Biology subjects

Peng, L.

Publications and source records attributed to Peng, L..

At least 73 records · Page 4Linked to original sources

bioRxiv2023

Therapeutic immune cell engineering with an mRNA : AAV-Sleeping Beauty composite system

Adoptive cell therapy has shown clinical success in patients with hematological malignancies. Immune cell engineering is critical for production, research, and development of cell therapy; however, current approaches for generation of therapeutic immune cells face various limitations. Here, we establish a composite gene delivery system for the highly efficient engineering of therapeutic immune cells. This system, termed MAJESTIC (mRNA AAV-Sleeping-Beauty Joint Engineering of Stable Therapeutic Immune Cells), combines the merits of mRNA, AAV vector, and transposon into one composite system. In MAJESTIC, the transient mRNA component encodes a transposase that mediates permanent genomic integration of the Sleeping Beauty (SB) transposon, which carries the gene-of-interest and is embedded within the AAV vector. This system can transduce diverse immune cell types with low cellular toxicity and achieve highly efficient and stable therapeutic cargo delivery. Compared with conventional gene delivery systems, such as lentiviral vector, DNA transposon plasmid, or minicircle electroporation, MAJESTIC shows higher cell viability, chimeric antigen receptor (CAR) transgene expression, therapeutic cell yield, as well as prolonged transgene expression. CAR-T cells generated by MAJESTIC are functional and have strong anti-tumor activity in vivo. This system also demonstrates versatility for engineering different cell therapy constructs such as canonical CAR, bi-specific CAR, kill switch CAR, and synthetic TCR; and for CAR delivery into various immune cells, including T cells, natural killer cells, myeloid cells, and induced pluripotent stem cells.

bioengineering↗

bioRxiv2023

Perturbomics of tumor-infiltrating NK cells

Natural killer (NK) cells are an innate immune cell type that serves at the first level of defense against pathogens and cancer. NK cells have clinical potential, however, multiple current limitations exist that naturally hinder the successful implementation of NK cell therapy against cancer, including their effector function, persistence, and tumor infiltration. To unbiasedly reveal the functional genetic landscape underlying critical NK cell characteristics against cancer, we perform perturbomics mapping of tumor infiltrating NK cells by joint in vivo AAV-CRISPR screens and single cell sequencing. We establish a strategy with AAV-SleepingBeauty(SB)- CRISPR screening leveraging a custom high-density sgRNA library targeting cell surface genes, and perform four independent in vivo tumor infiltration screens in mouse models of melanoma, breast cancer, pancreatic cancer, and glioblastoma. In parallel, we characterize single-cell transcriptomic landscapes of tumor-infiltrating NK cells, which identifies previously unexplored sub-populations of NK cells with distinct expression profiles, a shift from immature to mature NK (mNK) cells in the tumor microenvironment (TME), and decreased expression of mature marker genes in mNK cells. CALHM2, a calcium homeostasis modulator that emerges from both screen and single cell analyses, shows both in vitro and in vivo efficacy enhancement when perturbed in chimeric antigen receptor (CAR)-NK cells. Differential gene expression analysis reveals that CALHM2 knockout reshapes cytokine production, cell adhesion, and signaling pathways in CAR- NKs. These data directly and systematically map out endogenous factors that naturally limit NK cell function in the TME to offer a broad range of cellular genetic checkpoints as candidates for future engineering to enhance NK cell-based immunotherapies.

immunology↗

bioRxiv2023

Spatially Resolved Proteomic Profiling Uncovers Structural and Functional Regulators of the Axon Initial Segment

The axon initial segment (AIS) is a specialized neuronal compartment required for action potential generation and neuronal polarity. However, understanding the mechanisms regulating AIS structure and function has been hindered by an incomplete knowledge of its molecular composition. Here, using immuno-proximity biotinylation we further define the AIS proteome and its dynamic changes during neuronal maturation. Among the many AIS proteins identified, we show that SCRIB is highly enriched in the AIS both in vitro and in vivo, and exhibits a periodic architecture like the axonal spectrin-based cytoskeleton. We found that ankyrinG interacts with and recruits SCRIB to the AIS. However, loss of SCRIB has no effect on ankyrinG. This powerful and flexible approach further defines the AIS proteome and provides a rich resource to elucidate the mechanisms regulating AIS structure and function.

