Müller glia, the primary glial cell in the retina, provide structural and metabolic support for neurons and are essential for retinal integrity. Müller cells are closely involved in many retinal degenerative diseases, including macular telangiectasia type 2, in which impairment of central vision may be linked to a primary defect in Müller glia. Here, we used an engineered, Müller-specific variant of AAV, called ShH10, to deliver a photo-inducibly toxic protein, KillerRed, to Müller cells in the mouse retina. We characterized the results of specific ablation of these cells on visual function and retinal structure. ShH10-KillerRed expression was obtained following intravitreal injection and eyes were then irradiated with green light to induce toxicity. Induction of KillerRed led to loss of Müller cells and a concomitant decrease of Müller cell markers glutamine synthetase and cellular retinaldehyde-binding protein, reduction of rhodopsin and cone opsin, and upregulation of glial fibrillary acidic protein. Loss of Müller cells also resulted in retinal disorganization, including thinning of the outer nuclear layer and the photoreceptor inner and outer segments. High resolution imaging of thin sections revealed displacement of photoreceptors from the ONL, formation of rosette-like structures and the presence of phagocytic cells. Furthermore, Müller cell ablation resulted in increased area and volume of retinal blood vessels, as well as the formation of tortuous blood vessels and vascular leakage. Electrophysiologic measures demonstrated reduced retinal function, evident in decreased photopic and scotopic electroretinogram amplitudes. These results show that loss of Müller cells can cause progressive retinal degenerative disease, and suggest that AAV delivery of an inducibly toxic protein in Müller cells may be useful to create large animal models of retinal dystrophies.
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.
The sequence of a promoter within a genome does not uniquely determine gene expression levels and their variability; rather, promoter sequence can additionally interact with its location in the genome, or genomic context, to shape eukaryotic gene expression. Retroviruses, such as human immunodeficiency virus-1 (HIV), integrate their genomes into those of their host and thereby provide a biomedically-relevant model system to quantitatively explore the relationship between promoter sequence, genomic context, and noise-driven variability on viral gene expression. Using an in vitro model of the HIV Tat-mediated positive-feedback loop, we previously demonstrated that fluctuations in viral Tat-transactivating protein levels generate integration-site-dependent, stochastically-driven phenotypes, in which infected cells randomly ‘switch’ between high and low expressing states in a manner that may be related to viral latency. Here we extended this model and designed a forward genetic screen to systematically identify genetic elements in the HIV LTR promoter that modulate the fraction of genomic integrations that specify ‘Switching’ phenotypes. Our screen identified mutations in core promoter regions, including Sp1 and TATA transcription factor binding sites, which increased the Switching fraction several fold. By integrating single-cell experiments with computational modeling, we further investigated the mechanism of Switching-fraction enhancement for a selected Sp1 mutation. Our experimental observations demonstrated that the Sp1 mutation both impaired Tat-transactivated expression and also altered basal expression in the absence of Tat. Computational analysis demonstrated that the observed change in basal expression could contribute significantly to the observed increase in viral integrations that specify a Switching phenotype, provided that the selected mutation affected Tat-mediated noise amplification differentially across genomic contexts. Our study thus demonstrates a methodology to identify and characterize promoter elements that affect the distribution of stochastic phenotypes over genomic contexts, and advances our understanding of how promoter mutations may control the frequency of latent HIV infection.
The sequence of a gene within a cellular genome does not uniquely determine its expression level, even for a single type of cell under fixed conditions. Numerous other factors, including gene location on the chromosome and random gene-expression “noise,” can alter expression patterns and cause differences between otherwise identical cells. This poses new challenges for characterizing the genotype–phenotype relationship. Infection by the human immunodeficiency virus-1 (HIV-1) provides a biomedically important example in which transcriptional noise and viral genomic location impact the decision between viral replication and latency, a quiescent but reversible state that cannot be eliminated by anti-viral therapies. Here, we designed a forward genetic screen to systematically identify mutations in the HIV promoter that alter the fraction of genomic integrations that specify noisy/reactivating expression phenotypes. The mechanisms by which the selected mutations specify the observed phenotypic enrichments are investigated through a combination of single-cell experiments and computational modeling. Our study provides a framework for identifying genetic sequences that alter the distribution of stochastic expression phenotypes over genomic locations and for characterizing their mechanisms of regulation. Our results also may yield further insights into the mechanisms by which HIV sequence evolution can alter the propensity for latent infections.
