Cell replacement therapies have broad biomedical potential; however, low cell survival and poor functional integration post-transplantation are major hurdles that hamper clinical benefit. For example, following striatal transplantation of midbrain dopaminergic (mDA) neurons for the treatment of Parkinson's disease (PD), only 1–5% of the neurons typically survive in preclinical models and in clinical trials. In general, resource-intensive generation and implantation of larger numbers of cells are used to compensate for the low post-transplantation cell-survival. Poor graft survival is often attributed to adverse biochemical, mechanical, and/or immunological stress that cells experience during and after implantation. To address these challenges, we developed a functionalized hyaluronic acid (HA)-based hydrogel for in vitro maturation and central nervous system (CNS) transplantation of human pluripotent stem cell (hPSC)-derived neural progenitors. Specifically, we functionalized the HA hydrogel with RGD and heparin (hep) via click-chemistry and tailored its stiffness to encourage neuronal maturation, survival, and long-term maintenance of the desired mDA phenotype. Importantly, ∼5 times more hydrogel-encapsulated mDA neurons survived after transplantation in the rat striatum, compared to unencapsulated neurons harvested from commonly used 2D surfaces. This engineered biomaterial may therefore increase the therapeutic potential and reduce the manufacturing burden for successful neuronal implantation.

•A defined and scalable 3D system accelerates the differentiation of OPCs from hPSCs•A NKX2.2-EGFP hESC reporter line enables optimization of OPC differentiation•3D-derived OPCs engraft, migrate, and mature after implantation into NOD/SCID miceOligodendrocyte precursor cells (OPCs) offer considerable potential for the treatment of demyelinating diseases and injuries of the CNS. However, generating large quantities of high-quality OPCs remains a substantial challenge that impedes their therapeutic application. Here, we show that OPCs can be generated from human pluripotent stem cells (hPSCs) in a three-dimensional (3D), scalable, and fully defined thermoresponsive biomaterial system. We used CRISPR/Cas9 to create a NKX2.2-EGFP human embryonic stem cell reporter line that enabled fine-tuning of early OPC specification and identification of conditions that markedly increased the number of OLIG2+ and NKX2.2+ cells generated from hPSCs. Transplantation of 50-day-old OPCs into the brains of NOD/SCID mice revealed that progenitors generated in 3D without cell selection or purification subsequently engrafted, migrated, and matured into myelinating oligodendrocytes in vivo. These results demonstrate the potential of harnessing lineage reporter lines to develop 3D platforms for rapid and large-scale production of OPCs.Download high-res image (222KB)Download full-size image

•Neural stem cells (NSCs) are a promising resource for tissue engineering and regenerative medicine.•Various mechanical cues can affect NSC differentiation, proliferation, and maturation.•Biomaterial advancements are driving new discoveries in NSC mechanotransduction.•Understanding biophysical regulation of NSCs is crucial for designing novel cell replacement strategies.Neural stem cells (NSCs) are a valuable cell source for tissue engineering, regenerative medicine, disease modeling, and drug screening applications. Analogous to other stem cells, NSCs are tightly regulated by their microenvironmental niche, and prior work utilizing NSCs as a model system with engineered biomaterials has offered valuable insights into how biophysical inputs can regulate stem cell proliferation, differentiation, and maturation. In this review, we highlight recent exciting studies with innovative material platforms that enable narrow stiffness gradients, mechanical stretching, temporal stiffness switching, and three-dimensional culture to study NSCs. These studies have significantly advanced our knowledge of how stem cells respond to an array of different biophysical inputs and the underlying mechanosensitive mechanisms. In addition, we discuss efforts to utilize engineered material scaffolds to improve NSC-based translational efforts and the importance of mechanobiology in tissue engineering applications.Download high-res image (283KB)Download full-size image

