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. In this review, we discuss how computationally designed AAV libraries are enabling directed evolution. Specifically, we highlight approaches that harness sequences outputted by next-generation sequencing (NGS) coupled with machine learning (ML) to generate new functional AAV capsids and related regulatory elements, pushing the frontier of what vector engineering and gene therapy may achieve.

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. However, tissues in vivo are not purely elastic and have varying degrees of viscous character. Relatively little is known about how the viscoelastic properties of the substrate impact NSC fate commitment. Here, we introduce a polyacrylamide-based cell culture platform that incorporates mismatched DNA oligonucleotide-based cross-links as well as covalent cross-links. This platform allows for tunable viscous stress relaxation properties via variation in the number of mismatched base pairs. We find that NSCs exhibit increased astrocytic differentiation as the degree of stress relaxation is increased. Furthermore, culturing NSCs on increasingly stress-relaxing substrates impacts cytoskeletal dynamics by decreasing intracellular actin flow rates and stimulating cyclic activation of the mechanosensitive protein RhoA. Additionally, inhibition of motor-clutch model components such as myosin II and focal adhesion kinase partially or completely reverts cells to lineage distributions observed on elastic substrates. Collectively, our results introduce a unique system for controlling matrix stress relaxation properties and offer insight into how NSCs integrate viscoelastic cues to direct fate commitment.

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. Morphogen dynamics were controlled via optogenetic activation of canonical Wnt/ß-catenin signaling (optoWnt), which drove broad transcriptional changes and mesendoderm differentiation at high efficiency (>99% cells). When activated within cell subpopulations, optoWnt induced cell self-organization into distinct epithelial and mesenchymal domains, mediated by changes in cell migration, an epithelial to mesenchymal-like transition and TGFß signaling. Furthermore, we demonstrate that such optogenetic control of cell subpopulations can be used to uncover signaling feedback mechanisms between neighboring cell types. These findings reveal that cell-to-cell variability in Wnt signaling is sufficient to generate tissue-scale patterning and establish a hESC model system for investigating feedback mechanisms relevant to early human embryogenesis.

Network news: innovations in 21st century systems biology.

A decade ago, seminal perspectives and papers set a strong vision for the field of systems biology, and a number of these themes have flourished. Here, we describe key technologies and insights that have elucidated the evolution, architecture, and function of cellular networks, ultimately leading to the first predictive genome-scale regulatory and metabolic models of organisms. Can systems approaches bridge the gap between correlative analysis and mechanistic insights?

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. Here, we report that ECM stiffness cues alter not only the magnitude but also the temporal frequency of RhoA and Cdc42 activation in adult neural stem cells (NSCs). Using optogenetics to control the frequency of RhoA and Cdc42 activation, we further demonstrate that these dynamics are functionally significant, where high- vs. low-frequency activation of RhoA and Cdc42 drives astrocytic vs. neuronal differentiation, respectively. In addition, high-frequency Rho GTPase activation induces sustained phosphorylation of the TGFß pathway effector SMAD1, which in turn drives the astrocytic differentiation. By contrast, under low-frequency Rho GTPase stimulation, cells fail to accumulate SMAD1 phosphorylation and instead undergo neurogenesis. Our findings reveal the temporal patterning of Rho GTPase signaling and the resulting accumulation of an SMAD1 signal as a critical mechanism through which ECM stiffness cues regulate NSC fate.

A microfluidic system that replicates pharmacokinetic (PK) profiles in vitro improves prediction of in vivo efficacy in preclinical models.