The sophistication and success of recently reported microfabricated organs-on-chips and human organ constructs have made it possible to design scaled and interconnected organ systems that may significantly augment the current drug development pipeline and lead to advances in systems biology. Physiologically realistic live microHuman (µHu) and milliHuman (mHu) systems operating for weeks to months present exciting and important engineering challenges such as determining the appropriate size for each organ to ensure appropriate relative organ functional activity, achieving appropriate cell density, providing the requisite universal perfusion media, sensing the breadth of physiological responses, and maintaining stable control of the entire system, while maintaining fluid scaling that consists of ~5 mL for the mHu and ~5 µL for the µHu. We believe that successful mHu and µHu systems for drug development and systems biology will require low-volume microdevices that support chemical signaling, micro-fabricated pumps, valves and microformulators, automated optical microscopy, electrochemical sensors for rapid metabolic assessment, ion mobility-mass spectrometry for real-time molecular analysis, advanced bioinformatics, and machine learning algorithms for automated model inference and integrated electronic control. Toward this goal, we are building functional prototype components and are working toward top-down system integration.
Artificial biological organs; biological systems; biotechnology; systems biology
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.
Most past studies of the biophysical regulation of stem cell differentiation have focused on initial lineage commitment or proximal differentiation events. It would be valuable to understand whether biophysical inputs also influence distal endpoints more closely associated with physiological function, such as subtype specification in neuronal differentiation. To explore this question, we cultured adult neural stem cells (NSCs) on variable stiffness ECMs under conditions that promote neuronal fate commitment for extended time periods to allow neuronal subtype differentiation. We find that ECM stiffness does not modulate the expression of NeuroD1 and TrkA/B/C or the percentages of pan-neuronal, GABAergic, or glutamatergic neuronal subtypes. Interestingly, however, an ECM stiffness of 700 Pa maximizes expression of pan-neuronal markers. These results suggest that a wide range of stiffnesses fully permit pan-neuronal NSC differentiation, that an intermediate stiffness optimizes expression of pan-neuronal genes, and that stiffness does not impact commitment to particular neuronal subtypes.
This work builds upon our findings that proteins secreted by hESCs exhibit pro-regenerative activity, and determines that hESC-conditioned medium robustly enhances the proliferation of both muscle and neural progenitor cells. Importantly, this work establishes that it is the proteins that bind heparin which are responsible for the pro-myogenic effects of hESC-conditioned medium, and indicates that this strategy is suitable for enriching the potentially therapeutic factors. Additionally, this work shows that hESC-secreted proteins act independently of the mitogen FGF-2, and suggests that FGF-2 is unlikely to be a pro-aging molecule in the physiological decline of old muscle repair. Moreover, hESC-secreted factors improve the viability of human cortical neurons in an Alzheimer's disease (AD) model, suggesting that these factors can enhance the maintenance and regeneration of multiple tissues in the aging body.
rejuvenation; embryonic stem cell; myoblast; satellite cell
Neurogenesis in the adult hippocampus involves activation of quiescent neural stem cells (NSCs) to yield transiently amplifying NSCs and progenitors, and ultimately neurons that affect learning and memory. This process is tightly controlled by microenvironmental cues, though few endogenous factors are known to regulate neuronal differentiation. While astrocytes have been implicated, their role in juxtacrine (i.e. cell-cell contact-dependent) signaling within NSC niches has not been investigated. We show that ephrin-B2 presented from rodent hippocampal astrocytes regulates neurogenesis in vivo. Furthermore, clonal analysis in NSC fate-mapping studies reveals a novel role for ephrin-B2 in instructing neuronal differentiation. Additionally, ephrin-B2 signaling, transduced by EphB4 receptors on NSCs, activates β-catenin in vitro and in vivo independent of Wnt signaling and upregulates proneural transcription factors. Ephrin-B2+ astrocytes thus promote neuronal differentiation of adult NSCs through juxtacrine signaling, findings that advance our understanding of adult neurogenesis and may have future regenerative medicine implications.