•A novel AAV directed evolution approach to target parental and HIV-1-infected cells.•Triple infection of H9 T cells with AAV, adenovirus, and HIV-1 was successful.•Novel AAV capsids were isolated that show increased tropism toward the target cells.A directed evolution approach was used to select for Adeno-associated virus (AAV) capsids that would exhibit more tropism toward an HIV-1 producer T cell line with the long-term goal of developing improved gene transfer vectors. A library of AAV variants was used to infect H9 T cells previously infected or uninfected by HIV-1 followed by AAV amplification with wild-type adenovirus. Six rounds of biological selection were performed, including negative selection and diversification after round three. The H9 T cells were successfully infected with all three wild-type viruses (AAV, adenovirus, and HIV-1). Four AAV cap mutants best representing the small number of variants emerging after six rounds of selection were chosen for further study. These mutant capsids were used to package an AAV vector and subsequently used to infect H9 cells that were previously infected or uninfected by HIV-1. A quantitative polymerase chain reaction assay was performed to measure cell-associated AAV genomes. Two of the four cap mutants showed a significant increase in the amount of cell-associated genomes as compared to wild-type AAV2. This study shows that directed evolution can be performed successfully to select for mutants with improved tropism for a T cell line in the presence of HIV-1.

Adeno-associated virus (AAV) vectors have achieved clinical efficacy in treating several diseases. However, enhanced vectors are required to extend these landmark successes to other indications and protein engineering approaches may provide the necessary vector improvements to address such unmet medical needs. To generate new capsid variants with potentially enhanced infectious properties and to gain insights into AAV’s evolutionary history, we computationally designed and experimentally constructed a putative ancestral AAV library. Combinatorial variations at 32 amino acid sites were introduced to account for uncertainty in their identities. We then analyzed the evolutionary flexibility of these residues, the majority of which have not been previously studied, by subjecting the library to iterative selection on a representative cell line panel. The resulting variants exhibited transduction efficiencies comparable to the most efficient extant serotypes and, in general, ancestral libraries were broadly infectious across the cell line panel, indicating that they favored promiscuity over specificity. Interestingly, putative ancestral AAVs were more thermostable than modern serotypes and did not use sialic acids, galactose or heparan sulfate proteoglycans for cellular entry. Finally, variants mediated 19- to 31-fold higher gene expression in the muscle compared with AAV1, a clinically used serotype for muscle delivery, highlighting their promise for gene therapy.

Gene delivery vectors based on adeno-associated viruses (AAV) have exhibited promise in both preclinical disease models and human clinical trials for numerous disease targets, including the retinal degenerative disorders Leber’s congenital amaurosis and choroideremia. One general challenge for AAV is that preexisting immunity, as well as subsequent development of immunity following vector administration, can severely inhibit systemic AAV vector gene delivery. However, the role of neutralizing antibodies (NABs) in AAV transduction of tissues considered to be immune privileged, such as the eye, is unclear in large animals. Intravitreal AAV administration allows for broad retinal delivery, but is more susceptible to interactions with the immune system than subretinal administration. To assess the effects of systemic anti-AAV antibody levels on intravitreal gene delivery, we quantified the anti-AAV antibodies present in sera from non-human primates before and after intravitreal injections with various AAV capsids. Analysis showed that intravitreal administration resulted in an increase in anti-AAV antibodies regardless of the capsid serotype, transgene or dosage of virus injected. For monkeys injected with wild-type AAV2 and/or an AAV2 mutant, the variable that most significantly affected the production of anti-AAV2 antibodies was the amount of virus delivered. In addition, post-injection antibody titers were highest against the serotype administered, but the antibodies were also cross-reactive against other AAV serotypes. Furthermore, NAB levels in serum correlated with those in vitreal fluid, demonstrating both that this route of administration exposes AAV capsid epitopes to the adaptive immune system and that serum measurements are predictive of vitreous fluid NAB titers. Moreover, the presence of preexisting NAB titers in the serum of monkeys correlated strongly (R=0.76) with weak, decaying or no transgene expression following intravitreal administration of AAV. Investigating anti-AAV antibody development will aid in understanding the interactions between gene therapy vectors and the immune system during ocular administration and can form a basis for future clinical studies applying intravitreal gene delivery.