neuroscience↗

bioRxiv2023

An expanded GCaMP reporter toolkit for functional imaging in C. elegans

In living organisms, changes in calcium flux are integral to many different cellular functions and are especially critical for the activity of neurons and myocytes. Genetically encoded calcium indicators (GECIs) have been popular tools for reporting changes in calcium levels in vivo. In particular, GCaMP, derived from GFP, are the most widely used GECIs and have become an invaluable toolkit for neurophysiological studies. Recently, new variants of GCaMP, which offer a greater variety of temporal dynamics and improved brightness, have been developed. However, these variants are not readily available to the Caenorhabditis elegans research community. This work reports a set of GCaMP6 and jGCaMP7 reporters optimized for C. elegans studies. Our toolkit provides reporters with improved dynamic range, varied kinetics, and targeted subcellular localizations. Besides optimized routine uses, this set of reporters are also well-suited for studies requiring fast imaging speeds and low magnification or low-cost platforms.

neuroscience↗

bioRxiv2023

RNPS1 stabilizes NAT10 protein to facilitate translation in cancer via tRNA ac4C modification

NAT10 is dysregulated and plays an essential role in various types of cancers. However, the exact machenism of how NAT10 regulates cancer progression remains debatable. In this report, we show that NAT10 affects tumorigeneis mainly based on its acetylation function on tRNA. In addition, we found NAT10 regulate the ac4C of tRNA in cancer via interaction with RNPS1, which in turn protect NAT10 from degradation by E3 ubiquitin ligase ZSWIM6. We developed TRMC-seq method to compreshensively profile tRNA ac4C sites and uncovered the presence of ac4C in a broader range of tRNA isoacceptors than previous studies. Multi-omics analysis identified AP-1 signaling pathway as a major downstream mediator of NAT10. Mechanistically, we found NAT10 is responsible for the translation efficiency genes which contain higher ac4C-tRNA codon. Importantly, our genetic mouse model validated our in vitro findings of NAT10 in cancer. Our study highlights a role of NAT10 in mediating tRNA ac4C to regulate the translation and tumorigenesis of cancer.

molecular biology↗

bioRxiv2022

Growth of Mesenchymal Stem Cells at the Surface of Silicone, Mineral and Plant-Based Oils

Bioemulsions are attractive platforms for the expansion of adherent cells in bioreactors. Their design relies on the self-assembly of protein nanosheets at liquid-liquid interfaces, displaying strong interfacial mechanical properties and promoting integrin-mediated cell adhesion. However, most systems developed to date have focused on fluorinated oils, which are unlikely to be accepted for direct implantation of resulting cell products for regenerative medicine, and protein nanosheets self-assembly at other interfaces has not been investigated. In this report, the composition of aliphatic pro-surfactants palmitoyl chloride and sebacoyl chloride, on the assembly kinetics of poly(L-lysine) at silicone oil interfaces and characterisation of ultimate interfacial shear mechanics and viscoelasticity is presented. The impact of the resulting nanosheets on the adhesion of mesenchymal stem cells (MSCs) is investigated via immunostaining and fluorescence microscopy, demonstrating the engagement of the classic focal adhesion-actin cytoskeleton machinery. The ability of MSCs to proliferate at the corresponding interfaces is quantified. In addition, expansion of MSCs at other non-fluorinated oil interfaces, based on mineral and plant-based oils is investigated. Finally, the proof-of-concept of such non-fluorinated oil systems for the formulation of bioemulsions supporting stem cell adhesion and expansion is demonstrated.

bioengineering↗

bioRxiv2022

Function and Cryo-EM structures of broadly potent bispecific antibodies against multiple SARS-CoV-2 Omicron sublineages