Multiple extracellular factors have been shown to modulate adult hippocampal neural progenitor cell (NPC) proliferation and self-renewal, and we have previously shown that Akt is an important mediator of the effects of these extracellular factors on NPC proliferation and differentiation. However, very little work has investigated how and whether Akt is involved in maintaining the multipotency of these cells. Here we demonstrate that Akt promotes expression of Sox2, a core transcription factor important for the self-renewal of NPCs. Retroviral-mediated overexpression of wild-type Akt increased Sox2 protein expression, particularly under conditions that promote cell differentiation, whereas Akt inhibition decreased Sox2. Similarly, quantitative reverse transcription (RT)–PCR in differentiating cultures indicated that Akt rescued Sox2 mRNA to levels present under conditions that promote cell proliferation. Additionally, pharmacological inhibition of Akt did not affect Sox2 protein levels in cells constitutively expressing Sox2 from a retroviral vector, indicating that Akt does not affect Sox2 protein stability. Further, in contrast to Akt overexpression, Sox2 overexpression does not increase NPC viable cell number or proliferation yet does inhibit differentiation. Collectively, these results indicate that Akt promotes cell proliferation and maintenance of a multipotent state via two downstream paths.
We previously used directed evolution in human airway epithelia to create adeno-associated virus 2.5T (AAV2.5T), a highly infectious chimera of AAV2 and AAV5 with one point mutation (A581T). We hypothesized that the mechanism for its increased infection may be a higher binding affinity to the surface of airway epithelia than its parent AAV5. Here, we show that, like AAV5, AAV2.5T, uses 2,3N-linked sialic acid as its primary receptor; however, AAV2.5T binds to the apical surface of human airway epithelia at higher levels and has more receptors than AAV5. Furthermore, its binding affinity is similar to that of AAV5. An alternative hypothesis is that AAV2.5T interaction with 2,3N-linked sialic acid may instead be required for cellular internalization. Consistent with this, AAV2.5T binds but fails to be internalized by CHO cells that lack surface expression of sialic acid. Moreover, whereas AAV2.5T binds similarly to human (rich in 2,3N-linked sialic acid) and pig airway epithelia (2,6N-linked sialic acid), significantly more virus was internalized by human airway. Subsequent transduction correlated with the level of internalized rather than surface-bound virus. We also found that human airway epithelia internalized significantly more AAV2.5T than AAV5. These data suggest that AAV2.5T has evolved to utilize specific 2,3N-linked sialic acid residues on the surface of airway epithelia that mediate rapid internalization and subsequent infection. Thus, sialic acid serves as not just an attachment factor but is also required for AAV2.5T internalization, possibly representing an important rate-limiting step for other viruses that use sialic acids.
Stem cells are often cultured on substrates that present extracellular matrix (ECM) proteins; however, the heterogeneous and poorly defined nature of ECM proteins presents challenges both for basic biological investigation of cell-matrix investigations and translational applications of stem cells. Therefore, fully synthetic, defined materials conjugated with bioactive ligands, such as adhesive peptides, are preferable for stem cell biology and engineering. However, identifying novel ligands that engage cellular receptors can be challenging, and we have thus developed a high throughput approach to identify new adhesive ligands. We selected an unbiased bacterial peptide display library for the ability to bind adult neural stem cells (NSCs), and 44 bacterial clones expressing peptides were identified and found to bind to NSCs with high avidity. Of these clones, four contained RGD motifs commonly found in integrin binding domains, and three exhibited homology to ECM proteins. Three peptide clones were chosen for further analysis, and their synthetic analogs were adsorbed on tissue culture polystyrene (TCPS) or grafted onto an interpenetrating polymer network (IPN) for cell culture. These three peptides were found to support neural stem cell self-renewal in defined medium as well as multi-lineage differentiation. Therefore, bacterial peptide display offers unique advantages to isolate bioactive peptides from large, unbiased libraries for applications in biomaterials engineering.
