Human Cell Membrane Research Articles

Prospects of pharmacological regulation of aquaporin function in CNS diseases

The review analyzes the results of scientific research on the role of aquaporins in the pathogenesis of CNS diseases and the possibility of their pharmacological regulation.
 Aquaporins (AQP) are proteins involved in the transmembrane transport of water and other substances. They form the water channels of cell membranes and are widely represented in various mammalian cells, including the membranes of human brain and spinal cord cells. To date, about 300 types of proteins of the aquaporin family have been discovered, of which 13 (AQP0AQP12) have been identified in human cells. Localization of different types of AQP in CNS structures, their functional activity and involvement in the development of CNS diseases differ. There are mainly three types of AQPs in the central nervous system: AQP1, AQP4 and AQP9. The results of scientific research indicate the most important role of AQP in maintaining water-salt homeostasis and ensuring physiological processes in the central nervous system, and also confirm the role of AQP in the pathogenesis of a number of diseases of the central nervous system (cerebral edema of various genesis, invasion of tumor cells and the formation of peritumorous edema, in the development of autoimmune diseases opticomyelitis, Alzheimers disease). Pharmacological regulation of the functional activity of aquaporins can influence the course of these diseases. Therefore, there is a natural interest in drugs that can change the expression of AQP.
 Proteins of the aquaporin family provide transmembrane transport of water and play an essential role in the development of pathological conditions of the central nervous system. They can be potential targets for pharmacological effects in a number of diseases of the central nervous system. The search and study of drugs affecting the expression and functional activity of AQP is pathogenetically justified and is a promising direction in the development of pharmacotherapy strategies for cerebral edema, malignant brain tumors and other CNS diseases.

A cell-based electrochemical taste sensor for detection of Hydroxy-α-sanshool

The Transient Receptor Potential Vanilloid 1 (TRPV1) has been identified as a suitable candidate for a spicy taste (Zanthoxylum plant) sensor. In this study, we investigated the response of TRPV1 expressed on human HepG2 cell membranes following stimulation with Hydroxy-α-sanshool. A three-dimensional (3D) cell-based electrochemical sensor was fabricated by layering cells expressing hTRPV1. l-cysteine/AuNFs electrodes were functionalized on indium tin oxide-coated glass (ITO) to enhance the sensor's selectivity and sensitivity. HepG2 cells were encapsulated in sodium alginate/gelatin hydrogel to create a 3D cell cultivation system, which was immobilized on the l-cysteine/AuNFs/ITO to serve as biorecognition elements. Using differential pulse voltammetry (DPV), the developed biosensor was utilized to detect Hydroxy-α-sanshool, a representative substance in Zanthoxylum bungeanum Maxim. The result obtained from DPV was linear with Hydroxy-α-sanshool concentrations ranging from 0 to 70 μmol/L, with a detection limit of 2.23 μmol/L. This biosensor provides a sensitive and novel macroscopic approach for TRPV1 detection.

Modulating Lysosomal pH through Innovative Multimerized Succinic Acid-Based Nucleolipid Derivatives.

The multimerization of active compounds has emerged as a successful approach, mainly to address the multivalency of numerous biological targets. Regarding the pharmaceutical prospect, carrying several active ingredient units on the same synthetic scaffold was a practical approach to enhance drug delivery or biological activity with a lower global concentration. Various examples have highlighted better in vivo stability and therapeutic efficiency through sustained action over monomeric molecules. The synthesis strategy aims to covalently connect biologically active monomers to a central core using simple and efficient reaction steps. Despite extensive studies reporting carbohydrate or even peptide multimerization developed for therapeutic activities, very few are concerned with nucleic acid derivatives. In the context of our efforts to build non-viral nucleolipid (NL)-based nanocarriers to restore lysosomal acidification defects, we report here a straightforward synthesis of tetrameric NLs, designed as prodrugs that are able to release no more than one but four biocompatible succinic acid units. The use of oil-in-water nanoemulsion-type vehicles allowed the development of lipid nanosystems crossing the membranes of human neuroblastoma cells. Biological evaluations have proved the effective release of the acid within the lysosome of a genetic and cellular model of Parkinson's disease through the recovery of an optimal lysosomal pH associated with a remarkably fourfold lower concentration of active ingredients than with the corresponding monomers. Overall, these results suggest the feasibility, the therapeutic opportunity, and the better tolerance of multimeric compounds compared to only monomer molecules.