The SARS-CoV-2 variant, Omicron (B.1.1.529), rapidly swept the world since its emergence. Compared with previous variants, Omicron has a high number of mutations, especially those in its spike glycoprotein that drastically dampen or abolish the efficacy of currently available vaccines and therapeutic antibodies. Several major sublineages of Omicron evolved, including BA.1, BA.1.1, BA.2, BA.2.12.1, BA.3, BA.4/5, and BA.2.75, which rapidly changing the global and regional landscape of the pandemic. Although vaccines are available, therapeutic antibodies remain critical for infected and especially hospitalized patients. To address this, we have designed and generated a panel of human/humanized therapeutic bispecific antibodies against Omicron and its sub-lineage variants, with activity spectrum against other lineages. Among these, the top clone CoV2-0213 has broadly potent activities against multiple SARS-CoV-2 ancestral and Omicron lineages, including BA.1, BA.1.1, BA.2, BA.2.12.1, BA.3, BA.4/5, and BA.2.75. We have solved the cryo-EM structure of the lead bi-specific antibody CoV-0213 and its major Fab arm MB.02. Three-dimensional structural analysis shows distinct epitope of antibody - spike receptor binding domain (RBD) interactions and reveals that both Fab fragments of CoV2-0213 can simultaneously target one single spike RBD or two adjacent ones in the same spike trimer, further corroborating its mechanism of action. CoV2-0213 represents a unique and potent broad-spectrum SARS-CoV-2 neutralizing bispecific antibody (nbsAb) against the currently circulating major Omicron variants (BA.1, BA.1.1, BA.2, BA.2.12.1, BA.2.75, BA.3, and BA.4/5). CoV2-0213 is primarily human and ready for translational testing as a countermeasure against the ever-evolving pathogen.

immunology↗

bioRxiv2022

Simultaneous and sequential multi-species coronavirus vaccination

Although successful COVID-19 vaccines have been developed, multiple pathogenic coronavirus species exist, urging for development of multi-species coronavirus vaccines. Here we developed prototype LNP-mRNA vaccine candidates against SARS-CoV-2 (Delta variant), SARS-CoV and MERS-CoV, and test how multiplexing of these LNP-mRNAs can induce effective immune responses in animal models. A triplex scheme of LNP-mRNA vaccination induced antigen-specific antibody responses against SARS-CoV-2, SARS-CoV and MERS-CoV, with a relatively weaker MERS-CoV response in this setting. Single cell RNA-seq profiled the global systemic immune repertoires and the respective transcriptome signatures of multiplexed vaccinated animals, which revealed a systemic increase in activated B cells, as well as differential gene expression signatures across major adaptive immune cells. Sequential vaccination showed potent antibody responses against all three species, significantly stronger than simultaneous vaccination in mixture. These data demonstrated the feasibility, antibody responses and single cell immune profiles of multi-species coronavirus vaccination. The direct comparison between simultaneous and sequential vaccination offers insights on optimization of vaccination schedules to provide broad and potent antibody immunity against three major pathogenic coronavirus species. One sentence summaryMultiplexed mRNA vaccination in simultaneous and sequential modes provide broad and potent immunity against pathogenic coronavirus species.

immunology↗

bioRxiv2022

Broadly neutralizing antibodies target the coronavirus fusion peptide

The potential for future coronavirus outbreaks highlights the need to develop strategies and tools to broadly target this group of pathogens. Here, using an epitope-agnostic approach, we identified six monoclonal antibodies that bound to spike proteins from all seven human-infecting coronaviruses. Epitope mapping revealed that all six antibodies target the conserved fusion peptide region adjacent to the S2 cleavage site. Two antibodies, COV44-62 and COV44-79, broadly neutralize a range of alpha and beta coronaviruses, including SARS-CoV-2 Omicron subvariants BA.1 and BA.2, albeit with lower potency than RBD-specific antibodies. In crystal structures of Fabs COV44-62 and COV44-79 with the SARS-CoV-2 fusion peptide, the fusion peptide epitope adopts a helical structure and includes the arginine at the S2 cleavage site. Importantly, COV44-79 limited disease caused by SARS-CoV-2 in a Syrian hamster model. These findings identify the fusion peptide as the target of the broadest neutralizing antibodies in an epitope-agnostic screen, highlighting this site as a candidate for next-generation coronavirus vaccine development. One-Sentence SummaryRare monoclonal antibodies from COVID-19 convalescent individuals broadly neutralize coronaviruses by targeting the fusion peptide.