Human pluripotent stem cells (hPSCs)—including embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs)—are very promising candidates for cell therapies, tissue engineering, high throughput pharmacology screens, and toxicity testing. These applications require large numbers of high quality cells; however, scalable production of human pluripotent stem cells and their derivatives at a high density and under well-defined conditions has been a challenge. We recently reported a simple, efficient, fully defined, scalable, and good manufacturing practice (GMP) compatible 3D culture system based on a thermoreversible hydrogel for hPSC expansion and differentiation. Here, we describe additional design rationale and characterization of this system. For instance, we have determined that culturing hPSCs as a suspension in a liquid medium can exhibit lower volumetric yields due to cell agglomeration and possible shear force-induced cell loss. By contrast, using hydrogels as 3D scaffolds for culturing hPSCs reduces aggregation and may insulate from shear forces. Additionally, hydrogel-based 3D culture systems can support efficient hPSC expansion and differentiation at a high density if compatible with hPSC biology. Finally, there are considerable opportunities for future development to further enhance hydrogel-based 3D culture systems for producing hPSCs and their progeny.

A genetically encoded optogenetic system was constructed that activates mRNA translation in mammalian cells in response to light. Blue light induces the reconstitution of an RNA binding domain and a translation initiation domain, thereby activating target mRNA translation downstream of the binding sites.

We developed a strategy to create novel genetically encoded switches based on translational repression. We illustrated its efficacy by incorporating two copies of an RNA hairpin in the 5′-untranslated region (UTR) of a target mRNA and demonstrating 7-fold translational repression upon expression of a ligand – the BIV Tat peptide.

Vesicular stomatitis virus G glycoprotein (VSV-G) is the most widely used envelope protein for retroviral and lentiviral vector pseudotyping; however, serum inactivation of VSV-G pseudotyped vectors is a significant challenge for in vivo gene delivery. To address this problem, we conducted directed evolution of VSV-G to increase its resistance to human serum neutralization. After six selection cycles, numerous common mutations were present. On the basis of their location within VSV-G, we analyzed whether substitutions in several surface exposed residues could endow viral vectors with higher resistance to serum. S162T, T230N and T368A mutations enhanced serum resistance, and additionally K66T, T368A and E380K substitutions increased the thermostability of VSV-G pseudotyped retroviral vectors, an advantageous byproduct of the selection strategy. Analysis of a number of combined mutants revealed that VSV-G harboring T230N+T368A or K66T+S162T+T230N+T368A mutations exhibited both higher in vitro resistance to human serum and higher thermostability, as well as enhanced resistance to rabbit and mouse serum. Finally, lentiviral vectors pseudotyped with these variants were more resistant to human serum in a murine model. These serum-resistant and thermostable VSV-G variants may aid the application of retroviral and lentiviral vectors to gene therapy.

Human pluripotent stem cells (hPSCs), including human embryonic stem cells and induced pluripotent stem cells, are promising for numerous biomedical applications, such as cell replacement therapies, tissue and whole-organ engineering, and high-throughput pharmacology and toxicology screening. Each of these applications requires large numbers of cells of high quality; however, the scalable expansion and differentiation of hPSCs, especially for clinical utilization, remains a challenge. We report a simple, defined, efficient, scalable, and good manufacturing practice-compatible 3D culture system for hPSC expansion and differentiation. It employs a thermoresponsive hydrogel that combines easy manipulation and completely defined conditions, free of any human- or animal-derived factors, and entailing only recombinant protein factors. Under an optimized protocol, the 3D system enables long-term, serial expansion of multiple hPSCs lines with a high expansion rate (∼20-fold per 5-d passage, for a 1072-fold expansion over 280 d), yield (∼2.0 × 107 cells per mL of hydrogel), and purity (∼95% Oct4+), even with single-cell inoculation, all of which offer considerable advantages relative to current approaches. Moreover, the system enabled 3D directed differentiation of hPSCs into multiple lineages, including dopaminergic neuron progenitors with a yield of ∼8 × 107 dopaminergic progenitors per mL of hydrogel and ∼80-fold expansion by the end of a 15-d derivation. This versatile system may be useful at numerous scales, from basic biological investigation to clinical development.

Injection of a new gene therapy vector into the easily accessible vitreous transduced the entire retina and rescued disease phenotypes.

