Engineered virus-like particles (VLPs) are a promising technology for in vivo gene editing of human hematopoietic stem and progenitor cells (HSPCs). Here we design and test two different VLP envelopes for human HSPC editing in vitro and in vivo. The first is an optimized version of the baboon envelope BaEVTR, which efficiently transduces human HSPCs in vitro. We show that the optimized BaEVTR VLP enables in vivo editing of β2 microglobulin in long-term human HSPCs (31

Introduction: Chimeric antigen receptor T cell therapy (CAR T) is a successful treatment for B cell malignancies; however, the time, complexity and cost of manufacturing autologous CAR T products limits the availability of these therapies to patients. Furthermore, ex vivo manipulation of T cells is likely to have a negative impact on quality. In vivo gene delivery of CAR T transgenes by systemic infusion of standard lentiviral vectors may increase therapeutic accessibility but is limited by off-target transduction and the requirement for T cell activation. Here, we demonstrate that a paramyxovirus-based integrating vector (fusosome) engineered with a CD4 re-targeted envelop (CD4 fusogen) can efficiently and specifically transduce resting and activated CD4+ T cells to generate functional CD4+ CD19-specific CAR T cells capable of eliminating CD19+ lymphoma cells.

The uptake of receptors by clathrin-mediated endocytosis underlies signaling, nutrient import, and recycling of transmembrane proteins and lipids. In the complex, crowded environment of the plasma membrane, receptors are internalized when they bind to components of the clathrin coat, such as the major adaptor protein, AP2. Receptors with higher affinity for AP2 are known to be more strongly internalized compared to receptors with lower affinity. However, it remains unclear how receptors with different affinities compete for space within crowded endocytic structures. To address this question, we constructed receptors with varying affinities for AP2 and allowed them to compete against one another during internalization. As expected, the internalization of a receptor with high affinity for AP2 was reduced when it was coexpressed with a competing receptor of similar affinity. However, receptors of low affinity for AP2 were surprisingly difficult to displace from endocytic structures, even when expressed alongside receptors with much higher affinity. To understand how these low-affinity receptors are protected from competition, we looked at AP2 heterogeneity across clathrin-coated structures. When we examined structures with lower-than-average AP2 content, we found that they were relatively enriched in cargo of low affinity for AP2 and depleted of cargo with high affinity. These findings suggest that the heterogeneity of adaptor protein content across the population of endocytic structures enables the internalization of diverse receptors. Given the critical role that internalization plays in signaling, this effect may help to prevent strongly internalized receptors from interfering with the cell's ability to process signals from weakly internalized receptors.

Endocytic uptake of receptors is a fundamental process that underlies multiple cellular events from signal transduction and nutrient import to recycling transmembrane proteins and lipids. Since receptor recycling plays a role in most signaling pathways, it is important to understand how receptors are selected for internalization. In order to be internalized, receptors must bind to protein components of the endocytic coat, such as the major adaptor protein, AP2. Receptors with greater affinities for AP2 are more strongly internalized compared to receptors with lower affinity. How do receptors with different affinities compete for space within crowded endocytic structures? To address this question, we constructed receptors with varying affinities for AP2 and allowed them to compete against each other during internalization. Internalization of a receptor with high affinity for AP2 was reduced when co-expressed with a competing receptor of high affinity. Based on these results, we expected that the weaker the affinity of a receptor, the easier it would be for a strongly internalized receptor to compete it out of coated pits. Instead, we found that the fractional change in internalization of our model receptors upon competition with a strongly internalized receptor decreased with decreasing affinity for AP2. These observations suggest that weakly internalized receptors are protected from competition with more strongly internalized competitors. This protection could be due to a small fraction of the coated vesicle capacity being occupied by weakly internalized receptors. Therefore, to drive them out, a strongly internalized competitor has to saturate nearly the entire coated vesicle. Given the critical role that internalization plays in receptor signaling, this effect may serve as a protection mechanism that prevents overexpression of strongly internalized receptors from interfering with the cell's ability to process signals from weakly internalized receptors.

Introduction: The invasiveness of standard treatments for peripheral vascular disease (PVD) precludes their use in some patients and often does not provide a long term solution to continuing PVD. A...