immunology↗

bioRxiv2022

Mesenchymal Stem Cells Sense the Toughness of Nanomaterials and Interfaces

Stem cells are known to sense and respond to a broad range of physical stimuli arising from their extra-cellular environment. In particular, the role of the mechanical properties (Youngs or shear modulus, viscoelasticity) of biomaterials has extensively been shown to have a significant impact on the adhesion, spreading, expansion and differentiation of stem cells. In turn, cells exert forces on their environment that can lead to striking changes in shape, size and contraction of associated tissues, and may result in mechanical disruption and functional failure. However, no study has so far correlated stem cell phenotype and biomaterials toughness. Indeed, disentangling toughness-mediated cell response from other mechanosensing processes has remained elusive as it is particularly challenging to uncouple Youngs or shear moduli from toughness, within a range relevant to cell-generated forces. In this report, we show how the design of macromolecular architecture of polymer nanosheets regulates interfacial toughness, independently to interfacial shear storage modulus, and how this, in turn, controls the expansion of mesenchymal stem cells at liquid interfaces.

bioengineering↗

bioRxiv2022

Heterotypic vaccination responses against SARS-CoV-2 Omicron BA.2

The Omicron sub-lineage BA.2 of SARS-CoV-2 has recently become dominant across many areas in the world in the on-going waves of COVID-19. Compared to the ancestral/wild-type (WT) virus, Omicron lineage variants, both BA.1 and BA.2, contain high number of mutations, especially in the spike protein, causing significant immune escape that leads to substantial reduction of vaccine and antibody efficacy. Because of this antigenic drift, BA.2 exhibited differential resistance profile to monoclonal antibodies than BA.1. Thus, it is important to understand whether the immunity elicited by currently available vaccines are effective against the BA.2 subvariant. We directly tested the heterotypic vaccination responses against Omicron BA.2, using vaccinated serum from animals receiving WT- and variant-specific mRNA vaccine in lipid nanoparticle (LNP) formulations. Omicron BA.1 and BA.2 antigen showed similar reactivity to serum antibodies elicited by two doses of WT, B.1.351 and B.1.617 LNP-mRNAs. Neutralizing antibody titers of B.1.351 and B.1.617 LNP-mRNA were ~2-fold higher than that of WT LNP-mRNA. Both homologous boosting with WT LNP-mRNA and heterologous boosting with BA.1 LNP-mRNA substantially increased waning immunity of WT vaccinated mice against both BA.1 and BA.2 subvariants. The BA.1 LNP-mRNA booster was ~3-fold more efficient than WT LNP-mRNA at elevating neutralizing antibody titers of BA.2. Together, these data provided a direct preclinical evaluation of WT and variant-specific LNP-mRNAs in standard two-dose and as boosters against BA.1 and BA.2 subvariants.

immunology↗

bioRxiv2022

Potent and specific human monoclonal antibodies against SARS-CoV-2 Omicron variant by rapid mRNA immunization of humanized mice

The Omicron variant (B.1.1.529) of SARS-CoV-2 rapidly becomes dominant globally. Its extensive mutations confer severe efficacy reduction to most of existing antibodies or vaccines. Here, we developed RAMIHM, a highly efficient strategy to generate fully human monoclonal antibodies (mAbs), directly applied it with Omicron-mRNA immunization, and isolated three potent and specific clones against Omicron. Rapid mRNA immunization elicited strong anti-Omicron antibody response in humanized mice, along with broader anti-coronavirus activity. Customized single cell BCR sequencing mapped the clonal repertoires. Top-ranked clones collectively from peripheral blood, plasma B and memory B cell populations showed high rate of Omicron-specificity (93.3%) from RAMIHM-scBCRseq. Clone-screening identified three highly potent neutralizing antibodies that have low nanomolar affinity for Omicron RBD, and low ng/mL level IC50 in neutralization, more potent than majority of currently approved or authorized clinical RBD-targeting mAbs. These lead mAbs are fully human and ready for downstream IND-enabling and/or translational studies.