Nucleus-Targeting Manganese Dioxide Nanoparticles Coated with the Human Umbilical Cord Mesenchymal Stem Cell Membrane for Cancer Cell Therapy.

Recently, development of drug delivery systems for accurate delivery of antitumor drugs to tumor sites to improve their antitumor efficacy has attracted great interest in the area of cancer immunotherapy. In this report, an intelligent biodegradable hollow manganese dioxide (HMnO2) nanoparticle (NP) with a human umbilical cord mesenchymal stem cell (hUC-MSC) membrane coating was designed to exert efficient chemo-immunotherapy for cancer treatment. A TAT peptide-modified membrane structure was constructed for nuclear targeting. Our findings showed that this new nanoreactor inherited the active targeting capability of MSCs and exhibited tumoritropic accumulation significantly at the cancerous parts. Compared with other formulations, intravenous injection of the NPs markedly inhibited tumor growth, relapse, and metastasis. Moreover, we found that the NPs effectively boosted dendritic cell maturation and recruited effector T cells into tumors. Overall, this work demonstrates the great potential of applying MSC membrane-coated manganese dioxide NPs as nucleus-targeting nanocarriers in cancer chemo-immunotherapy.

Interaction of Some Asymmetrical Porphyrins with U937 Cell Membranes-In Vitro and In Silico Studies.

The aim of the present study was to assess the effects exerted in vitro by three asymmetrical porphyrins (5-(2-hydroxyphenyl)-10,15,20-tris-(4-acetoxy-3-methoxyphenyl)porphyrin, 5-(2-hydroxyphenyl)-10,15,20-tris-(4-acetoxy-3-methoxyphenyl)porphyrinatozinc(II), and 5-(2-hydroxyphenyl)-10,15,20-tris-(4-acetoxy-3-methoxyphenyl)porphyrinatocopper(II)) on the transmembrane potential and the membrane anisotropy of U937 cell lines, using bis-(1,3-dibutylbarbituric acid)trimethine oxonol (DiBAC4(3)) and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH), respectively, as fluorescent probes for fluorescence spectrophotometry. The results indicate the hyperpolarizing effect of porphyrins in the concentration range of 0.5, 5, and 50 μM on the membrane of human U937 monocytic cells. Moreover, the tested porphyrins were shown to increase membrane anisotropy. Altogether, the results evidence the interaction of asymmetrical porphyrins with the membrane of U937 cells, with potential consequences on cellular homeostasis. Molecular docking simulations, and Molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) free energy of binding calculations, supported the hypothesis that the investigated porphyrinic compounds could potentially bind to membrane proteins, with a critical role in regulating the transmembrane potential. Thus, both the free base porphyrins and the metalloporphyrins could bind to the SERCA2b (sarco/endoplasmic reticulum ATPase isoform 2b) calcium pump, while the metal complexes may specifically interact and modulate calcium-dependent (large conductance calcium-activated potassium channel, Slo1/KCa1.1), and ATP-sensitive (KATP), potassium channels. Further studies are required to investigate these interactions and their impact on cellular homeostasis and functionality.

