISPR system in half. Half of the system, inert on its own, was an enzyme that can mutate DNA in the vHPC. The other half, a guide RNA, was sent to all cells that project to the NAc and tells the enzyme where to bind and the specific gene to mutate. Only those cells specific to the circuit from the vHPC to the NAc got both halves, triggering the enzyme to bind with and turn off a single gene: FosB.

When the FosB gene was turned off in the neurons, we were able to get a circuit-specific behavioral effect relevant to a disease like depression, said Robison about the landmark discovery. When we put it back, or rescued it within the circuit, the effect was erased.

Claire Manning was a key contributor to the groundbreaking study and is now a postdoctoral researcher at Stanford University. Credit: Ken Moon

One of the most exciting findings from our investigations was the circuit-specific role of the FosB protein in conferring resilience to stress, Eagle said. We also discovered that FosB altered the excitability of hippocampal circuit neurons and may be affecting long-term downstream changes that lead to changes in the activity of this circuit. But removing DFosB permanently altered the expression of a suite of genes, in effect removing the conductor from the orchestra. To that end, the paper goes on to report in-depth experiments on DFosB largely done by the members of theRobison Labincluding co-first authorsClaire Manning, a 2019 neuroscience graduate, now a postdoc at Stanford University; andAndrew Eagle, a former postdoctoral researcher, now an assistant professor in the MSU Department of Physiology.

Andrew Eagle, shown here imaging a mouse brain, played a major role in conducting experiments to further probe the function of DFosB. Credit: Research@MSU.

Based on the findings in the paper, the Robison Lab will continue to develop highly collaborative and cutting-edge techniques, accelerated by MSUs newly completed Interdisciplinary Science and Technology Building. This work is important because it elucidates a potential mechanism, namely FosB, for how stress may contribute to depression, Eagle continued. Future clinical work may find ways to directly manipulate FosB, or more likely one of its gene targets, to provide resilience to stress and decrease the incidence of depression in vulnerable people.

The end of this paper, which shows us measuring the changes of expression in hundreds of genes when we remove DFosB, is only the beginning of years of work for our lab, Robison said. Which genes are important and what are they doing in the brain? This is the challenge of a lifetime for me and my lab.

This article is repurposed content originally featured on the College of Natural Sciences website.

Link:
Illuminating the opaque pathways of depression - MSUToday

-- Webcast conference call to be held on Monday, Sept. 28, 2020 at 8:30 a.m. Eastern Time --

-- Additional poster presentations at WMS will highlight data from Sareptas RNA and gene therapy programs --

CAMBRIDGE, Mass., Sept. 14, 2020 (GLOBE NEWSWIRE) -- Sarepta Therapeutics, Inc. (NASDAQ:SRPT), the leader in precision genetic medicine for rare diseases, today announced that new data from its most advanced gene therapy programs will be presented at the WMS25 Virtual Congress, the 25th International Annual Congress of the World Muscle Society, being held Sept. 28 Oct. 2.

Sarepta will host a webcast and conference call on Monday, Sept. 28, 2020 at 8:30 a.m. ET, to discuss the results, which include two-year functional data from Study 101 of SRP-9001 for Duchenne muscular dystrophy and 18-month functional results from Cohort 1 in the study of SRP-9003 for Limb-girdle muscular dystrophy Type 2E.

This will be webcast live under the investor relations section of Sarepta's website at https://investorrelations.sarepta.com/events-presentationsand will be archived there following the call for one year. Please connect to Sarepta's website several minutes prior to the start of the broadcast to ensure adequate time for any software download that may be necessary. The conference call may be accessed by dialing (844) 534-7313 for domestic callers and (574) 990-1451 for international callers. The passcode for the call is 6793650. Please specify to the operator that you would like to join the "Long-term Functional Data from Sareptas Gene Therapy Programs call.

In total, Sarepta will present 16 abstracts at this years meeting. All posters will be available on-demand throughout the Congress beginning on Monday, Sept. 28 at 7:00 a.m. EST. The full WMS25 Virtual Congress program is available here: https://www.wms2020.com/programme/.

Gene Therapy:

RNA Platform:

Natural history and other presentations:

Presentations will be archived under the events and presentations section of the Sarepta Therapeutics website at http://www.sarepta.comforone year following their presentation at WMS25.