Test compounds used on in vitro model systems are conventionally delivered to cell culture wells as fixed concentration bolus doses; however, this poorly replicates the pharmacokinetic (PK) concentration changes seen in vivo and reduces the predictive value of the data. Herein, proof-of-concept experiments were performed using a novel microfluidic device, the Microformulator, which allows in vivo like PK profiles to be applied to cells cultured in microtiter plates and facilitates the investigation of the impact of PK on biological responses. We demonstrate the utility of the device in its ability to reproduce in vivo PK profiles of different oncology compounds over multiweek experiments, both as monotherapy and drug combinations, comparing the effects on tumour cell efficacy in vitro with efficacy seen in in vivo xenograft models. In the first example, an ERK1/2 inhibitor was tested using fixed bolus dosing and Microformulator-replicated PK profiles, in 2 cell lines with different in vivo sensitivities. The Microformulator-replicated PK profiles were able to discriminate between cell line sensitivities, unlike the conventional fixed bolus dosing. In a second study, murine in vivo PK profiles of multiple Poly(ADP-Ribose) Polymerase 1/2 (PARP) and DNA-dependent protein kinase (DNA-PK) inhibitor combinations were replicated in a FaDu cell line resulting in a reduction in cell growth in vitro with similar rank ordering to the in vivo xenograft model. Additional PK/efficacy insight into theoretical changes to drug exposure profiles was gained by using the Microformulator to expose FaDu cells to the DNA-PK inhibitor for different target coverage levels and periods of time. We demonstrate that the Microformulator enables incorporating PK exposures into cellular assays to improve in vitro-in vivo translation understanding for early therapeutic insight.

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. Here, we show that inactivation of MAAP from AAV2 by a single point mutation that is silent in the VP1 open reading frame (ORF) (AAV2-ΔMAAP) decreased exosome-associated and secreted vector genome production. We hypothesized that novel MAAP variants could be evolved to increase AAV production and thus subjected a library encoding over 1 × 106 MAAP protein variants to five rounds of packaging selection into the AAV2-ΔMAAP capsid. Between each successive packaging round, we observed a progressive increase in both overall titer and ratio of secreted vector genomes conferred by the bulk-selected MAAP library population. Next-generation sequencing uncovered enriched mutational features, and a resulting selected MAAP variant containing missense mutations and a frameshifted C-terminal domain increased overall GFP transgene packaging in AAV2, AAV6, and AAV9 capsids.

CRISPR-guided DNA polymerases enable diversification of all nucleotides in a tunable window.

The capacity to diversify genetic codes advances our ability to understand and engineer biological systems1,2. A method for continuously diversifying user-defined regions of a genome would enable forward genetic approaches in systems that are not amenable to efficient homology-directed oligonucleotide integration. It would also facilitate the rapid evolution of biotechnologically useful phenotypes through accelerated and parallelized rounds of mutagenesis and selection, as well as cell-lineage tracking through barcode mutagenesis. Here we present EvolvR, a system that can continuously diversify all nucleotides within a tunable window length at user-defined loci. This is achieved by directly generating mutations using engineered DNA polymerases targeted to loci via CRISPR-guided nickases. We identified nickase and polymerase variants that offer a range of targeted mutation rates that are up to 7,770,000-fold greater than rates seen in wild-type cells, and editing windows with lengths of up to 350 nucleotides. We used EvolvR to identify novel ribosomal mutations that confer resistance to the antibiotic spectinomycin. Our results demonstrate that CRISPR-guided DNA polymerases enable multiplexed and continuous diversification of user-defined genomic loci, which will be useful for a broad range of basic and biotechnological applications.

Presentation counts: microenvironmental regulation of stem cells by biophysical and material cues.

Stem cells reside in adult and embryonic tissues in a broad spectrum of developmental stages and lineages, and they are thus naturally exposed to diverse microenvironments or niches that modulate their hallmark behaviors of self-renewal and differentiation into one or more mature lineages. Within each such microenvironment, stem cells sense and process multiple biochemical and biophysical cues, which can exert redundant, competing, or orthogonal influences to collectively regulate cell fate and function. The proper presentation of these myriad regulatory signals is required for tissue development and homeostasis, and their improper appearance can potentially lead to disease. Whereas these complex regulatory cues can be challenging to dissect using traditional cell culture paradigms, recently developed engineered material systems offer advantages for investigating biochemical and biophysical cues, both static and dynamic, in a controlled, modular, and quantitative fashion. Advances in the development and use of such systems have helped elucidate novel regulatory mechanisms controlling stem cell behavior, particularly the importance of solid-phase mechanical and immobilized biochemical microenvironmental signals, with implications for basic stem cell biology, disease, and therapeutics.

ß-Catenin signaling dynamics regulate cell fate in differentiating neural stem cells.