HCN channels at the cell soma ensure the rapid electrical reactivity of fast-spiking interneurons in human neocortex

Accumulating evidence indicates that there are substantial species differences in the properties of mammalian neurons, yet theories on circuit activity and information processing in the human brain are based heavily on results obtained from rodents and other experimental animals. This knowledge gap may be particularly important for understanding the neocortex, the brain area responsible for the most complex neuronal operations and showing the greatest evolutionary divergence. Here, we examined differences in the electrophysiological properties of human and mouse fast-spiking GABAergic basket cells, among the most abundant inhibitory interneurons in cortex. Analyses of membrane potential responses to current input, pharmacologically isolated somatic leak currents, isolated soma outside-out patch recordings, and immunohistochemical staining revealed that human neocortical basket cells abundantly express hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channel isoforms HCN1 and HCN2 at the cell soma membrane, whereas these channels are sparse at the rodent basket cell soma membrane. Antagonist experiments showed that HCN channels in human neurons contribute to the resting membrane potential and cell excitability at the cell soma, accelerate somatic membrane potential kinetics, and shorten the lag between excitatory postsynaptic potentials and action potential generation. These effects are important because the soma of human fast-spiking neurons without HCN channels exhibit low persistent ion leak and slow membrane potential kinetics, compared with mouse fast-spiking neurons. HCN channels speed up human cell membrane potential kinetics and help attain an input-output rate close to that of rodent cells. Computational modeling demonstrated that HCN channel activity at the human fast-spiking cell soma membrane is sufficient to accelerate the input-output function as observed in cell recordings. Thus, human and mouse fast-spiking neurons exhibit functionally significant differences in ion channel composition at the cell soma membrane to set the speed and fidelity of their input-output function. These HCN channels ensure fast electrical reactivity of fast-spiking cells in human neocortex.

Exploring the dimer-dimer association of the ebolavirus matrix protein VP40 at the human plasma membrane.

Ebolavirus (EBOV) infections cause the deadly Ebola Virus Disease (EVD) which has a high fatality rate in both human and non-human primates. The EBOV genome codes for only seven proteins that can change structures and have multiple functions during the viral assembly. The matrix protein VP40 is the primary component that makes up the cylindrical structure of the Ebola virus. In addition to its other functions during the viral life cycle, it forms the virion's viral matrix layer below the lipid envelope. Once the VP40 dimers are trafficked to the plasma membrane (PM), they assemble into linear filaments which further associate to form the viral matrix. The end-to-end association of the dimers via their C-terminal interfaces is a crucial step during the formation of the matrix filaments. A crystal structure of the CTD-CTD assembly is available and it provides insight into the nature of the interfacial interactions. In this study, we investigated the lipid interactions of the dimer-dimer filament at the inner leaflet of the PM. We used all-atom molecular dynamics simulations of the dimer-dimer structure of the VP40 associated with the PM and identified the major amino acid residues involved in the lipid interactions. We also compared the differences in the lipid contacts of VP40 of a single dimer versus the lipid contacts with the dimer-dimer conformation.

Role of lipid composition on human plasma membrane interactions with the Ebola virus matrix protein VP40.

The Ebola virus is a very dangerous zoonotic virus and its infection can cause hemorrhagic fever with high fatality rates. The Ebola virus matrix protein VP40 is the major protein responsible for the formation of the viral matrix by localizing and assembling it at the inner leaflet of the human plasma membrane (PM). This assembly leads to the formation of virus-like-particles (VLP) and eventual budding. Anionic lipids PI(4,5)P2 (PIP2) and Phosphatidylserine (PS) have been shown to facilitate VP40-membrane binding through electrostatic interactions with cationic VP40 residues. It has been determined that Phosphatidic Acid (PA), resulting from the enzyme Phospholipase-D (PLD), may also play an active role in this process. We performed coarse-grained molecular dynamics (CGMD) simulations to investigate the effects various lipids have on VP40-lipid interactions. We used a triple-dimer structure of VP40 and a PM composed of various lipid types. Contact analyses show interactions with specific cationic VP40 residues that facilitate most of the lipid interactions. Interaction strengths between VP40 residues and lipid headgroups are found to vary when PA was included in the PM compared to when it was not. The radial distribution functions also show differences in local lipid clustering between the two systems. These simulations provide new insights on VP40 interactions with the human PM and how different lipids may influence the overall membrane association and assembly of the VP40 dimers.

