A systems pharmacology model for gene therapy in sickle cell disease.

We developed a mathematical model for autologous stem cell therapy to cure sickle cell disease (SCD). Experimental therapies using this approach seek to engraft stem cells containing a curative gene. These stem cells are expected to produce a lifelong supply of red blood cells (RBCs) containing an anti-sickling hemoglobin. This complex, multistep treatment is expensive, and there is limited patient data available from early clinical trials. Our objective was to quantify the impact of treatment parameters, such as initial stem cell dose, efficiency of lentiviral transduction, and degree of bone marrow preconditioning on engraftment efficiency, peripheral RBC numbers, and anti-sickling hemoglobin levels over time. We used ordinary differential equations to model RBC production from progenitor cells in the bone marrow, and hemoglobin assembly from its constituent globin monomers. The model recapitulates observed RBC and hemoglobin levels in healthy and SCD phenotypes. Treatment simulations predict dynamics of stem cell engraftment and RBC containing the therapeutic gene product. Post-treatment dynamics show an early phase of reconstitution due to short lived stem cells, followed by a sustained RBC production from stable engraftment of long-term stem cells. This biphasic behavior was previously reported in the literature. Sensitivity analysis of the model quantified relationships between treatment parameters and efficacy. The initial dose of transduced stem cells, and the intensity of myeloablative bone marrow preconditioning are predicted to most positively impact long-term outcomes. The quantitative systems pharmacology approach used here demonstrates the value of model-assisted therapeutic design for gene therapies in SCD.

Phosphatidylserine dictates the assembly and dynamics of caveolae in the plasma membrane.

Caveolae are bulb-shaped nanodomains of the plasma membrane that are enriched in cholesterol and sphingolipids. They have many physiological functions, including endocytic transport, mechanosensing, and regulation of membrane and lipid transport. Caveola formation relies on integral membrane proteins termed caveolins (Cavs) and the cavin family of peripheral proteins. Both protein families bind anionic phospholipids, but the precise roles of these lipids are unknown. Here, we studied the effects of phosphatidylserine (PtdSer), phosphatidylinositol 4-phosphate (PtdIns4P), and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) on caveolar formation and dynamics. Using live-cell, single-particle tracking of GFP-labeled Cav1 and ultrastructural analyses, we compared the effect of PtdSer disruption or phosphoinositide depletion with caveola disassembly caused by cavin1 loss. We found that PtdSer plays a crucial role in both caveola formation and stability. Sequestration or depletion of PtdSer decreased the number of detectable Cav1-GFP puncta and the number of caveolae visualized by electron microscopy. Under PtdSer-limiting conditions, the co-localization of Cav1 and cavin1 was diminished, and cavin1 degradation was increased. Using rapamycin-recruitable phosphatases, we also found that the acute depletion of PtdIns4P and PtdIns(4,5)P2 has minimal impact on caveola assembly but results in decreased lateral confinement. Finally, we show in a model of phospholipid scrambling, a feature of apoptotic cells, that caveola stability is acutely affected by the scrambling. We conclude that the predominant plasmalemmal anionic lipid PtdSer is essential for proper Cav clustering, caveola formation, and caveola dynamics and that membrane scrambling can perturb caveolar stability.

Integrins Form an Expanding Diffusional Barrier that Coordinates Phagocytosis.

Phagocytosis is initiated by lateral clustering of receptors, which in turn activates Src-family kinases (SFKs). Activation of SFKs requires depletion of tyrosine phosphatases from the area of particle engagement. We investigated how the major phosphatase CD45 is excluded from contact sites, using single-molecule tracking. The mobility of CD45 increased markedly upon engagement of Fcγ receptors. While individual CD45 molecules moved randomly, they were displaced from the advancing phagocytic cup by an expanding diffusional barrier. By micropatterning IgG, the ligand of Fcγ receptors, we found that the barrier extended well beyond the perimeter of the receptor-ligand engagement zone. Second messengers generated by Fcγ receptors activated integrins, which formed an actin-tethered diffusion barrier that excluded CD45. The expanding integrin wave facilitates the zippering of Fcγ receptors onto the target and integrates the information from sparse receptor-ligand complexes, coordinating the progression and ultimate closure of the phagocytic cup.

Actin cytoskeleton reorganization by Syk regulates Fcγ receptor responsiveness by increasing its lateral mobility and clustering.

Clustering of immunoreceptors upon association with multivalent ligands triggers important responses including phagocytosis, secretion of cytokines, and production of immunoglobulins. We applied single-molecule detection and tracking methods to study the factors that control the mobility and clustering of phagocytic Fcγ receptors (FcγR). While the receptors exist as monomers in resting macrophages, two distinct populations were discernible based on their mobility: some diffuse by apparent free motion, while others are confined within submicron boundaries that reduce the frequency of spontaneous collisions. Src-family and Syk kinases determine the structure of the actin cytoskeleton, which is fenestrated, accounting for the heterogeneous diffusion of the FcγR. Stimulation of these kinases during phagocytosis induces reorganization of the cytoskeleton both locally and distally in a manner that alters receptor mobility and clustering, generating a feedback loop that facilitates engagement of FcγR at the tip of pseudopods, directing the progression of phagocytosis.