Recent advances in synthetic biology have created genetic tools with the potential to enhance the specificity, dynamic control, efficacy, and safety of medical treatments. Interfacing these genetic devices with human patients may thus bring about more efficient treatments or entirely new solutions to presently intractable maladies. Here we review engineered circuits with clinical potential and discuss their design, implementation, and validation.Highlights► Synthetic biology tools are being applied towards the clinical development of enhanced therapeutics. ► Engineered genetic circuits can create ‘smart’ drugs with sensing and actuating capabilities. ► Therapeutic devices may be delivered through viral vectors, encapsulated cells, or bacteria. ► With initial clinical trials underway, safety will be the first priority.

Human pluripotent stem cells (hPSCs) are of great interest in biology and medicine due to their ability to self-renew and differentiate into any adult or fetal cell type. Important efforts have identified biochemical factors, signaling pathways, and transcriptional networks that regulate hPSC biology. However, recent work investigating the effect of biophysical cues on mammalian cells and adult stem cells suggests that the mechanical properties of the microenvironment, such as stiffness, may also regulate hPSC behavior. While several studies have explored this mechanoregulation in mouse embryonic stem cells (mESCs), it has been challenging to extrapolate these findings and thereby explore their biomedical implications in hPSCs. For example, it remains unclear whether hPSCs can be driven down a given tissue lineage by providing tissue-mimetic stiffness cues. Here we address this open question by investigating the regulation of hPSC neurogenesis by microenvironmental stiffness. We find that increasing extracellular matrix (ECM) stiffness in vitro increases hPSC cell and colony spread area but does not alter self-renewal, in contrast to past studies with mESCs. However, softer ECMs with stiffnesses similar to that of neural tissue promote the generation of early neural ectoderm. This mechanosensitive increase in neural ectoderm requires only a short 5-day soft stiffness “pulse”, which translates into downstream increases in both total neurons as well as therapeutically relevant dopaminergic neurons. These findings further highlight important differences between mESCs and hPSCs and have implications for both the design of future biomaterials as well as our understanding of early embryonic development.

Higher order chromatin structure in eukaryotes can lead to differential gene expression in response to the same transcription factor; however, how transcription factor inputs integrate with quantitative features of the chromatin environment to regulate gene expression is not clear. In vitro models of HIV gene regulation, in which repressive mechanisms acting locally at an integration site keep proviruses transcriptionally silent until appropriately stimulated, provide a powerful system to study gene expression regulation in different chromatin environments. Here we quantified HIV expression as a function of activating transcription factor nuclear factor-κB RelA/p65 (RelA) levels and chromatin features at a panel of viral integration sites. Variable RelA overexpression demonstrated that the viral genomic location sets a threshold RelA level necessary to induce gene expression. However, once the induction threshold is reached, gene expression increases similarly for all integration sites. Furthermore, we found that higher induction thresholds are associated with repressive histone marks and a decreased sensitivity to nuclease digestion at the LTR promoter. Increasing chromatin accessibility via inhibition of histone deacetylation or DNA methylation lowered the induction threshold, demonstrating that chromatin accessibility sets the level of RelA required to activate gene expression. Finally, a functional relationship between gene expression, RelA level, and chromatin accessibility accurately predicted synergistic HIV activation in response to combinatorial pharmacological perturbations. Different genomic environments thus set a threshold for transcription factor activation of a key viral promoter, which may point toward biological principles that underlie selective gene expression and inform strategies for combinatorial therapies to combat latent HIV.

Gene therapy vectors based on adeno-associated virus (AAV) are currently in clinical trials for numerous disease targets, such as muscular dystrophy, hemophilia, Parkinson's disease, Leber's congenital amaurosis and macular degeneration. Despite its considerable promise and emerging clinical success, several challenges impede the broader implementation of AAV gene therapy, including the prevalence of neutralizing antibodies in the human population, low transduction of a number of therapeutically relevant cell and tissue types, an inability to overcome physical and cellular barriers in vivo and a relatively limited carrying capacity. These challenges arise as the demands we place on AAV vectors are often different from or even at odds with the properties nature bestowed on their parent viruses. Viral-directed evolution—the iterative generation of large, diverse libraries of viral mutants and selection for variants with specific properties of interest—offers an approach to address these problems. Here we outline progress in creating novel classes of AAV variant libraries and highlight the successful isolation of variants with novel and advantageous in vitro and in vivo gene delivery properties.

