Biology subjects

Patel, M.

Publications and source records attributed to Patel, M..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

DINOcell: Learning Generalizable Perturbation Effects through Self-Distillation

AO_SCPLOWBSTRACTC_SCPLOWPredicting cellular responses to therapeutics is a promising approach for novel target discovery. However, state-of-the-art computational models designed to predict perturbation effects struggle to generalize and outperform simple baselines. We present DINOcell, a weakly supervised framework that adapts self-distillation to single-cell transcriptomics for predicting perturbation effects. We demonstrate that DINOcell outperforms baselines in predicting the effects of single gene perturbations. Furthermore, DINOcell accurately predicts non-additive effects of combination perturbations, indicating its capacity to model complex genetic interactions. Finally, we show that DINOcell learns representations that capture biological signals and is a promising, generalizable approach for in silico perturbation modeling, providing a valuable tool for accelerating therapeutic target discovery.

bioinformatics↗

bioRxiv2025

Targeting CREB remodels the immune microenvironment to enhance immunotherapy responses in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) remains a challenging disease in need of improved treatments. Cyclic adenosine monophosphate response element binding protein 1 (CREB) is an emerging therapeutic target whose oncogenic effects in PDAC have been largely attributed to a key molecular interplay between oncogenic KrasG12D/+ (Kras*) and chronic inflammation driving irreversible acinar to ductal reprogramming. Here, we demonstrate that CREB activation fosters tumor associated macrophage (TAM) mediated immunosuppression and promotes PDAC growth in an aggressive LSL-KrasG12D/+;Trp53R172H/+;Pdx1Cre/+(KPC) genetically engineered mouse model. Selective deletion of CREB (Crebfl/fl) in KPC(KPCC-/-) mice attenuates primary disease burden. Unbiased transcriptomic analysis and validation using diverse molecular, genetic and pharmacological approaches in vitro and in vivo identify CREB-mediated transcriptional regulation of leukemia inhibitory factor (Lif) as one of the potential mediators of tumor cell-macrophage crosstalk promoting a pro-tumor polarization of TAMs, thereby attenuating the infiltration of effector T cells. Mechanistically, cancer cell derived LIF facilitates an immunosuppressive, pro-tumorigenic state. Importantly, pharmacological targeting of the CREB-LIF signaling axis between cancer cells and macrophages, using a CREB-specific inhibitor (CREBi), significantly suppresses tumor growth and sensitizes PDAC to immunotherapy, highlighting the therapeutic potential of this treatment combination to improve outcomes in this aggressive disease.

cancer biology↗

bioRxiv2025

Trichome formation in Nicotiana benthamiana is induced by Agrobacterium

Plant trichomes are specialised epidermal appendages found on various organs of many plant species. They play important roles in plant protection against pests, pathogens, herbivores, resistance to abiotic stress, light perception, mechanosensitivity etc. They are of direct agricultural importance, providing fibre (e.g. cotton) and producing secondary metabolites essential for agriculture, medicine, and industry. Trichomes can be relatively sparsely distributed, making their biological investigation and commercial exploitation more challenging. Here we show that infiltration of Nicotiana benthamiana leaf tissue with certain nopaline-type Agrobacterium tumefacians strains induces extremely dense, localised formation of glandular trichomes 15 days post infiltration (dpi). This Agrobacterium-strain specific effect was shown to be due to the presence of a trans-zeatin synthase (tzs) gene located on some Ti plasmids, which results in the bacterial production of the cytokinin trans-zeatin. This simple procedure enables dramatically increased trichome density to be compared biologically with adjacent isogenic plant tissues on the same leaf.

plant biology↗

bioRxiv2025

NUAK2 is a therapeutically tractable regulator of RNA splicing and tumor progression in neuroendocrine prostate cancer

Prostate cancer remains a leading cause of cancer-related mortality in men, with aggressive, treatment-emergent androgen receptor (AR)-indifferent subtypes, including double-negative prostate cancer (DNPC) and neuroendocrine prostate cancer (NEPC), posing major clinical challenges due to limited therapeutic options. NUAK family kinase 2 (NUAK2), an AMPK-related kinase, has been implicated in tumor growth and metastatic progression; however, its functional significance and therapeutic potential in advanced prostate cancer remain largely unexplored. Here, we identify NUAK2 as a therapeutically actionable kinase dependency in AR-indifferent prostate cancer. Transcriptomic analyses across independent patient cohorts demonstrated progressive upregulation of NUAK2 with disease progression, with the highest expression in NEPC. Immunohistochemical analysis of clinical specimens further confirmed elevated NUAK2 protein expression in NEPC relative to prostate adenocarcinoma. Genetic loss- and gain-of-function studies established NUAK2 as a functional dependency that promotes tumor cell proliferation, clonogenic growth, and tumor growth in vivo. Mechanistically, integrated proteomic analyses revealed that NUAK2 associates with spliceosomal and RNA-processing machinery, while NUAK2 perturbation induced widespread alterations in pre-mRNA splicing programs involving genes linked to mitotic regulation and oncogenic signaling. Pharmacologic studies identified trilaciclib (G1T-28), a clinically approved CDK4/6 inhibitor, as a functionally relevant NUAK2 inhibitor that directly engages NUAK2, suppresses tumor growth, and enhances the efficacy of platinum-based chemotherapy across multiple preclinical models. Collectively, these findings uncover NUAK2 as a previously unrecognized regulator of RNA splicing and therapeutic vulnerability in AR-indifferent prostate cancer and provide a rationale for repurposing G1T-28 and developing NUAK2-directed therapeutic strategies for aggressive, treatment-refractory prostate cancer.