AboutSarepta TherapeuticsAt Sarepta, we are leading a revolution in precision genetic medicine and every day is an opportunity to change the lives of people living with rare disease. The Company has built an impressive position in Duchenne muscular dystrophy (DMD) and in gene therapies for limb-girdle muscular dystrophies (LGMDs), mucopolysaccharidosis type IIIA, Charcot-Marie-Tooth (CMT), and other CNS-related disorders, with more than 40 programs in various stages of development. The Companys programs and research focus span several therapeutic modalities, including RNA, gene therapy and gene editing. For more information, please visitwww.sarepta.com or follow us on Twitter, LinkedIn, Instagram and Facebook.

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We routinely post information that may be important to investors in the 'For Investors' section of our website atwww.sarepta.com. We encourage investors and potential investors to consult our website regularly for important information about us.

Source: Sarepta Therapeutics, Inc.

Sarepta Therapeutics, Inc.

Investors: Ian Estepan, 617-274-4052, iestepan@sarepta.com

Media: Tracy Sorrentino, 617-301-8566, tsorrentino@sarepta.com

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Long-term functional data from Sarepta Therapeutics' Most Advanced Gene Therapy Programs to be Presented at Upcoming Annual Congress of the World...

SAN DIEGO, Sept. 14, 2020 /PRNewswire/ -- Locanabio, Inc., a leader in RNA-targeted gene therapy, today announced that results from a preclinical study of the company's therapeutic systems for the potential treatment of myotonic dystrophy type 1 (DM1) were published in Nature Biomedical Engineering. For the full article, titled "The sustained expression of Cas9 targeting toxic RNAs reverses disease phenotypes in mouse models of myotonic dystrophy," please visit: https://www.nature.com/articles/s41551-020-00607-7

Scientists at Locanabio, working with academic collaborators at UC San Diego School of Medicine and the University of Florida, assessed whether an RNA-targeting CRISPR Cas9 system (RCas9) could provide molecular and functional rescue of dysfunctional RNA processing in a DM1 mouse model. The RCas9 system was administered with one dose of an AAV gene therapy vector. Results in both adult and neonatal mice and using both intramuscular and systemic delivery showed prolonged RCas9 expression even at three months post-injection with efficient reversal of molecular (elimination of toxic RNA foci, MBNL1 redistribution, reversal of splicing biomarkers) and physiological (myotonia) features of DM1.Importantly, there were no significant adverse responses to the treatment.

"These results are consistent with earlier findings from several in vitro studies in muscle cells derived from DM1 patients published by Locanabio's scientific co-founder Dr. Gene Yeo of UC San Diego and further indicate the significant potential of our RNA-targeting gene therapy as a DM1 treatment," said Jim Burns, Ph.D., Chief Executive Officer at Locanabio. "Data show that our RNA-targeting system is able to destroy the toxic RNA at the core of this devastating genetic disease and thereby correct the downstream molecular and biochemical changes that result in myotonia, which is a hallmark symptom of DM1. We are pleased that Nature Biomedical Engineering recognizes the value of these preclinical data and we look forward to further advancing this developmental program to the benefit of DM1 patients."

"Currently available treatments for DM1 can improve specific symptoms but do not target the underlying biology and cause of the disease. These data demonstrate that RNA-targeting systems may efficiently and specifically eliminate toxic RNA repeats that cause DM1 and potentially lead to a more effective treatment option for patients," said Dr. Yeo. "The results also indicate that RNA-targeting gene therapy has potential applications in the treatment of other diseases, such as Huntington's disease and certain genetic forms of ALS, which are also caused by a buildup of toxic RNA repeats."

These studies were funded in part by the Muscular Dystrophy Association (MDA). "We are delighted to support Locanabio's recent work in myotonic dystrophy. These preclinical results represent a promising advance and a novel scientific approach for a group of patients who represent a major unmet medical need," said Sharon Hesterlee, Ph.D., Chief Research Officer, MDA.

About Locanabio, Inc.