Stem cells undergo differentiation in complex and dynamic environments wherein instructive signals fluctuate on various timescales. Thus, cells must be equipped to properly respond to the timing of signals, for example, to distinguish sustained signaling from transient noise. However, how stem cells respond to dynamic variations in differentiation cues is not well characterized. Here, we use optogenetic activation of ß-catenin signaling to probe the dynamic responses of differentiating adult neural stem cells (NSCs). We discover that, while elevated, sustained ß-catenin activation sequentially promotes proliferation and differentiation, transient ß-catenin induces apoptosis. Genetic perturbations revealed that the neurogenic/apoptotic fate switch was mediated through cell-cycle regulation by Growth Arrest and DNA Damage 45 gamma (Gadd45γ). Our results thus reveal a role for ß-catenin dynamics in NSC fate decisions and may suggest a role for signal timing to minimize cell-fate errors, analogous to kinetic proofreading of stem-cell differentiation.

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 S/N) and thus with mass. An analysis of the HAR method with current instrumentation indicates that higher resolution can be obtained with the HAR method than the best resolution demonstrated for instruments with energy-selective optics for ions in the low MDa range and above. However, this gain is typically unrealized because the resolution obtainable with molecular systems in this mass range is limited by sample heterogeneity. This phenomenon is illustrated with both tobacco mosaic virus (0.6-2.7 MDa) and AAV9 (3.7-4.7 MDa) samples where mass spectral resolution is limited by the sample, including salt adducts, and not by instrument resolution. Nevertheless, the ratio of full to empty AAV9 capsids and the included genome mass can be accurately obtained in a few minutes from 1× PBS buffer solution and an elution buffer containing 300+ mM nonvolatile content despite extensive adduction and lower resolution. Empty and full capsids adduct similarly indicating that salts encrust the complexes during late stages of droplet evaporation and that mass shifts can be calibrated in order to obtain accurate analyte masses even from highly salty solutions.

A bistable, multiport valve enables microformulators creating microclinical analyzers that reveal aberrant glutamate metabolism in astrocytes derived from a tuberous sclerosis patient.

There is a need for valves and pumps that operate at the microscale with precision and accuracy, are versatile in their application, and are easily fabricated. To that end, we developed a new rotary planar multiport valve to faithfully select solutions (contamination = 5.22 ± 0.06 ppb) and a rotary planar peristaltic pump to precisely control fluid delivery (flow rate = 2.4 ± 1.7 to 890 ± 77 µL/min). Both the valve and pump were implemented in a planar format amenable to single-layer soft lithographic fabrication. These planar microfluidics were actuated by a rotary motor controlled remotely by custom software. Together, these two devices constitute an innovative microformulator that was used to prepare precise, high-fidelity mixtures of up to five solutions (deviation from prescribed mixture = ±|0.02 ± 0.02| %). This system weighed less than a kilogram, occupied around 500 cm3, and generated pressures of 255 ± 47 kPa. This microformulator was then combined with an electrochemical sensor creating a microclinical analyzer (µCA) for detecting glutamate in real time. Using the chamber of the µCA as an in-line bioreactor, we compared glutamate homeostasis in human astrocytes differentiated from human-induced pluripotent stem cells (hiPSCs) from a control subject (CC-3) and a Tuberous Sclerosis Complex (TSC) patient carrying a pathogenic TSC2 mutation. When challenged with glutamate, TSC astrocytes took up less glutamate than control cells. These data validate the analytical power of the µCA and the utility of the microformulator by leveraging it to assess disease-related alterations in cellular homeostasis.

Advanced Materials to Enhance Central Nervous System Tissue Modeling and Cell Therapy.

The progressively deeper understanding of mechanisms underlying stem cell fate decisions has enabled parallel advances in basic biology-such as the generation of organoid models that can further one's basic understanding of human development and disease-and in clinical translation-including stem cell based therapies to treat human disease. Both of these applications rely on tight control of the stem cell microenvironment to properly modulate cell fate, and materials that can be engineered to interface with cells in a controlled and tunable manner have therefore emerged as valuable tools for guiding stem cell growth and differentiation. With a focus on the central nervous system (CNS), a broad range of material solutions that have been engineered to overcome various hurdles in constructing advanced organoid models and developing effective stem cell therapeutics is reviewed. Finally, regulatory aspects of combined material-cell approaches for CNS therapies are considered.