Lipid-driven CFTR clustering is impaired in cystic fibrosis and restored by corrector drugs.

Some membrane proteins cluster in nanoscale cholesterol and sphingomyelin rich membrane domains, called lipid rafts, that transform into ceramide-rich rafts and coalesce into large ceramide-rich platforms during cell stress. However, the clustering mechanisms and their impact on membrane protein stability and function remain uncertain. We reported previously that the cystic fibrosis transmembrane conductance regulator (CFTR), which is mutated in the hyperinflammatory pulmonary disease cystic fibrosis (CF), forms clusters that are cholesterol-dependent and become incorporated into large and long-lived ceramide-rich platforms during hormonal stimulation. Using a combination of optimized fluorescence confocal imaging and quantitative analyses such as image correlation spectroscopy (ICS) and the k-space ICS (kICS), we report here that CFTR clustering at the plasma membrane of primary human bronchial epithelial cells does not involve known tethering interactions of CFTR with PDZ domain-containing proteins, filamin A or the actin cytoskeleton. It also does not require CFTR palmitoylation but is critically dependent on membrane lipid order and is induced by detergents that increase the phase separation of membrane lipids. Clustering and integration of CFTR into ceramide-rich platforms are impaired by the CF disease-causing mutations F508del and S13F and rescued by the CFTR modulators elexacaftor plus tezacaftor. These results indicate that CF therapeutics that correct mutant protein folding restore both CFTR trafficking and normal lipid interactions in the plasma membrane which results in its stability at the plasma membrane and its functional restoration.

Three-dimensional single-molecule super-resolution imaging of proteins involved in primary cilia formation.

Primary cilia are organelles that protrude from the cell membrane of most human cells where they perform critical sensory and signaling functions. Defects and mutations in proteins in the primary cilium cause a range of diseases and syndromes collectively termed ciliopathies. Intracellular primary cilium formation begins with the recruitment of Golgi-derived pre-ciliary vesicles to the distal appendages of the mother centriole, but the molecular mechanisms behind the recruitment of the distal appendage proteins are not fully understood. The proteins Rab34 and MyosinVa were found through structured illumination microscopy (SIM) to be closely associated with these pre-ciliary vesicles during this step. However, because of the sub-diffraction size and three-dimensional (3D) structures of the centriole and of the involved proteins, higher resolution multi-target 3D microscopy is required to resolve their organization. To address this limitation, 3D two-color direct stochastic optical reconstruction microscopy (dSTORM) was implemented, providing a single-molecule localization precision of roughly 20 nm in all dimensions. A two-channel 4f emissions system was constructed using a 2-μm axial range double helix phase mask for single-molecule localization in 3D through point spread function engineering. This system allowed for 3D single-molecule super-resolution imaging of Rab34 and MyosinVa in two different color-channels together with imaging of the centriolar protein FOP in both color-channels for context and precise channel registration. The resulting 3D super-resolved reconstructions revealed the nanoscale structures formed by these proteins at the pre-ciliary vesicles, the structural relation between Rab34 and MyosinVa, and their position in relation to the mother centriole.

Polymers of the candidalysin peptide become membrane pores.

C. albicans infection requires the action of the virulence factor candidalysin (CL), which damages the plasma membrane of target human cells (Moyes Nature 2016). However, the molecular mechanism that CL uses to permeabilize membranes was unknown. We have employed complementary biophysical, modeling, microscopy, and cell biology methods to reveal that CL forms membrane pores using a unique molecular mechanism (Russell eLife 2022). Unexpectedly, we observed that CL readily assembles into linear polymers in solution. We propose that the basic structural unit in polymer formation is a CL oligomer, which is sequentially added into a string configuration that can close into a loop. Our data indicate that CL loops spontaneously insert into the membrane to become membrane pores. We identified a CL mutation (G4W) that inhibited the formation of polymers in solution and prevented formation of pores in synthetic lipid membrane systems. Studies in epithelial cells showed that G4W CL failed to activate the danger response signaling pathway, a hallmark of the pathogenic effect of CL. These results indicate that CL polymerization in solution is a necessary step for the damage of cellular membranes. Analysis of thousands of CL pores by atomic force microscopy revealed co-existence of simple depressions and complex pores decorated with protrusions. Imaging and modeling indicate that the two types of pores are formed by CL oligomers assembled into alternate orientations. We propose that this structural rearrangement represents a maturation mechanism that stabilizes pore formation . The data show that CL uses a previously unknown mechanism to damage membranes, whereby pre-assembly of CL loops in solution directly leads to formation of membrane pores. Our investigation not only unravels a new paradigm for the formation of membrane pores, but additionally identifies CL polymerization as a novel therapeutic target to treat candidiasis.