Biomimetic material; ECM; Peptide; Stem cell
Neuronal degeneration and the deterioration of neuronal communication lie at the origin of many neuronal disorders, and there have been major efforts to develop cell replacement therapies for treating such diseases. One challenge, however, is that differentiated cells are challenging to transplant due to their sensitivity both to being uprooted from their cell culture growth support and to shear forces inherent in the implantation process. Here, we describe an approach to address these problems. We demonstrate that rat hippocampal neurons can be grown on colloidal particles or beads, matured and even transfected in vitro, and subsequently transplanted while adhered to the beads into the young adult rat hippocampus. The transplanted cells have a 76% cell survival rate one week post-surgery. At this time, most transplanted neurons have left their beads and elaborated long processes, similar to the host neurons. Additionally, the transplanted cells distribute uniformly across the host hippocampus. Expression of a fluorescent protein and the light-gated glutamate receptor in the transplanted neurons enabled them to be driven to fire by remote optical control. At 1-2 weeks after transplantation, calcium imaging of host brain slice shows that optical excitation of the transplanted neurons elicits activity in nearby host neurons, indicating the formation of functional transplant-host synaptic connections. After 6 months, the transplanted cell survival and overall cell distribution remained unchanged, suggesting that cells are functionally integrated. This approach, which could be extended to other cell classes such as neural stem cells and other regions of the brain, offers promising prospects for neuronal circuit repair via transplantation of in vitro differentiated, genetically engineered neurons.
Peptide-functionalized materials show promise in controlling stem cell behavior by mimicking cell-matrix interactions. Supported lipid bilayers are an excellent platform for displaying peptides due to their ease of fabrication and low non-specific interactions with cells. In this paper, we report on the behavior of adult hippocampal neural stem cells (NSCs) on phospholipid bilayers functionalized with different RGD-containing peptides: either GGGNGEPRGDTYRAY (‘bsp-RGD(15)’) or GRGDSP. Fluid supported bilayers were prepared on glass surfaces by adsorption and fusion of small lipid vesicles incorporating synthetic peptide amphiphiles. NSCs adhered to bilayers with either GRGDSP or bsp-RGD(15) peptide. After 5 days in culture, NSCs formed neurosphere-like aggregates on GRGDSP bilayers, whereas on bsp-RGD(15) bilayers a large fraction of single adhered cells were observed, comparable to monolayer growth seen on laminin controls. NSCs retained their ability to differentiate into neurons and astrocytes on both peptide surfaces. This work illustrates the utility of supported bilayers in displaying peptide ligands and demonstrates that RGD peptides may be useful in synthetic culture systems for stem cells.
Vectors based on adeno-associated viruses (AAV) have shown considerable promise in both preclinical models and increasingly in clinical trials. However, one formidable challenge is pre-existing immunity due to widespread exposure to numerous AAV variants and serotypes within the human population, which affect efficacy of clinical trials due to the accompanying high levels of anti-capsid neutralizing antibodies. Transient immunosuppression has promise in mitigating cellular and humoral responses induced by vector application in naïve hosts, but cannot overcome the problem that pre-existing neutralizing antibodies pose toward the goal of safe and efficient gene delivery. Shielding of AAV from antibodies, however, may be possible by covalent attachment of polymers to the viral capsid or by encapsulation of vectors inside biomaterials. In addition, there has been considerable progress in using rational mutagenesis, combinatorial libraries, and directed evolution approaches to engineer capsid variants that are not recognized by anti-AAV antibodies generally present in the human population. While additional progress must be made, such strategies, alone or in combination with immunosuppression to avoid de novo induction of antibodies, have strong potential to significantly enhance the clinical efficacy of AAV vectors.
adeno-associated virus; viral vector; immune response; neutralizing antibodies; bioconjugation; mutagenesis; directed evolution
Intravitreally injected AAV2 transduced inner retinal cells in a restricted region at the macaque fovea. Because macaque and human eyes are similar, the results suggest a need to improve transduction methods in gene therapy for the human inner retina.
Adeno-associated virus serotype 2 (AAV2) has been shown to be effective in transducing inner retinal neurons after intravitreal injection in several species. However, results in nonprimates may not be predictive of transduction in the human inner retina, because of differences in eye size and the specialized morphology of the high-acuity human fovea. This was a study of inner retina transduction in the macaque, a primate with ocular characteristics most similar to that of humans.
In vivo imaging and histology were used to examine GFP expression in the macaque inner retina after intravitreal injection of AAV vectors containing five distinct promoters.