Endocytic uptake of receptors from the cell surface plays an important role in diverse processes from cell signaling to nutrient internalization. Understanding the mechanisms by which endocytic structures select receptors for internalization is of fundamental importance to our understanding of cellular physiology. Binding of receptors to the endocytic protein machinery is known to facilitate receptor loading into endocytic structures. However, many receptor species use the same small set of biochemical motifs to interact with the endocytic machinery, suggesting that receptors may compete for a limited number of binding sites within endocytic structures. Previous studies have shown that such competition can substantially modify receptor uptake. However, a predictive biophysical understanding of this phenomenon is currently lacking. Toward addressing this gap, here we employ quantitative imaging and statistical thermodynamics to measure and predict the competition between two distinct receptor species that are internalized simultaneously from the cell surface. Our studies demonstrate that when receptors compete for the same interactions with the endocytic machinery, their uptake is fundamentally coupled. Importantly, we find that these trends can be quantitatively predicted by a simple thermodynamic analysis. These results suggest that multiple receptor species reach an equilibrium partitioning between endocytic structures and the surrounding plasma membrane as the receptors compete for occupancy within dynamic endocytic structures. More broadly, this work provides a quantitative framework for predicting the impact of competition on receptor uptake, an effect which has the potential to physically couple signaling pathways that impact diverse aspects of cellular physiology.

Recruitment of receptors into clathrin-coated structures is essential to signal transduction and nutrient uptake. Among the many receptors involved in these processes, a significant fraction forms dimers. Dimerization of identical partners has generally been thought to promote receptor recruitment for uptake because of increased affinity of the dimer for the endocytic machinery. But what happens when receptors with substantially different affinities for the endocytic machinery come together to form a heterodimer? Evidence from diverse receptor classes, including G-protein-coupled receptors and receptor tyrosine kinases, suggests that heterodimerization with a strongly recruited receptor can drive significant recruitment of a receptor that lacks direct interactions with the endocytic machinery. However, a systematic biophysical understanding of this effect has yet to be established. Motivated by the potential of such events to influence cell signaling, here, we investigate the impact of receptor heterodimerization on endocytic recruitment using a family of engineered model receptors. As expected, we find that dimerization of a weakly recruited receptor with a strongly recruited receptor promotes incorporation of the weakly recruited receptor to endocytic structures. However, the effectiveness of this collaborative mechanism depends heavily on the relative strengths of endocytic recruitment of the two receptors that make up the dimer. Specifically, as the strength of endocytic recruitment of the weakly recruited receptor approaches that of the strongly recruited receptor, monomers of each receptor compete with heterodimers for space within endocytic structures. In this regime, the presence of the strongly recruited receptor drives a reduction in incorporation of the weakly recruited receptor into clathrin-coated structures. Similarly, as the strength of the dimer bond between the two receptors is progressively weakened, competition begins to dominate over collaboration. Collectively, these results demonstrate that the impact of receptor heterodimerization on endocytic recruitment is controlled by a delicate balance between collaborative and competitive mechanisms.

The ability of proteins to sense membrane curvature is essential to cellular function. All known sensing mechanisms rely on protein domains with specific structural features such as wedge-like amphipathic helices and crescent-shaped BAR domains. Yet many proteins that contain these domains also contain large intrinsically disordered regions. Here we report that disordered domains are themselves potent sensors of membrane curvature. Comparison of Monte Carlo simulations with in vitro and live-cell measurements demonstrates that the polymer-like behavior of disordered domains found in endocytic proteins drives them to partition preferentially to convex membrane surfaces, which place fewer geometric constraints on their conformational entropy. Further, proteins containing both structured curvature sensors and disordered regions are more than twice as curvature sensitive as their respective structured domains alone. These findings demonstrate an entropic mechanism of curvature sensing that is independent of protein structure and illustrate how structured and disordered domains can synergistically enhance curvature sensitivity.