Production of Monoclonal antibodies to membrane components of human colorectal cancer HCT-116 cell line for diagnostic purposes

Colorectal cancer (CRC) is the second leading cause of cancer-related death worldwide. The survival rate of people diagnosed in the early stages of disease is 92 % whereas; less than 11 % of patients survive if reached stage IV. Moreover, only 24 % of cases are diagnosed during stage I. Thus, the early diagnosis of the CRC will save many lives, reduce suffering of patients. Biomarkers that are in use for the diagnosis of CRC such as Carcinoembryonic Antigen (CEA), Carbohydrate Antigen (CA 19–9) suffer from low sensitivity, expressed in other neoplasms, and not expressed in early stages of cancer. This study aims to find a highly sensitive and specific biomarker for early detection of CRC. Hybridoma clones were established by fusing spleen cells from mice hyperimmunized with human cell membrane homogenate of HCT116 with murine myeloma cells X63Ag8. One clone was selected after fusion and designated as D2C5. Isotyping of secretory antibody revealed an IgG1 kappa light chain and was reactive to a protein with molecular weight about 55 kDa using immunoblot. In situ reactivity of the D2C5 showed homogenous and cytoplasmic pattern staining in stage IV and III, discrete and heterogeneous pattern was seen in stage II samples. Moreover, nuclear staining pattern was seen in one slide of stage II samples. In addition, mucinous type was detected. Low peripheral staining was observed in normal colon tissue. These observations suggest the localization of the target protein in the cytoplasm of epithelial cells with high expression in more advanced cases, suggesting a cytokeratin pattern of staining.

Identification of Substrates of Secreted Bacterial Protease by APEX2-Based Proximity Labeling.

The interactions between bacterial virulence factors and host receptors play a critical role during bacterial infection. Therefore, the identification of interactions between host receptor and bacterial virulence factors can not only elucidate the molecular mechanisms of bacterial infection but also provide a framework for new therapeutic and prevention strategies. Herein, we report an APEX2-based live cell proximity labeling strategy in combination with high-resolution quantitative mass spectrometry to profile the substrates of Helicobacter pylori HtrA protease on the membrane of human stomach epithelial cells. This strategy can be further applied to identify other interactions between secreted bacterial virulence factors and host receptors on live cells.

Molecular modelling of shockwave-mediated delivery of paclitaxel aggregates across the neuronal plasma membrane.

Shock-assisted paclitaxel (PTX) transport across the blood-brain barrier offers a promising treatment strategy for brain tumors. Here, based on a realistically complex human brain plasma membrane (PM) model, we investigated the dynamic transmembrane behavior of a PTX cluster by shock induced bubble collapse, focusing on the effect of impulse (I), bubble diameter (D) and arrays. The results show that all three factors can control the transport depth (ΔDPM) of PTX. For a fixed D, the ΔDPM grows exponentially with I, ΔDPM ∼ exp (I), and eventually reaches a critical depth. But the depth, ΔDPM, can be adjusted linearly in a wider range of D. This mainly depends on the size of jets from bubble collapse. For bubble arrays, the bubbles in series can transport PTX deeper than a single bubble, while the parallel does the opposite. In addition, only PTX clusters in the range of jet action can be successfully transported. Finally, the absorption of PTX clusters was examined via recovery simulation. Not all PTX clusters across the membrane can be effectively absorbed by cells. The shallow PTX clusters are quickly attracted by the membrane and embedded into it. The critical depth at which PTX clusters can be effectively absorbed is about 20 nm. These molecular-level mechanisms and dynamic processes of PTX clusters crossing the PM membrane may be helpful in optimizing the application of shock-induced bubble collapse for the delivery of PTX to tumor cells.