bioengineering↗

bioRxiv2022

A broad and potent neutralization epitope in SARS-related coronaviruses

Many neutralizing antibodies (nAbs) elicited to ancestral SARS-CoV-2 through natural infection and vaccination generally have reduced effectiveness to SARS-CoV-2 variants. Here we show therapeutic antibody ADG20 is able to neutralize all SARS-CoV-2 variants of concern (VOCs) including Omicron (B.1.1.529) as well as other SARS-related coronaviruses. We delineate the structural basis of this relatively escape-resistant epitope that extends from one end of the receptor binding site (RBS) into the highly conserved CR3022 site. ADG20 can then benefit from high potency through direct competition with ACE2 in the more variable RBS and interaction with the more highly conserved CR3022 site. Importantly, antibodies that are able to target this site generally neutralize all VOCs, albeit with reduced potency against Omicron. Thus, this highly conserved and vulnerable site can be exploited for design of universal vaccines and therapeutic antibodies.

microbiology↗

bioRxiv2022

Broadly neutralizing anti-S2 antibodies protect against all three human betacoronaviruses that cause severe disease

Pan-betacoronavirus neutralizing antibodies may hold the key to developing broadly protective vaccines against coronaviruses that cause severe disease, for anticipating novel pandemic-causing viruses, and to respond more effectively to SARS-CoV-2 variants. The emergence of the Omicron variant of SARS-CoV-2 has illustrated the limitations of solely targeting the receptor binding domain (RBD) of the envelope Spike (S)-protein. Here, we isolated a large panel of broadly neutralizing antibodies (bnAbs) from SARS-CoV-2 recovered-vaccinated donors that target a conserved S2 region in the fusion machinery on betacoronavirus spikes. Select bnAbs show broad in vivo protection against all three pathogenic betacoronaviruses, SARS-CoV-1, SARS-CoV-2 and MERS-CoV, that have spilled over into humans in the past 20 years to cause severe disease. The bnAbs provide new opportunities for antibody-based interventions and key insights for developing pan-betacoronavirus vaccines.

immunology↗

bioRxiv2022

Cryo-EM structures reveal the activation and substrate recognition mechanism of human enteropeptidase

The enteropeptidase (EP) initiates the intestinal digestion by proteolytic processing of trypsinogen, generating catalytic active trypsin. The dysfunction of EP will cause a series of pancreatic diseases, the most severe of which is acute necrotizing pancreatitis. However, the molecular mechanism of EP activation and substrate recognition remain elusive due to the lack of structural information, hampering the structure-based research of EP and even further EP-targeted drug design. Here we report cryo-EM structures of human EP in multiple states, covering the functional cycle spanning from inactive to active state and eventually to the substrate binding state, with the inactive core region reached an atomic 2.7-[A]-resolution. The heavy chain of EP exhibits a clamping configuration with the CUB2 domain serving for substrate recognition. The N-terminus of light chain induces the surface loop remodeling from inactive to active conformation, resulting in a highly dynamic and active EP. Then the heavy chain performs like a hinge to ensure the flexibility of light chain for substrate recruitment and subsequent cleavage. Our study provides structural insights of EP remodeling and heavy chain dynamics while performing enzymatic function, facilitating our understanding of the pathogenies of EP-related pancreatitis and the EP-targeted treatment of pancreatitis.