AAV2 produced pronounced GFP expression in inner retinal cells of the fovea, no expression in the central retina beyond the fovea, and variable expression in the peripheral retina. AAV2 vector incorporating the neuronal promoter human connexin 36 (hCx36) transduced ganglion cells within a dense annulus around the fovea center, whereas AAV2 containing the ubiquitous promoter hybrid cytomegalovirus (CMV) enhancer/chicken-β-actin (CBA) transduced both Müller and ganglion cells in a dense circular disc centered on the fovea. With three shorter promoters—human synapsin (hSYN) and the shortened CBA and hCx36 promoters (smCBA and hCx36sh)—AAV2 produced visible transduction, as seen in fundus images, only when the retina was altered by ganglion cell loss or enzymatic vitreolysis.
The results in the macaque suggest that intravitreal injection of AAV2 would produce high levels of gene expression at the human fovea, important in retinal gene therapy, but not in the central retina beyond the fovea.
Reactive oxygen species (ROS) are conventionally classified as toxic consequences of aerobic life, and the brain is particularly susceptible to ROS-induced oxidative stress and damage owing to its high energy and oxygen demands. In this context, NAPDH oxidases (Nox) are a widespread source of brain ROS implicated in seizures, stroke, and neurodegeneration. A physiological role for ROS generation in normal brain function has not been established, despite the fact that mice and humans lacking functional Nox proteins exhibit cognitive deficits. Using molecular imaging with Peroxyfluor-6 (PF6), a new selective fluorescent indicator for hydrogen peroxide (H2O2), we show that adult hippocampal stem/progenitor cells (AHPs) generate H2O2 through Nox2 to regulate intracellular growth signaling pathways, which in turn maintains their normal proliferation in vitro and in vivo. Our results challenge the traditional view that brain ROS are solely deleterious by demonstrating that controlled ROS chemistry is needed for maintaining specific cell populations.
Adeno-associated viral vectors, which are undergoing broad exploration in clinical trials, have significant promise for therapeutic gene delivery due to their safety and delivery efficiency. Gene delivery technologies capable of mediating localized gene expression may further enhance AAV’s potential in a variety of therapeutic applications by reducing spread outside of 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 via a hexahistidine-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.
adeno-associated virus; localized gene delivery; substrate-mediated gene delivery; hexa-histidine
Gene therapies for retinal degeneration have relied on subretinal delivery of viral vectors carrying therapeutic DNA. The subretinal injection is clearly not ideal as it limits the viral transduction profile to a focal region at the injection site and negatively affects the neural retina by detaching it from the supportive retinal pigment epithelium (RPE). We assessed changes in adeno-associated virus (AAV) dispersion and transduction in the degenerating rat retina after intravitreal delivery. We observed a significant increase in AAV-mediated gene transfer in the diseased compared with normal retina, the extent of which depends on the AAV serotype injected. We also identified key structural changes that correspond to increased viral infectivity. Particle diffusion and transgene accumulation in normal and diseased retina were monitored via fluorescent labeling of viral capsids and quantitative PCR. Viral particles were observed to accumulate at the vitreoretinal junction in normal retina, whereas particles spread into the outer retina and RPE in degenerated tissue. Immunohistochemistry illustrates remarkable changes in the architecture of the inner limiting membrane, which are likely to underlie the increased viral transduction in diseased retina. These data highlight the importance of characterizing gene delivery vectors in diseased tissue as structural and biochemical changes can alter viral vector transduction patterns. Furthermore, these results indicate that gene delivery to the outer nuclear layer may be achieved by noninvasive intravitreal AAV administration in the diseased state.
Kolstad et al. evaluate the distribution of vector particles and transduction of AAV administered intravitreally in diseased versus healthy retinas. Whereas healthy retinas are not very receptive to vector penetration and transduction following intravitreal injection, in retinal degenerations the authors show improved and more extensive gene transfer.
