Design and Characterization of a Novel Intravitreal Dual-Transgene Genetic Medicine for Neovascular Retinopathies.

Purpose: Intravitreal delivery of therapeutic transgenes to the retina via engineered viral vectors can provide sustained local concentrations of therapeutic proteins and thus potentially reduce the treatment burden and improve long-term vision outcomes for patients with neovascular (wet) age-related macular degeneration (AMD), diabetic macular edema (DME), and diabetic retinopathy.

Methods: We performed directed evolution in nonhuman primates (NHP) to invent an adeno-associated viral (AAV) variant (R100) with the capacity to cross vitreoretinal barriers and transduce all regions and layers of the retina following intravitreal injection. We then engineered 4D-150, an R100-based genetic medicine carrying 2 therapeutic transgenes: a codon-optimized sequence encoding aflibercept, a recombinant protein that inhibits VEGF-A, VEGF-B, and PlGF, and a microRNA sequence that inhibits expression of VEGF-C.

Multimodal evaluation of network activity and optogenetic interventions in human hippocampal slices.

Seizures are made up of the coordinated activity of networks of neurons, suggesting that control of neurons in the pathologic circuits of epilepsy could allow for control of the disease. Optogenetics has been effective at stopping seizure-like activity in non-human disease models by increasing inhibitory tone or decreasing excitation, although this effect has not been shown in human brain tissue. Many of the genetic means for achieving channelrhodopsin expression in non-human models are not possible in humans, and vector-mediated methods are susceptible to species-specific tropism that may affect translational potential.

Engineering novel adeno-associated viruses (AAVs) for improved delivery in the nervous system.

Harnessing adeno-associated virus (AAV) vectors for therapeutic gene delivery has emerged as a progressively promising strategy to treat disorders of both the central nervous system (CNS) and peripheral nervous system (PNS), and there are many ongoing clinical trials. However, unique physiological and molecular characteristics of the CNS and PNS pose obstacles to efficient vector delivery, ranging from the blood-brain barrier to the diverse nature of nervous system disorders. Engineering novel AAV capsids may help overcome these ongoing challenges and maximize therapeutic transgene delivery.

Spectrin mediates 3D-specific matrix stress-relaxation response in neural stem cell lineage commitment.

While extracellular matrix (ECM) stress relaxation is increasingly appreciated to regulate stem cell fate commitment and other behaviors, much remains unknown about how cells process stress-relaxation cues in tissue-like three-dimensional (3D) geometries versus traditional 2D cell culture. Here, we develop an oligonucleotide-crosslinked hyaluronic acid-based ECM platform with tunable stress relaxation properties capable of use in either 2D or 3D. Strikingly, stress relaxation favors neural stem cell (NSC) neurogenesis in 3D but suppresses it in 2D.

Substrate stress relaxation regulates neural stem cell fate commitment.

Adult neural stem cells (NSCs) reside in the dentate gyrus of the hippocampus, and their capacity to generate neurons and glia plays a role in learning and memory. In addition, neurodegenerative diseases are known to be caused by a loss of neurons and glial cells, resulting in a need to better understand stem cell fate commitment processes. We previously showed that NSC fate commitment toward a neuronal or glial lineage is strongly influenced by extracellular matrix stiffness, a property of elastic materials.

Electrospray deposition of physical unclonable functions for drug anti-counterfeiting.

In recent years, pharmaceutical counterfeiting has become an increasingly dangerous situation. A patient who unknowingly consumes a counterfeit drug is at a serious health risk. To address this problem, a low-cost and robust approach for authentication that can be administered at the point-of-care is required.

Flow in temporally and spatially varying porous media: a model for transport of interstitial fluid in the brain.

Flow in a porous medium can be driven by the deformations of the boundaries of the porous domain. Such boundary deformations locally change the volume fraction accessible by the fluid, creating non-uniform porosity and permeability throughout the medium. In this work, we construct a deformation-driven porous medium transport model with spatially and temporally varying porosity and permeability that are dependent on the boundary deformations imposed on the medium.