cell biology↗

bioRxiv2022

Double knockout CRISPR screen in cancer resistance to T cell cytotoxicity

Immunotherapy has transformed cancer treatments; however, a large fraction of patients encounter resistance. Such resistance is mediated by complex factors, often involving interactions between multiple genes. Thus, it is crucially important to identify genetic interactions between genes that are significantly mutated in cancer patients and those involved in immune responses, ideally the ones that currently have chemical compounds for direct targeting. To systematically interrogate such genetic interactions that mediate cancer cells response to T cell killing, we designed an asymmetric CRISPR/Cas9 dual perturbation library targeting the matched combinations between significantly mutated tumor suppressors and immune resistance genes. We performed a combinatorial double knockout screen on 1,159 gene pairs and identified those where joint loss-of-function renders altered cellular response to T cell cytotoxicity. With individual double knockout constructs we validated these genetic interactions including Jak1-Trp53, Jak1-Kmt2d, and Ifngr1-Kmt2d. Interactions between significantly mutated tumor suppressors and potentially druggable immune resistance genes may offer insights on potential new concepts of how to target clinically relevant cancer mutations with currently available agents. This study also provides a technology platform and an asymmetric CRISPR double knockout library for interrogating genetic interactions between cancer mutations and immune resistance pathways under various settings.

genomics↗

bioRxiv2022

SARS-CoV-2 Omicron-specific mRNA vaccine induces potent and broad antibody responses in vivo

The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has high transmissibility and recently swept the globe. Due to the extensive number of mutations, this variant has high level of immune evasion, which drastically reduced the efficacy of existing antibodies and vaccines. Thus, it is important to test an Omicron-specific vaccine, evaluate its immune response against Omicron and other variants, and compare its immunogenicity as boosters with existing vaccine designed against the reference wildtype virus (WT). Here, we generated an Omicron-specific lipid nanoparticle (LNP) mRNA vaccine candidate, and tested its activity in animals, both alone and as a heterologous booster to existing WT mRNA vaccine. Our Omicron-specific LNP-mRNA vaccine elicited strong and specific antibody response in vaccination-naive mice. Mice that received two-dose WT LNP-mRNA, the one mimicking the commonly used Pfizer/Moderna mRNA vaccine, showed a >40-fold reduction in neutralization potency against Omicron variant than that against WT two weeks post second dose, which further reduced to background level >3 months post second dose. As a booster shot for two-dose WT mRNA vaccinated mice, a single dose of either a homologous booster with WT LNP-mRNA or a heterologous booster with Omicron LNP-mRNA restored the waning antibody response against Omicron, with over 40-fold increase at two weeks post injection as compared to right before booster. Interestingly, the heterologous Omicron LNP-mRNA booster elicited neutralizing titers 10-20 fold higher than the homologous WT booster against the Omicron variant, with comparable titers against the Delta variant. All three types of vaccination, including Omicron mRNA alone, WT mRNA homologous booster, and Omicron heterologous booster, elicited broad binding antibody responses against SARS-CoV-2 WA-1, Beta, and Delta variants, as well as other Betacoronavirus species such as SARS-CoV, but not Middle East respiratory syndrome coronavirus (MERS-CoV). These data provided direct proof-of-concept assessments of an Omicron-specific mRNA vaccination in vivo, both alone and as a heterologous booster to the existing widely-used WT mRNA vaccine form.

immunology↗

bioRxiv2022

Engineering SARS-CoV-2 neutralizing antibodies for increased potency and reduced viral escape

The rapid spread of SARS-CoV-2 variants poses a constant threat of escape from monoclonal antibody and vaccine countermeasures. Mutations in the ACE2 receptor binding site on the surface S protein have been shown to disrupt antibody binding and prevent viral neutralization. Here, we use a directed evolution-based approach to engineer three neutralizing antibodies for enhanced binding to S protein. The engineered antibodies showed increased in vitro functional activity in terms of neutralization potency and/or breadth of neutralization against viral variants. Deep mutational scanning revealed that higher binding affinity reduced the total number of viral escape mutations. Studies in the Syrian hamster model showed two examples where the affinity matured antibody provided superior protection compared to the parental antibody. These data suggest that monoclonal antibodies for anti-viral indications could benefit from in vitro affinity maturation to reduce viral escape pathways and appropriate affinity maturation in vaccine immunization could help resist viral variation.

bioengineering↗