Due to the natural tropism of most viral vectors, including adeno-associated viral (AAV) vectors, efficient gene delivery within the central nervous system and retina occurs primarily to neurons and epithelia. Despite the clinical relevance of glia for homeostasis in neural tissue, and as causal contributors in genetic disorders such as Alzheimer's and amyotrophic lateral sclerosis, efforts to develop more efficient gene delivery vectors for glia have met with limited success. Recently, viral vector engineering involving high-throughput random diversification and selection has enabled the rapid creation of novel AAV vectors with valuable new gene delivery properties. We have engineered novel AAV variants capable of efficient glia transduction by employing directed evolution with a panel of four distinct AAV libraries, including a new semi-random peptide replacement strategy. Several novel variants transduced both human and rat astrocytes in vitro up to 15-fold higher than their parent serotypes, and injection into the rat striatum led to astrocyte transduction levels up to 16% of the total transduced cell population. Furthermore, one variant exhibited a substantial shift in tropism towards Müller glia within the retina, further highlighting the general utility of these variants for efficient glia transduction within the CNS and retina.
The eradication of HIV-1 will likely require novel clinical approaches to purge the reservoir of latently infected cells from a patient. We hypothesize that this therapy should target a wide range of latent integration sites, act effectively against viral variants that have acquired mutations in their promoter regions, and function across multiple HIV-1 subtypes. By using primary CD4+ and Jurkat cell-based in vitro HIV-1 latency models, we observe that single-agent latency reactivation therapy is ineffective against most HIV-1 subtypes. However, we demonstrate that the combination of two clinically promising drugs—namely, prostratin and suberoylanilide hydroxamic acid (SAHA)—overcomes the limitations of single-agent approaches and can act synergistically for many HIV-1 subtypes, including A, B, C, D, and F. Finally, by identifying the proviral integration position of latent Jurkat cell clones, we demonstrate that this drug combination does not significantly enhance the expression of endogenous genes nearest to the proviral integration site, indicating that its effects may be selective.
With an estimated 38 million people worldwide currently infected with human immunodeficiency virus (HIV), and an additional 4.1 million people becoming infected each year, it is important to understand how this virus mutates and develops resistance in order to design successful therapies.
We report a novel experimental method for amplifying full-length HIV genomes without the use of sequence-specific primers for high throughput DNA sequencing, followed by assembly of full length viral genome sequences from the resulting large dataset. Illumina was chosen for sequencing due to its ability to provide greater coverage of the HIV genome compared to prior methods, allowing for more comprehensive characterization of the heterogeneity present in the HIV samples analyzed. Our novel amplification method in combination with Illumina sequencing was used to analyze two HIV populations: a homogenous HIV population based on the canonical NL4-3 strain and a heterogeneous viral population obtained from a HIV patient's infected T cells. In addition, the resulting sequence was analyzed using a new computational approach to obtain a consensus sequence and several metrics of diversity.
This study demonstrates how a lower bias amplification method in combination with next generation DNA sequencing provides in-depth, complete coverage of the HIV genome, enabling a stronger characterization of the quasispecies present in a clinically relevant HIV population as well as future study of how HIV mutates in response to a selective pressure.
Mammalian gene expression patterns, and their variability across populations of cells, are regulated by factors specific to each gene in concert with its surrounding cellular and genomic environment. Lentiviruses such as HIV integrate their genomes into semi-random genomic locations in the cells they infect, and the resulting viral gene expression provides a natural system to dissect the contributions of genomic environment to transcriptional regulation. Previously, we showed that expression heterogeneity and its modulation by specific host factors at HIV integration sites are key determinants of infected-cell fate and a possible source of latent infections. Here, we assess the integration context dependence of expression heterogeneity from diverse single integrations of a HIV-promoter/GFP-reporter cassette in Jurkat T-cells. Systematically fitting a stochastic model of gene expression to our data reveals an underlying transcriptional dynamic, by which multiple transcripts are produced during short, infrequent bursts, that quantitatively accounts for the wide, highly skewed protein expression distributions observed in each of our clonal cell populations. Interestingly, we find that the size of transcriptional bursts is the primary systematic covariate over integration sites, varying from a few to tens of transcripts across integration sites, and correlating well with mean expression. In contrast, burst frequencies are scattered about a typical value of several per cell-division time and demonstrate little correlation with the clonal means. This pattern of modulation generates consistently noisy distributions over the sampled integration positions, with large expression variability relative to the mean maintained even for the most productive integrations, and could contribute to specifying heterogeneous, integration-site-dependent viral production patterns in HIV-infected cells. Genomic environment thus emerges as a significant control parameter for gene expression variation that may contribute to structuring mammalian genomes, as well as be exploited for survival by integrating viruses.