Biomimetic Nanosonosensitizers Combined with Noninvasive Ultrasound Actuation to Reverse Drug Resistance and Sonodynamic-Enhanced Chemotherapy against Orthotopic Glioblastoma

Glioblastoma (GBM) is the most devastating brain tumor and highly resistant to conventional chemotherapy. Herein, we introduce biomimetic nanosonosensitizer systems (MDNPs) combined with noninvasive ultrasound (US) actuation for orthotopic GBM-targeted delivery and sonodynamic-enhanced chemotherapy. MDNPs were fabricated with biodegradable and pH-sensitive polyglutamic acid (PGA) and the chemotherapeutic agent and sonosensitizer doxorubicin (DOX), camouflaged with human GBM U87 cell membranes. MDNPs presented homologous targeting accumulation and in vivo long-term circulation ability. They effectively passed through the blood-brain barrier (BBB) under US assistance and reached the orthotopic GBM site. MDNPs exhibited controllable US-elicited sonodynamic effect by generation of reactive oxygen species (ROS). ROS not only induced cancer cell apoptosis but also downregulated drug-resistance-related factors to disrupt chemoresistance and increase sensitivity to chemotherapy. The in vivo study of orthotopic GBM treatments further proved that MDNPs exhibited US-augmented synergistic antitumor efficacy and strongly prolonged the survival rate of mice. The use of low-dose DOX and the safety of US enabled repeated treatment (4 times) without obvious cardiotoxicity. This effective and safe US-enhanced chemotherapy strategy with the advantages of noninvasive brain delivery and high drug sensitivity holds great promise for deep-seated and drug-resistant tumors.

Dual Role of ACBD6 in the Acylation Remodeling of Lipids and Proteins.

The transfer of acyl chains to proteins and lipids from acyl-CoA donor molecules is achieved by the actions of diverse enzymes and proteins, including the acyl-CoA binding domain-containing protein ACBD6. N-myristoyl-transferase (NMT) enzymes catalyze the covalent attachment of a 14-carbon acyl chain from the relatively rare myristoyl-CoA to the N-terminal glycine residue of myr-proteins. The interaction of the ankyrin-repeat domain of ACBD6 with NMT produces an active enzymatic complex for the use of myristoyl-CoA protected from competitive inhibition by acyl donor competitors. The absence of the ACBD6/NMT complex in ACBD6.KO cells increased the sensitivity of the cells to competitors and significantly reduced myristoylation of proteins. Protein palmitoylation was not altered in those cells. The specific defect in myristoyl-transferase activity of the ACBD6.KO cells provided further evidence of the essential functional role of the interaction of ACBD6 with the NMT enzymes. Acyl-CoAs bound to the acyl-CoA binding domain of ACBD6 are acyl donors for the lysophospholipid acyl-transferase enzymes (LPLAT), which acylate single acyl-chain lipids, such as the bioactive molecules LPA and LPC. Whereas the formation of acyl-CoAs was not altered in ACBD6.KO cells, lipid acylation processes were significantly reduced. The defect in PC formation from LPC by the LPCAT enzymes resulted in reduced lipid droplets content. The diversity of the processes affected by ACBD6 highlight its dual function as a carrier and a regulator of acyl-CoA dependent reactions. The unique role of ACBD6 represents an essential common feature of (acyl-CoA)-dependent modification pathways controlling the lipid and protein composition of human cell membranes.