Incidence and Early Course of Retinopathy of Prematurity.

In the Multicenter Trial of Cryotherapy for Retinopathy of Prematurity (ROP), 4099 infants weighing less than 1251 g at birth underwent sequential ophthalmic examinations, beginning at age 4 to 6 weeks, to monitor the incidence and course of ROP. Overall, 65.8% of the infants developed ROP to some degree; 81.6% for infants of less than 1000 g birth weight. As expected, ROP incidence and severity were higher in lower birth weight and gestational age categories. Black infants appeared less susceptible to ROP, of all severity categories, than nonblack infants. The timing of retinal vascular events correlated more closely with postconceptional age than with postnatal age, implicating the level of maturity more than postnatal environmental influences in governing the timing of these vascular events. These results include the current incidence of various severity stages of ROP found in the United States and provide new. insight into the development of ROP.

Gene Editing to Generate Versatile Human Pluripotent Stem Cell Reporter Lines for Analysis of Differentiation and Lineage Tracing.

Transcription factors (TFs) are potent proteins that control gene expression and can thereby drive cell fate decisions. Fluorescent reporters have been broadly knocked into endogenous TF loci to investigate the biological roles of these factors; however, the sensitivity of such analyses in human pluripotent stem cells (hPSCs) is often compromised by low TF expression levels and/or reporter silencing. Complementarily, we report an inducible and quantitative reporter platform based on the Cre-LoxP recombination system that enables robust, quantifiable, and continuous monitoring of live hPSCs and their progeny to investigate the roles of TFs during human development and disease. Stem Cells 2019;37:1556-1566.

Engineering adeno-associated viruses for clinical gene therapy.

Clinical gene therapy has been increasingly successful owing both to an enhanced molecular understanding of human disease and to progressively improving gene delivery technologies. Among these technologies, delivery vectors based on adeno-associated viruses (AAVs) have emerged as safe and effective and, in one recent case, have led to regulatory approval. Although shortcomings in viral vector properties will render extension of such successes to many other human diseases challenging, new approaches to engineer and improve AAV vectors and their genetic cargo are increasingly helping to overcome these barriers.

Kawasaki Disease Features and Myocarditis in a Patient with COVID-19.

A 10-year-old male with prolonged fever, rash, and conjunctivitis presented to the emergency department with concern for Kawasaki disease, found to have myocarditis and PCR positive for SARS-CoV-2.

High-throughput identification of factors promoting neuronal differentiation of human neural progenitor cells in microscale 3D cell culture.

Identification of conditions for guided and specific differentiation of human stem cell and progenitor cells is important for continued development and engineering of in vitro cell culture systems for use in regenerative medicine, drug discovery, and human toxicology. Three-dimensional (3D) and organotypic cell culture models have been used increasingly for in vitro cell culture because they may better model endogenous tissue environments. However, detailed studies of stem cell differentiation within 3D cultures remain limited, particularly with respect to high-throughput screening. Herein, we demonstrate the use of a microarray chip-based platform to screen, in high-throughput, individual and paired effects of 12 soluble factors on the neuronal differentiation of a human neural progenitor cell line (ReNcell VM) encapsulated in microscale 3D Matrigel cultures. Dose-response analysis of selected combinations from the initial combinatorial screen revealed that the combined treatment of all-trans retinoic acid (RA) with the glycogen synthase kinase 3 inhibitor CHIR-99021 (CHIR) enhances neurogenesis while simultaneously decreases astrocyte differentiation, whereas the combined treatment of brain-derived neurotrophic factor and the small azide neuropathiazol enhances the differentiation into neurons and astrocytes. Subtype specification analysis of RA- and CHIR-differentiated cultures revealed that enhanced neurogenesis was not biased toward a specific neuronal subtype. Together, these results demonstrate a high-throughput screening platform for rapid evaluation of differentiation conditions in a 3D environment, which will aid the development and application of 3D stem cell culture models.