Viruses continuously evolve to contend with an ever-changing environment that involves transmission between hosts and sometimes species, immune responses, and in some cases therapeutic interventions. Given the high mutation rate of viruses relative to the timescales of host evolution and drug development, novel drug classes that are readily screened and translated to the clinic are needed. RNA interference (RNAi)—a natural mechanism for specific degradation of target RNAs that is conserved from plants to invertebrates and vertebrates—can potentially be harnessed to yield therapies with extensive specificity, ease of design, and broad application. In this review, we discuss basic mechanisms of action and therapeutic applications of RNAi, including design considerations and areas for future development in the field.

Adeno-associated viral (AAV) vectors, which are undergoing broad exploration in clinical trials, have significant promise for therapeutic gene delivery because of their safety and delivery efficiency. Gene delivery technologies capable of mediating localized gene expression may further enhance the potential of AAV in a variety of therapeutic applications by reducing spread outside a target region, which may thereby reduce off-target side effects. We have genetically engineered an AAV variant capable of binding to surfaces with high affinity through a hexa-histidine metal-binding interaction. This immobilized AAV vector system mediates high-efficiency delivery to cells that contact the surface and thus may have promise for localized gene delivery, which may aid numerous applications of AAV delivery to gene therapy.

Retroviral vectors offer benefits of efficient delivery and stable gene expression; however, their clinical use raises the concerns of insertional mutagenesis and potential oncogenesis due to genomic integration preferences in transcriptional start sites (TSS). We have shifted the integration preferences of retroviral vectors by generating a library of viral variants with a DNA-binding domain inserted at random positions throughout murine leukemia virus Gag-Pol, then selecting for variants that are viable and exhibit altered integration properties. We found seven permissive zinc finger domain (ZFD) insertion sites throughout Gag-Pol, including within p12, reverse transcriptase, and integrase. Comprehensive genome integration analysis showed that several ZFD insertions yielded retroviral vector variants with shifted integration patterns that did not favor TSS. Furthermore, integration site analysis revealed selective integration for numerous mutants. For example, two retroviral variants with a given ZFD at appropriate positions in Gag-Pol strikingly integrated primarily into four common sites out of 3.1 × 109 possible human genome locations (P = 4.6 × 10-29). Our findings demonstrate that insertion of DNA-binding motifs into multiple locations in Gag-Pol can make considerable progress toward engineering safer retroviral vectors that integrate into a significantly narrowed pool of sites on human genome and overcome the preference for TSS.

Respiratory viruses evolve to maintain infectivity levels that permit spread yet prevent host and virus extinction, resulting in surprisingly low infection rates. Respiratory viruses harnessed as gene therapy vectors have illustrated this limitation. We used directed evolution in an organotypic human airway model to generate a highly infectious adeno-associated virus. This virus mediated gene transfer more than 100-fold better than parental strains and corrected the cystic fibrosis epithelial Cl− transport defect. Thus, under appropriate selective pressures, viruses can evolve to be more infectious than observed in nature, a finding that holds significant implications for designing vectors for gene therapy and for understanding emerging pathogens.

Gene delivery vectors based on adeno-associated virus (AAV) are highly promising due to several desirable features of this parent virus, including a lack of pathogenicity, efficient infection of dividing and non-dividing cells, and sustained maintenance of the viral genome. However, several problems should be addressed to enhance the utility of AAV vectors, particularly those based on AAV2, the best characterized AAV serotype. First, altering viral tropism would be advantageous for broadening its utility in various tissue or cell types. In response to this need, vector pseudotyping, mosaic capsids, and targeting ligand insertion into the capsid have shown promise for altering AAV specificity. In addition, library selection and directed evolution have recently emerged as promising approaches to modulate AAV tropism despite limited knowledge of viral structure–function relationships. Second, pre-existing immunity to AAV must be addressed for successful clinical application of AAV vectors. “Shielding” polymers, site-directed mutagenesis, and alternative AAV serotypes have shown success in avoiding immune neutralization. Furthermore, directed evolution of the AAV capsid is a high throughput approach that has yielded vectors with substantial resistance to neutralizing antibodies. Molecular engineering and directed evolution of AAV vectors therefore offer promise for generating ‘designer’ gene delivery vectors with enhanced properties.