Locanabio is the global leader in developing a new class of genetic medicines. Our unique and multi-dimensional approach uses gene therapy to deliver RNA binding protein-based systems to correct the message of disease-causing RNA and thereby change the lives of patients with devastating genetic diseases. These broad capabilities delivered via gene therapy enable Locanabio to potentially address a wide range of severe diseases with a single administration. The company is currently advancing programs in neuromuscular, neurodegenerative and retinal diseases. For more information, visit http://www.locanabio.com.

About Myotonic Dystrophy

Myotonic dystrophy type 1 (DM1) is an autosomal dominant genetic disorder affecting skeletal muscle, cardiac muscle, the gastrointestinal tract, and the central nervous system. DM1 is caused by a mutation in the myotonic dystrophy protein kinase (DMPK) gene. This mutation leads to a repeat expansion of the CTG (cytosine-thymine-guanine) trinucleotide. The expanded CTG is transcribed into toxic CUG (cytosine-uracil-guanine) repeats in the DMPK messenger RNA (mRNA). These toxic mRNA repeats lead to disease symptoms including progressive muscle wasting, weakness and myotonia (delayed relaxation of skeletal muscle), a hallmark of DM1. The incidence of myotonic dystrophy has historically been estimated at one in 8,000 individuals worldwide or approximately 40,000 people in the United States.

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Treatment with RNA-Targeting Gene Therapy Reverses Molecular and Functional Features of Myotonic Dystrophy Type 1 in Mice - PRNewswire

CAMBRIDGE, Mass., Sept. 09, 2020 (GLOBE NEWSWIRE) -- Sarepta Therapeutics, Inc. (NASDAQ:SRPT), the leader in precision genetic medicine for rare diseases, today announced that it has completed a Type C written response only meeting with the Office of Tissues and Advanced Therapies (OTAT), part of the Center for Biologics Evaluation and Research (CBER) at the U.S. Food and Drug Administration (FDA), to obtain OTATs concurrence on the commencement of its next clinical trial for SRP-9001 using commercial process material. SRP-9001 (AAVrh74.MHCK7.micro-dystrophin) is Sareptas investigational gene transfer therapy for the treatment of Duchenne muscular dystrophy.

Among other items, OTAT has requested that Sarepta utilize an additional potency assay for release of SRP-9001 commercial process material prior to dosing in a clinical study. Sarepta has several existing assays and data that it believes could be employed in response to OTATs request. However, additional dialogue with the Agency is required to determine the acceptability of the potency assay approach.

We look forward to working with OTAT to potentially satisfy their requests and to obtain clarity on the timing of the commencement of our commercial supply study. We will provide further updates as we are able, said Doug Ingram, president and chief executive officer, Sarepta Therapeutics. Every day, thousands of children degenerate from the irreversible damage caused by Duchenne muscular dystrophy. It is for that reason that we will work relentlessly with the Division to satisfy any requests of OTAT and continue the advancement of a potentially transformative therapy for these patients.

About SRP-9001 (AAVrh74.MHCK7.micro-dystrophin)SRP-9001 is an investigational gene transfer therapy intended to deliver the micro-dystrophin-encoding gene to muscle tissue for the targeted production of the micro-dystrophin protein. Sarepta is responsible for global development and manufacturing for SRP-9001 and plans to commercialize SRP-9001 in the United States. In December 2019, the Company announced a licensing agreement granting Roche the exclusive right to launch and commercialize SRP-9001 outside the United States. Sarepta has exclusive rights to the micro-dystrophin gene therapy program initially developed at the Abigail Wexner Research Institute at Nationwide Childrens Hospital.

AboutSarepta TherapeuticsAt Sarepta, we are leading a revolution in precision genetic medicine and every day is an opportunity to change the lives of people living with rare disease. The Company has built an impressive position in Duchenne muscular dystrophy (DMD) and in gene therapies for limb-girdle muscular dystrophies (LGMDs), mucopolysaccharidosis type IIIA, Charcot-Marie-Tooth (CMT), and other CNS-related disorders, with more than 40 programs in various stages of development. The Companys programs and research focus span several therapeutic modalities, including RNA, gene therapy and gene editing. For more information, please visitwww.sarepta.com or follow us on Twitter, LinkedIn, Instagram and Facebook.