G Protein-Coupled Receptor 37L1 Modulates Epigenetic Changes in Human Renal Proximal Tubule Cells.

Renal luminal sodium transport is essential for physiological blood pressure control, and abnormalities in this process are strongly implicated in the pathogenesis of essential hypertension. Renal G protein-coupled receptors (GPCRs) are critical for the regulation of the reabsorption of essential nutrients, ions, and water from the glomerular filtrate. Recently, we showed that GPCR 37L1 (GPR37L1) is expressed on the apical membrane of renal proximal tubules (RPT) and regulates luminal sodium transport and blood pressure by modulating the function of the sodium proton exchanger 3 (NHE3). However, little is known about GPR37L1 intracellular signaling. Here, we show that GPR37L1 is localized to the nuclear membrane, in addition to the plasma membrane in human RPT cells. Furthermore, GPR37L1 signals via the PI3K/AKT/mTOR pathway to decrease the expression of DNA (cytosine-5)-methyltransferase 1 (DNMT1) and enhance NHE3 transcription. Overall, we demonstrate the direct role of a nuclear membrane GPCR in the regulation of renal sodium through epigenetic gene regulation.

Clinical Development of Senza5TM CART5: a Novel Dual Population CD5 CRISPR-Cas9 Knocked out Anti-CD5 Chimeric Antigen Receptor T Cell Product for Relapsed and Refractory CD5+ Nodal T-Cell Lymphomas

Introduction: Chimeric antigen receptor T-cell (CART) immunotherapy is FDA-approved for B-cell lymphoblastic leukemia, B-cell non-Hodgkin lymphomas, and multiple myeloma. However, CART immunotherapy for T-cell non-Hodgkin lymphomas (T-NHL) is lagging, with only limited clinical results. T-NHL have a poor prognosis and there is an unmet need for effective and long-lasting treatments. We previously presented preliminary preclinical results of anti-CD5 CAR T cells (CART5) that had CD5 knocked out (KO) (Blood (2020) 136 (Supplement 1): 51-52.). CD5 KO not only prevented CART fratricide but also dramatically enhanced CART function. Critical challenges to developing CART cells for T-NHL are CART fratricide and normal T-cell toxicity. In this study, we addressed these challenges by improving manufacturing and including a dual-population technology to mitigate toxicity. We present the development of Senza5TM CART5 an autologous good-manufacturing practice (GMP)-grade product (Fig.1) that will be tested in a phase I clinical trial for relapsed and refractory CD5+ nodal T-cell lymphomas. Methods and Results: We first identified a single guide RNA (sgRNA) that deletes CD5 in T cells with >90% KO efficiency by flow cytometry and ICE analysis. We then assessed the specificity of the KO by performing iGUIDE sequencing. We confirmed high on-target CD5 cleavage with minimal off-targets (CALCP, C20orf85, INPP4B, XPO7, and SLC10A7). These genes have little to no expression in naïve CD4+ and CD8+ T cells by RNA-seq (DICE). Targeted functional assays are ongoing. For the CAR construct, we selected a lead CAR5 among six 4-1BB-constructs based on its superior anti-tumor function in NOD-SCID IL2Rgnull (NSG) mice engrafted with a Jurkat T-lymphoblastic leukemia cell line. Furthermore, to confirm the specificity of the CAR5 for CD5, we screened CAR5 T cells for interactions with HEK293 cells expressing 5,828 human plasma membrane proteins or cell-surface tethered secreted proteins and 398 human heterodimers (Retrogenix assay). The lead CAR5 did not bind to any targets besides CD5. For an optimized GMP-grade product, we modified our manufacturing protocol, shortening it from 10 to 5 days. Moreover, KO efficiency and safety were optimized to reduce off-target activity without loss of efficiency. We also optimized the activation strategy to decrease cell loss. Functional studies were performed to demonstrate the comparability of these methods. With these improvements, we were able to achieve >90% CD5 KO with >30% CAR transduction, thereby allowing the generation of two main populations: one of CD5KO CART5 cells and the other of CD5KO normal untransduced T cells. CD5KO CART5 are expected to target CD5+ T-NHL cells but could also lead to normal T-cell toxicity. This toxicity will be mitigated by the CD5KO normal T cells that will have a survival advantage after infusion (Fig.1) and have been demonstrated to maintain reactivity against opportunistic pathogens such as CMV and EBV. To evaluate Senza5TMCART5 safety, we performed cytokine-independent CART proliferation and long- and short-term xenograft models. Senza5TM CART5 cells did not grow aberrantly without growth/survival-associated cytokines (IL2/7/15). We then used NSG mice to exclude uncontrolled Senza5TM CART5 growth and observed enhanced expansion and proliferation of Senza5TM CART5 with a xenogeneic GVHD rate similar to that of control mice with similar wild-type CART5 expansion. Since the CAR5 does not cross-react with murine CD5, we tested non-specific toxicity in tissues using an NSG xenograft model of Jurkat. On Day 10 post-CAR T injections, animals were euthanized, and a complete necropsy was performed on all animals. Senza5TM CART5 demonstrated tumor clearance with no detection of treatment-related lesions. Mice treated with untransduced T cells or CART19 demonstrated significant overall and organ-specific weight loss compared to Senza5TM CART5. Blood analyses determined no differences in their clinical chemistry profile or complete blood counts. Conclusion: In this study, we developed a novel dual-population, full-scale GMP-ready CD5 KO anti-CD5 CART product for T-cell lymphomas characterized by enhanced anti-tumor effect and a strategy to potentially mitigate toxicity. Autologous Senza5TM CART5 will be tested for CD5+ nodal T-cell lymphomas in a phase I clinical trial. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Bio-distribution and longevity of mesenchymal stromal cell derived membrane particles