Receptor internalization by endocytosis regulates diverse cellular processes, from the rate of nutrient uptake to the timescale of essential signaling events. The established view is that internalization is tightly controlled by specific protein-binding interactions. However, recent work suggests that physical aspects of receptors influence the process in ways that cannot be explained by biochemistry alone. Specifically, work from several groups suggests that increasing the steric bulk of receptors may inhibit their uptake by multiple types of trafficking vesicles. How do biochemical and biophysical factors work together to control internalization? Here, we show that receptor uptake is well described by a thermodynamic trade-off between receptor-vesicle binding energy and the entropic cost of confining receptors within endocytic vesicles. Specifically, using large ligands to acutely increase the size of engineered variants of the transferrin receptor, we demonstrate that an increase in the steric bulk of a receptor dramatically decreases its probability of uptake by clathrin-coated structures. Further, in agreement with a simple thermodynamic analysis, all data collapse onto a single trend relating fractional occupancy of the endocytic structure to fractional occupancy of the surrounding plasma membrane, independent of receptor size. This fundamental scaling law provides a simple tool for predicting the impact of receptor expression level, steric bulk, and the size of endocytic structures on receptor uptake. More broadly, this work suggests that bulky ligands could be used to drive the accumulation of specific receptors at the plasma membrane surface, providing a biophysical tool for targeted modulation of signaling and metabolism from outside the cell.

Clathrin-mediated endocytosis, essential to signal transduction from the cellular surface, gives cells precise temporal and spatial control over hundreds of receptors on the plasma membrane surface. The biochemical motifs responsible for recruiting individual receptors into clathrin-coated pits have been thoroughly mapped. However to initiate a signaling cascade, membrane receptors such as G-protein coupled receptors (GPRCRs) often assemble into heterogenous dimers and clusters. How does assembly of heterogeneous receptor clusters impact the uptake of individual receptor species? To address this question, we created a simplified model system consisting of 2 chimeric transmembrane receptors. While both receptors are constitutively endocytosed, one receptor contains a mutated AP2 binding motif, leading to severely reduced localization to clathrin-coated pits. Using quantitative imaging to precisely measure the distribution of receptors on the plasma membrane, we demonstrate that when two receptors form a biochemical bond, the receptor with high affinity for endocytic structures can drive efficient internalization of the receptor of low affinity. Specifically, localization of low affinity receptors to clathrin-coated pits increases as a function of high affinity receptor expression levels, suggesting that the binding interaction between the two receptors is the driving force behind internalization of the low affinity receptor. In particular, our data demonstrates that the recruitment of low affinity receptors can be further enhanced by increasing the overall binding affinity between the two receptors, achieved by incorporating serial repeats of the receptor-receptor interaction domain into our model receptors. Collectively, these results suggest that receptor uptake depends on both recruitment of individual receptors and recruitment of heterogeneous protein complexes. Moving forward, this work suggests novel approaches for amplifying signaling in therapeutically relevant pathways, such as GPCR and apoptotic signaling, by promoting interactions between target receptors and receptors undergoing active internalization.

Receptor internalization by endocytosis controls diverse cellular processes from the rate of nutrient uptake to the timescale of essential signaling events. While many of the biochemical motifs that enable uptake have been thoroughly investigated, the physical factors that influence receptor internalization remain poorly understood. Specifically, recent work from several groups has demonstrated that the steric bulk of receptors inhibits their uptake by multiple types of trafficking vesicles including caveolae, COPII vesicles, and clathrin coated pits. However, the mechanism by which endocytic structures differentiate among receptors of different sizes to control their uptake remains unclear. Using bulky ligands to acutely increase receptor size, here we show that receptor uptake is governed by a thermodynamic balance between receptor-vesicle binding energy and the entropic cost of confining receptors within nascent vesicles. Specifically, we used quantitative fluorescence imaging to precisely map the distribution of receptors among clathrin-coated pits at the plasma membrane surface. In agreement with a simple thermodynamic model, these data show that the increased entropic cost of internalizing bulky ligands inhibits receptor uptake, driving accumulation of ligated receptors at the plasma membrane. Further, by examining the differential uptake of two receptors competing for space within the same population of endocytic structures, our results reveal the relative importance of receptor size and receptor affinity for endocytic sites. From a biological perspective, these results can predict how processes such as ligand binding and dimerization, which alter both the effective size of receptors and their biochemical affinity for endocytic structures, will impact receptor uptake rate. From an applied perspective, this work guides the design of bulky ligands that can drive the accumulation of specific receptors at the plasma membrane surface, a tool that may be useful for modulating diverse pathways in cell physiology and signaling.