The advent of induced pluripotent stem cells—generated via the ectopic overexpression of reprogramming factors such as OCT4, SOX2, KLF4, and C-MYC (OSKM) in a differentiated cell type—has enabled groundbreaking research efforts in regenerative medicine, disease modeling, and drug discovery. Although initial studies have focused on the roles of nuclear factors, increasing evidence highlights the importance of signal transduction during reprogramming. By utilizing a quantitative, medium-throughput screen to initially identify signaling pathways that could potentially replace individual transcription factors during reprogramming, we initially found that several pathways—such as Notch, Smoothened, and cyclic AMP (cAMP) signaling—were capable of generating alkaline phosphatase positive colonies in the absence of OCT4, the most stringently required Yamanaka factor. After further investigation, we discovered that cAMP signal activation could functionally replace OCT4 to induce pluripotency, and results indicate that the downstream exchange protein directly activated by cAMP (EPAC) signaling pathway rather than protein kinase A (PKA) signaling is necessary and sufficient for this function. cAMP signaling may reduce barriers to reprogramming by contributing to downstream epithelial gene expression, decreasing mesenchymal gene expression, and increasing proliferation. Ultimately, these results elucidate mechanisms that could lead to new reprogramming methodologies and advance our understanding of stem cell biology.

Complete eradication of HIV-1 infection is impeded by the existence of cells that harbor chromosomally integrated but transcriptionally inactive provirus. These cells can persist for years without producing viral progeny, rendering them refractory to immune surveillance and antiretroviral therapy and providing a permanent reservoir for the stochastic reactivation and reseeding of HIV-1. Strategies for purging this latent reservoir are thus needed to eradicate infection. Here, we show that engineered transcriptional activation systems based on CRISPR/Cas9 can be harnessed to activate viral gene expression in cell line models of HIV-1 latency. We further demonstrate that complementing Cas9 activators with latency-reversing compounds can enhance latent HIV-1 transcription and that epigenome modulation using CRISPR-based acetyltransferases can also promote viral gene activation. Collectively, these results demonstrate that CRISPR systems are potentially effective tools for inducing latent HIV-1 expression and that their use, in combination with antiretroviral therapy, could lead to improved therapies for HIV-1 infection.

In addition to their broad potential for therapeutic gene delivery, adeno-associated virus (AAV) vectors possess the innate ability to stimulate homologous recombination in mammalian cells at high efficiencies. This process—referred to as AAV-mediated gene targeting—has enabled the introduction of a diverse array of genomic modifications both in vitro and in vivo. With the recent emergence of targeted nucleases, AAV-mediated genome engineering is poised for clinical translation. Here, we review key properties of AAV vectors that underscore its unique utility in genome editing. We highlight the broad range of genome engineering applications facilitated by this technology and discuss the strong potential for unifying AAV with targeted nucleases for next-generation gene therapy.

•Fully defined and xeno-free culture components have been developed.•Synthetic 2D substrates have achieved high expansion rates.•Improved long-term passage on 2D or in 3D suspension will be useful.•Thermoresponsive gels allow long-term, high viability passaging and expansion.•Demonstrating capacity for large-scale automation in 3D is needed.Large numbers of human pluripotent stem cells (hPSCs) are needed to meet the high demands of a range of biomedical applications, including cell replacement therapies and drug screening. Recent advances in media formulations and cell culture platforms have addressed many previous challenges that have hindered efficient expansion. Understanding and addressing the remaining challenges will further facilitate the development of technologies for large-scale hPSC expansion and, to an increasing extent, differentiation.Download high-res image (190KB)Download full-size image