Vesicle-based medicines hold great promise for therapy development but essential knowledge on the bio-distribution and longevity of vesicles after administration is lacking. We generated vesicles from the membranes of human mesenchymal stromal cells (MSC) and we demonstrated earlier that these so-called membrane particles (MP) mediate immunomodulatory and regenerative responses in target cells. In the present study we examined the bio-distribution and longevity of MP after intravenous administration in mice. While most vesicle tracking methods are based on imaging techniques, which require labeling of vesicles and can only detect dense accumulations of vesicles, we used proteomics analysis to detect the presence of MP-derived proteins in multiple organs and tissues. MP proteins were mainly present in plasma and leukocytes at 1h after injection, indicating that MP - in contrast to whole MSC - do not accumulate in the lungs upon first passage but remain in circulation. After 24h, MP proteins were still present in plasma but were most abundant in the liver. RNA sequencing of livers demonstrated that MP impact liver function and in particular induce metabolic pathways. These data provide a clear view of the bio-distribution and longevity of MP, which is likely extrapolatable to other types of vesicles, and demonstrate that MP circulate for up to 24h and may be a tool for targeting the liver.

ER-Golgi-localized proteins TMED2 and TMED10 control the formation of plasma membrane lipid nanodomains.

To promote infections, pathogens exploit host cell machineries such as structural elements of the plasma membrane. Studying these interactions and identifying molecular players are ideal for gaining insights into the fundamental biology of the host cell. Here, we used the anthrax toxin to screen a library of 1,500 regulatory, cell-surface, and membrane trafficking genes for their involvement in the intoxication process. We found that endoplasmic reticulum (ER)-Golgi-localized proteins TMED2 and TMED10 are required for toxin oligomerization at the plasma membrane of human cells, an essential step dependent on localization to cholesterol-rich lipid nanodomains. Biochemical, morphological, and mechanistic analyses showed that TMED2 and TMED10 are essential components of a supercomplex that operates the exchange of both cholesterol and ceramides at ER-Golgi membrane contact sites. Overall, this study of anthrax intoxication led to the discovery that lipid compositional remodeling at ER-Golgi interfaces fully controls the formation of functional membrane nanodomains at the cell surface.