Cellular gene expression is a fundamentally stochastic process due to the intrinsic randomness of the underlying biochemical reactions involved. The resulting stochastically generated expression heterogeneities have important biological consequences and also encode information about the underlying dynamics that generate them. A fundamental goal of transcriptional biology is to understand the quantitative regulation of gene-expression dynamics, which in eukaryotes depends on factors specific to each gene in concert with its surrounding cellular and genomic environment. We investigated the regulatory effects of variable genomic environments by quantitatively measuring expression heterogeneity from diverse single genomic integrations of the HIV promoter in cultured cells. Systematically fitting a model of stochastic gene expression to our measurements reveals transcript production in bursts as the underlying dynamic that accounts for the large heterogeneities observed within single-integration clonal cell populations, with the size of transcriptional bursts as the primary feature that varies over genomic integrations. Our findings implicate genomic environment as an important quantitative control parameter that eukaryotic cells might use to shape gene-expression patterns, and that lentviruses such as HIV, whose genomes are semi-randomly integrated into the genomes of the host cells they infect, may exploit to sample diverse and heterogeneous expression patterns that evade treatment.
There has been an increasing interest in understanding how the mechanical properties of the microenvironment influence stem cell fate. We describe studies of the proliferation and differentiation of neural stem cells (NSCs) encapsulated within three-dimensional scaffolds – alginate hydrogels – whose elastic moduli were varied over two orders of magnitude. The rate of proliferation of neural stem cells decreased with increase in the modulus of the hydrogels. Moreover, we observed the greatest enhancement in expression of the neuronal marker β-tubulin III within the softest hydrogels, which had an elastic modulus comparable to that of brain tissues. To our knowledge, this work represents the first demonstration of the influence of modulus on NSC differentiation in three-dimensional scaffolds. Three-dimensional scaffolds that control stem cell fate would be broadly useful for applications in regenerative medicine and tissue engineering.
Gene therapy is an emerging alternative to conventional anti-HIV-1 drugs, and can potentially control the virus while alleviating major limitations of current approaches. Yet, HIV-1's ability to rapidly acquire mutations and escape therapy presents a critical challenge to any novel treatment paradigm. Viral escape is thus a key consideration in the design of any gene-based technique. We develop a computational model of HIV's evolutionary dynamics in vivo in the presence of a genetic therapy to explore the impact of therapy parameters and strategies on the development of resistance. Our model is generic and captures the properties of a broad class of gene-based agents that inhibit early stages of the viral life cycle. We highlight the differences in viral resistance dynamics between gene and standard antiretroviral therapies, and identify key factors that impact long-term viral suppression. In particular, we underscore the importance of mutationally-induced viral fitness losses in cells that are not genetically modified, as these can severely constrain the replication of resistant virus. We also propose and investigate a novel treatment strategy that leverages upon gene therapy's unique capacity to deliver different genes to distinct cell populations, and we find that such a strategy can dramatically improve efficacy when used judiciously within a certain parametric regime. Finally, we revisit a previously-suggested idea of improving clinical outcomes by boosting the proliferation of the genetically-modified cells, but we find that such an approach has mixed effects on resistance dynamics. Our results provide insights into the short- and long-term effects of gene therapy and the role of its key properties in the evolution of resistance, which can serve as guidelines for the choice and optimization of effective therapeutic agents.
A primary obstacle to the success of any anti-HIV treatment is HIV's ability to rapidly resist it by generating new viral strains whose vulnerability to the treatment is reduced. Gene therapies represent a novel class of treatments for HIV infection that may supplement or replace present therapies, as they alleviate some of their major shortcomings. The design of gene therapeutic agents that effectively reduce viral resistance can be aided by a quantitative elucidation of the processes by which resistance is acquired following therapy initiation. We developed a computational model that describes a patient's response to therapy and used it to quantify the influence of therapy parameters and strategies on the development of viral resistance. We find that gene therapy induces different clinical conditions and a much slower viral response than present therapies. These dictate different design principles such as a greater significance to the virus' competence in the absence of therapy. We also show that one can effectively delay emergence of resistance by delivering distinct therapeutic genes into separate cell populations. Our results highlight the differences between traditional and gene therapies and provide a basic understanding of how key controllable parameters and strategies affect resistance development.