Dynamic light-responsive RhoA activity regulates mechanosensitive stem cell fate decision in 3D matrices.

The behavior of stem cells is regulated by mechanical cues in their niche that continuously vary due to extracellular matrix (ECM) remodeling, pulsated mechanical stress exerted by blood flow, and/or cell migration. However, it is still unclear how dynamics of mechanical cues influence stem cell lineage commitment, especially in a 3D microenvironment where mechanosensing differs from that in a 2D microenvironment. In the present study, we investigated how temporally varying mechanical signaling regulates expression of the early growth response 1 gene (Egr1), which we recently discovered to be a 3D matrix-specific mediator of mechanosensitive neural stem cell (NSC) lineage commitment.

Computationally guided AAV engineering for enhanced gene delivery.

Gene delivery vehicles based on adeno-associated viruses (AAVs) are enabling increasing success in human clinical trials, and they offer the promise of treating a broad spectrum of both genetic and non-genetic disorders. However, delivery efficiency and targeting must be improved to enable safe and effective therapies. In recent years, considerable effort has been invested in creating AAV variants with improved delivery, and computational approaches have been increasingly harnessed for AAV engineering.

Gravity-perfused airway-on-a-chip optimized for quantitative BSL-3 studies of SARS-CoV-2 infection: barrier permeability, cytokine production, immunohistochemistry, and viral load assays.

Human microphysiological systems, such as organs on chips, are an emerging technology for modeling human physiology in a preclinical setting to understand the mechanism of action of drugs, to evaluate the efficacy of treatment options for human disease and impairment, and to assess drug toxicity. By using human cells co-cultured in three-dimensional constructs, organ chips can provide greater fidelity to the human cellular condition than their two-dimensional predecessors. However, with the rise of SARS-CoV-2 and the global COVID-19 pandemic, it became clear that many microphysiological systems were not compatible with or optimized for studies of infectious disease and operation in a Biosafety Level 3 (BSL-3) environment.

Optimal trade-off control in machine learning-based library design, with application to adeno-associated virus (AAV) for gene therapy.

Adeno-associated viruses (AAVs) hold tremendous promise as delivery vectors for gene therapies. AAVs have been successfully engineered-for instance, for more efficient and/or cell-specific delivery to numerous tissues-by creating large, diverse starting libraries and selecting for desired properties. However, these starting libraries often contain a high proportion of variants unable to assemble or package their genomes, a prerequisite for any gene delivery goal.

Evolving membrane-associated accessory protein variants for improved adeno-associated virus production.

Manufacturing sufficient adeno-associated virus (AAV) to meet current and projected clinical needs is a significant hurdle to the growing gene therapy industry. The recently discovered membrane-associated accessory protein (MAAP) is encoded by an alternative open reading frame in the AAV cap gene that is found in all presently reported natural serotypes. Recent evidence has emerged supporting a functional role of MAAP in AAV egress, although the underlying mechanisms of MAAP function remain unknown.

From garages to ecosystems: the coevolution of life science incubators and accelerators.

Incubators and accelerators catalyze the launch of life science startups and have evolved from simple facilities to vibrant ecosystems offering research infrastructure, programs, and funding. Analysis of financing activities indicates the outperformance of incubator companies relative to accelerators in fundraising, mergers and acquisitions (M&As), and initial public offerings (IPOs), attributed to extended interactions with investors and peers.

Optogenetic control of Wnt signaling models cell-intrinsic embryogenic patterning using 2D human pluripotent stem cell culture.

In embryonic stem cell (ESC) models for early development, spatially and temporally varying patterns of signaling and cell types emerge spontaneously. However, mechanistic insight into this dynamic self-organization is limited by a lack of methods for spatiotemporal control of signaling, and the relevance of signal dynamics and cell-to-cell variability to pattern emergence remains unknown. Here, we combine optogenetic stimulation, imaging and transcriptomic approaches to study self-organization of human ESCs (hESC) in two-dimensional (2D) culture.