•We generated cortical glutamatergic neurons from human pluripotent stem cells.•We exposed glutamatergic neurons to an oligomeric Aβ to model Alzheimer's disease.•We observed cell age-dependent binding of Aβ and glutamatergic neuronal cell death.•Aβ-induced toxicity was selective for glutamatergic rather than GABAeric neurons.Alzheimer's disease (AD) is among the most prevalent forms of dementia affecting the aging population, and pharmacological therapies to date have not been successful in preventing disease progression. Future therapeutic efforts may benefit from the development of models that enable basic investigation of early disease pathology. In particular, disease-relevant models based on human pluripotent stem cells (hPSCs) may be promising approaches to assess the impact of neurotoxic agents in AD on specific neuronal populations and thereby facilitate the development of novel interventions to avert early disease mechanisms. We implemented an efficient paradigm to convert hPSCs into enriched populations of cortical glutamatergic neurons emerging from dorsal forebrain neural progenitors, aided by modulating Sonic hedgehog (Shh) signaling. Since AD is generally known to be toxic to glutamatergic circuits, we exposed glutamatergic neurons derived from hESCs to an oligomeric pre-fibrillar forms of Aβ known as “globulomers”, which have shown strong correlation with the level of cognitive deficits in AD. Administration of such Aβ oligomers yielded signs of the disease, including cell culture age-dependent binding of Aβ and cell death in the glutamatergic populations. Furthermore, consistent with previous findings in postmortem human AD brain, Aβ-induced toxicity was selective for glutamatergic rather than GABAeric neurons present in our cultures. This in vitro model of cortical glutamatergic neurons thus offers a system for future mechanistic investigation and therapeutic development for AD pathology using human cell types specifically affected by this disease.

•AAV can be endowed with robust retrograde functionality through directed evolution•Up to two orders of magnitude increase in retrograde transport over existing variants•Efficiency comparable to synthetic tracers•Sufficient payload expression for circuit interrogation and gene manipulationEfficient retrograde access to projection neurons for the delivery of sensors and effectors constitutes an important and enabling capability for neural circuit dissection. Such an approach would also be useful for gene therapy, including the treatment of neurodegenerative disorders characterized by pathological spread through functionally connected and highly distributed networks. Viral vectors, in particular, are powerful gene delivery vehicles for the nervous system, but all available tools suffer from inefficient retrograde transport or limited clinical potential. To address this need, we applied in vivo directed evolution to engineer potent retrograde functionality into the capsid of adeno-associated virus (AAV), a vector that has shown promise in neuroscience research and the clinic. A newly evolved variant, rAAV2-retro, permits robust retrograde access to projection neurons with efficiency comparable to classical synthetic retrograde tracers and enables sufficient sensor/effector expression for functional circuit interrogation and in vivo genome editing in targeted neuronal populations.Video AbstractDownload video (84MB)Help with mp4 files

Although biochemical signals that modulate stem cell self-renewal and differentiation were extensively studied, only recently were the mechanical properties of a stem cell's microenvironment shown to regulate its behavior. It would be desirable to have independent control over biochemical and mechanical cues, to analyze their relative and combined effects on stem-cell function. We developed a synthetic, interfacial hydrogel culture system, termed variable moduli interpenetrating polymer networks (vmIPNs), to assess the effects of soluble signals, adhesion ligand presentation, and material moduli from 10–10,000 Pa on adult neural stem-cell (aNSC) behavior. The aNSCs proliferated when cultured in serum-free growth media on peptide-modified vmIPNs with moduli of ≥100 Pa. In serum-free neuronal differentiation media, a peak level of the neuronal marker, β-tubulin III, was observed on vmIPNs of 500 Pa, near the physiological stiffness of brain tissue. Furthermore, under mixed differentiation conditions with serum, softer gels (∼100–500 Pa) greatly favored neurons, whereas harder gels (∼1,000–10,000 Pa) promoted glial cultures. In contrast, cell spreading, self-renewal, and differentiation were inhibited on substrata with moduli of ∼10 Pa. This work demonstrates that the mechanical and biochemical properties of an aNSC microenvironment can be tuned to regulate the self-renewal and differentiation of aNSCs.

A genetically encoded optogenetic system was constructed that activates mRNA translation in mammalian cells in response to light. Blue light induces the reconstitution of an RNA binding domain and a translation initiation domain, thereby activating target mRNA translation downstream of the binding sites.

We developed a strategy to create novel genetically encoded switches based on translational repression. We illustrated its efficacy by incorporating two copies of an RNA hairpin in the 5′-untranslated region (UTR) of a target mRNA and demonstrating 7-fold translational repression upon expression of a ligand – the BIV Tat peptide.