Sarepta Forward-Looking Statements

This press release contains "forward-looking statements." Any statements contained in this press release that are not statements of historical fact may be deemed to be forward-looking statements. Words such as "believes," "anticipates," "plans," "expects," "will," "intends," "potential," "possible" and similar expressions are intended to identify forward-looking statements. These forward-looking statements include statements regarding Sareptas belief that its existing assays and data could be employed in response to OTATs request; the acceptability of Sareptas potency assay approach by the FDA; our plan to work with OTAT to potentially satisfy their requests and to obtain clarity on the timing of the commencement of our commercial supply study; and the potential of SRP-9001 to be a transformative therapy for DMD patients.

These forward-looking statements involve risks and uncertainties, many of which are beyond Sareptas control. Known risk factors include, among others: delays in the commencement of Sareptas next clinical study for SRP-9001 could delay, prevent or limit our ability to gain regulatory approval for SRP-9001; any inability to complete successfully clinical development could result in additional costs to Sarepta or impair Sareptas ability to generate revenues from product sales, regulatory and commercialization milestones and royalties; SRP-9001 may not result in a viable treatment suitable for commercialization due to a variety of reasons, including the results of future research may not be consistent with past positive results or may fail to meet regulatory approval requirements for the safety and efficacy of product candidates; Sarepta may not be able to execute on its business plans and goals, including meeting its expected or planned regulatory milestones and timelines, clinical development plans, and bringing its product candidates to market, due to a variety of reasons, many of which may be outside of Sareptas control, including possible limitations of company financial and other resources, manufacturing limitations that may not be anticipated or resolved for in a timely manner, regulatory, court or agency decisions, such as decisions by the United States Patent and Trademark Office with respect to patents that cover Sareptas product candidates and the COVID-19 pandemic; and those risks identified under the heading Risk Factors in Sareptas most recent Annual Report on Form 10-K for the year ended December 31, 2019, and most recent Quarterly Report on Form 10-Q filed with the Securities and Exchange Commission (SEC) as well as other SEC filings made by Sarepta which you are encouraged to review.

Any of the foregoing risks could materially and adversely affect Sareptas business, results of operations and the trading price of Sareptas common stock. For a detailed description of risks and uncertainties Sarepta faces, you are encouraged to review the SEC filings made by Sarepta. We caution investors not to place considerable reliance on the forward-looking statements contained in this press release. Sarepta does not undertake any obligation to publicly update its forward-looking statements based on events or circumstances after the date hereof.

Internet Posting of Information

We routinely post information that may be important to investors in the 'For Investors' section of our website atwww.sarepta.com. We encourage investors and potential investors to consult our website regularly for important information about us.

Source: Sarepta Therapeutics, Inc.

Sarepta Therapeutics, Inc.

Investors:Ian Estepan, 617-274-4052iestepan@sarepta.com

Media:Tracy Sorrentino, 617-301-8566tsorrentino@sarepta.com

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Sarepta Therapeutics Provides Program Update for SRP-9001, its Investigational Gene Therapy for the Treatment of Duchenne Muscular Dystrophy -...

PLEASANTON, Calif., Sept. 15, 2020 (GLOBE NEWSWIRE) --10x Genomics (Nasdaq: TXG) today announced it has begun shipping its Chromium Single Cell Multiome ATAC + Gene Expression solution to customers, marking the first commercial release of a product capable of simultaneously profiling the epigenome and transcriptome from the same single cell. This multi-omic approach provides customers with the ability to link a cells epigenetic program to its transcriptional output, enabling a better understanding of cell functionality and bypassing the need to infer relationships through computer simulations.

This is one of our most ambitious undertakings at the company, said Ben Hindson, co-founder and Chief Scientific Officer of 10x Genomics. By introducing the first solution that captures ATAC and gene expression simultaneously, researchers can gain even more clarity by combining two already powerful methods to profile biological systems at single cell resolution simultaneously for the first time.

The new solution builds on an array of new products launched by the company this year for both its Chromium platform for single cell analysis as well as its Visium platform for spatial genomics. Early customers already working with Chromium Single Cell Multiome ATAC + Gene Expression include Stanford University School of Medicine, Icahn School of Medicine at Mt. Sinai and Spains Centro Nacional de Anlisis Genmico.