Mechanosensitive stem cell fate choice is instructed by dynamic fluctuations in activation of Rho GTPases.

During the intricate process by which cells give rise to tissues, embryonic and adult stem cells are exposed to diverse mechanical signals from the extracellular matrix (ECM) that influence their fate. Cells can sense these cues in part through dynamic generation of protrusions, modulated and controlled by cyclic activation of Rho GTPases. However, it remains unclear how extracellular mechanical signals regulate Rho GTPase activation dynamics and how such rapid, transient activation dynamics are integrated to yield long-term, irreversible cell fate decisions.

Application of a Human Blood Brain Barrier Organ-on-a-Chip Model to Evaluate Small Molecule Effectiveness against Venezuelan Equine Encephalitis Virus.

The blood brain barrier (BBB) is a multicellular microenvironment that plays an important role in regulating bidirectional transport to and from the central nervous system (CNS). Infections by many acutely infectious viruses such as alphaviruses and flaviviruses are known to impact the integrity of the endothelial lining of the BBB. Infection by Venezuelan Equine Encephalitis Virus (VEEV) through the aerosol route causes significant damage to the integrity of the BBB, which contributes to long-term neurological sequelae.

N-Cadherin adhesive ligation regulates mechanosensitive neural stem cell lineage commitment in 3D matrices.

During differentiation, neural stem cells (NSCs) encounter diverse cues from their niche, including not only biophysical cues from the extracellular matrix (ECM) but also cell-cell communication. However, it is still poorly understood how these cues cumulatively regulate mechanosensitive NSC fate commitment, especially in 3D matrices that better mimic systems. Here, we develop a click chemistry-based 3D hydrogel material system to fully decouple cell-cell and cell-ECM interactions by functionalizing small peptides: the HAVDI motif from N-cadherin and RGD motif from fibronectin.

Apodization Specific Fitting for Improved Resolution, Charge Measurement, and Data Analysis Speed in Charge Detection Mass Spectrometry.

Short-time Fourier transforms with short segment lengths are typically used to analyze single ion charge detection mass spectrometry (CDMS) data either to overcome effects of frequency shifts that may occur during the trapping period or to more precisely determine the time at which an ion changes mass or charge, or enters an unstable orbit. The short segment lengths can lead to scalloping loss unless a large number of zero-fills are used, making computational time a significant factor in real-time analysis of data. Apodization specific fitting leads to a 9-fold reduction in computation time compared to zero-filling to a similar extent of accuracy.

A scalable and tunable thermoreversible polymer for 3D human pluripotent stem cell biomanufacturing.

Human pluripotent stem cells (hPSCs) are an exciting and promising source to enable cell replacement therapies for a variety of unmet medical needs. Though hPSCs can be successfully derived into numerous physiologically relevant cell types, effective translation to the clinic is limited by challenges in scalable production of high-quality cells, cellular immaturity following the differentiation process, and the use of animal-derived components in culture. To address these limitations, we have developed a fully defined, reproducible, and tunable thermoreversible polymer for high-quality, scalable 3D cell production.

Effects of Molecular Size on Resolution in Charge Detection Mass Spectrometry.

Instrumental resolution of Fourier transform-charge detection mass spectrometry instruments with electrostatic ion trap detection of individual ions depends on the precision with which ion energy is determined. Energy can be selected using ion optic filters or from harmonic amplitude ratios (HARs) that provide Fellgett's advantage and eliminate the necessity of ion transmission loss to improve resolution. Unlike the ion energy-filtering method, the resolution of the HAR method increases with charge (improved /) and thus with mass.

A reductionist approach to determine the effect of cell-cell contact on human epidermal stem cell differentiation.

The balance between stem cell renewal and differentiation is determined by the interplay between intrinsic cellular controls and extrinsic factors presented by the microenvironment, or 'niche'. Previous studies on cultured human epidermis have utilised suspension culture and restricted cell spreading to investigate regulation of differentiation in single keratinocytes. However, keratinocytes are typically adherent to neighbouring cells in vivo.