My lab is interested in understanding why some immune cell types fail to fight the cancer, said Dr. Ansuman Satpathy, Assistant Professor of Pathology, Stanford University School of Medicine. We plan to use 10x Genomics' new assay to understand the epigenetic and transcriptional regulation of immune cell dysfunction directly in patient samples, and to use this information to precisely engineer more effective immunotherapies in the future.

Until now, we have relied on computational prediction to match a cell's epigenome to a single-cell gene expression profile, said Dr. Holger Heyn, leader of the single cell genomics team at Spains Centro Nacional de Anlisis Genmico that is working on delineating the dynamics underlying B-cell differentiation and activation. 10x Genomics new multiome assay will allow us to directly measure what before could only be predicted, and offers a new gold standard that will confirm how accurate these predictions had been.

"With this new technology, we can better understand the mechanisms affected by the non-coding risk genetic variation across a wide range of neuropsychiatric diseases, including Alzheimers, Parkinsons, Schizophrenia, bipolar disorder and major depression, along with different severity of neuropathology and clinical symptomatology," added Dr. Panagiotis Roussos, Associate Professor of Genetics and Genomics Sciences, Icahn School of Medicine at Mount Sinai.

By using Chromium Single Cell Multiome ATAC + Gene Expression, researchers can:

Chromium Single Cell Multiome ATAC + Gene Expression is shipping to customers. To learn more, visit https://www.10xgenomics.com/products/single-cell-multiome-atac-plus-gene-expression.

About 10x Genomics10x Genomics is a life science technology company building products to interrogate, understand and master biology to advance human health. The companys integrated solutions include instruments, consumables and software for analyzing biological systems at a resolution and scale that matches the complexity of biology. 10x Genomics products have been adopted by researchers around the world including 97 of the top 100 global research institutions and 19 of the top 20 global pharmaceutical companies, and have been cited in over 1,500 research papers on discoveries ranging from oncology to immunology and neuroscience. The companys patent portfolio comprises more than 775 issued patents and patent applications.

Forward Looking StatementsThis press release contains forward-looking statements within the meaning of the Private Securities Litigation Reform Act of 1995 as contained in Section 27A of the Securities Act of 1933, as amended, and Section 21E of the Securities Exchange Act of 1934, as amended. Forward-looking statements generally can be identified by the use of forward-looking terminology such as may, will, should, expect, plan, anticipate, could, intend, target, project contemplate, believe, estimate, predict, potential or continue or the negatives of these terms or variations of them or similar terminology. These forward-looking statements include statements regarding 10x Genomics, Inc.s partnership activities, which involve risks and uncertainties that could cause 10x Genomics, Inc.s actual results to differ materially from the anticipated results and expectations expressed in these forward-looking statements. These statements are based on managements current expectations, forecasts, beliefs, assumptions and information currently available to management, and actual outcomes and results could differ materially from these statements due to a number of factors. These and additional risks and uncertainties that could affect 10x Genomics, Inc.s financial and operating results and cause actual results to differ materially from those indicated by the forward-looking statements made in this press release include those discussed under the captions "Risk Factors" and "Management's Discussion and Analysis of Financial Condition and Results of Operations" and elsewhere in the documents 10x Genomics, Inc. files with the Securities and Exchange Commission from time to time. The forward-looking statements in this press release are based on information available to 10x Genomics, Inc. as of the date hereof, and 10x Genomics, Inc. disclaims any obligation to update any forward-looking statements provided to reflect any change in its expectations or any change in events, conditions, or circumstances on which any such statement is based, except as required by law. These forward-looking statements should not be relied upon as representing 10x Genomics, Inc.s views as of any date subsequent to the date of this press release.

Disclosure Information10x Genomics uses filings with the Securities and Exchange Commission, its website (www.10xgenomics.com), press releases, public conference calls, public webcasts and its social media accounts as means of disclosing material non-public information and for complying with its disclosure obligations under Regulation FD.

ContactsMedia:media@10xgenomics.comInvestors:investors@10xgenomics.com

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10x Genomics First to Market With Product to Simultaneously Capture Epigenome and TranscriptomeChromium Single Cell Multiome ATAC + Gene Expression...