cancer biology↗

bioRxiv2025

Oncogenic and immunomodulatory functions of SUV420H1 in HPV-negative head and neck squamous cell carcinoma.

Despite the advent of immunotherapy, human-papilloma-virus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) carries a high morbidity and mortality rate, thus novel therapies are urgently needed. Suppressor Of Variegation 4-20 Homolog 1 (SUV420H1) is a protein lysine methyltransferase that writes H4K20me3. Approximately 35% of HPV-negative HNSCC tumors carry gains/amplifications of SUV420H1. Gene Set Enrichment Analysis (GSEA) showed enrichment of proliferation-, epithelial-mesenchymal transition (EMT)- and immune-response pathways in SUV420H1-overexpressing HPV-negative HNSCC tumors. Depletion of SUV420H1 led to decreased proliferation, cell cycling and invasion in human HPV-negative HNSCC cell lines, while enzymatic inhibition decreased the invasive capacity but not the proliferation and cell cycling of HPV-negative HNSCC cell lines, supporting the presence of catalytically-independent and -dependent functions of SUV420H1. In a syngeneic mouse model of mouse oral carcinoma 1 tumors (MOC1), Suv420h1 knockout (KO) in MOC1 cancer cells halted tumor growth and synergized with anti-PD-1 therapy. In the tumor immune microenvironment (TIME) of Suv420h1 KO MOC1 tumors, a significant increase in the macrophage compartment with a concurrent decrease in the protumorigenic granulocytic myeloid derived suppressor cells (gMDSCs) was observed. Genome-wide mapping of SUV420H1-mediated H4K20me3 revealed enrichment of EMT-, IFN-response, and myeloid-attracting chemokines. This work provides rationale for the depletion/degradation of SUV420H1 as a novel therapeutic strategy to hinder proliferation and invasion, and to impart sensitization to anti-PD-1 immunotherapy for patients with HPV-negative HNSCC tumors with SUV420H1 gain/amplification.

cancer biology↗

bioRxiv2025

Preclinical evaluation of Targeted IL-1β Knockdown via CD44-Immunoliposomes: A Nano-therapy against the Inflammatory Microenvironment

Chronic inflammation, characterized by the infiltration of macrophages and the heightened release of pro-inflammatory cytokines, is the underlying cause of the pathogenesis of many critical diseases. Therapeutic interventions for controlling inflammation via gene knockdown of inflammatory mediators have emerged as a promising approach for regulating uncontrolled inflammation. This study explores the potential of siIL-1{beta}-anti-CD44-Liposomes (SIL) as a potent anti-inflammatory therapy against pro-inflammatory RAW264.7 macrophages via gene specific knockdown of IL-1{beta} mRNA through RNAi, and the subsequent down-regulation of the pro-inflammatory cytokine loop. The designed SIL exhibited a uniform size of 131.1 {+/-} 0.5 nm with a quasi-spherical morphology and sustained release of siIL-1{beta} within 24 hours. The reduction in pro-inflammatory cytokines like IL-1{beta}, TNF-, and IL-6 and inflammatory enzymes iNOS and COX-2; and the simultaneous increase in the anti-inflammatory cytokine IL-4, is indicative of the formulations therapeutic efficacy in reducing inflammation at a cellular level. The effects of SIL on the Macrophage-T cell crosstalk also uncovers the liposomes efficacy in reducing cytokine-mediated T cell effector functions. The nuanced effects of siIL-1{beta}-anti-CD44-Liposomes on in-vivo model of chronic inflammation underscore their potential for precise therapeutic interventions in inflammatory conditions, with multifaceted anti-inflammatory effects on tissue levels and cytokine levels. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/683015v1_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@f4d0f9org.highwire.dtl.DTLVardef@c3c24aorg.highwire.dtl.DTLVardef@8c115org.highwire.dtl.DTLVardef@bc863d_HPS_FORMAT_FIGEXP M_FIG Schematic representation of the study: CD44 receptors are elevated in LPS activated macrophages, which can be targeted by using anti-CD44 Liposomes, for RNA therapy. IL-1 knockdown via siRNA leads to lower inflammatory nature of macrophages, compromising its the antigen presentation and T-cell activation. This lowers the cytokine storm in inflammatory milieu and lower tissue damage can be achieved. Created with BioRender.com. C_FIG

pharmacology and toxicology↗

bioRxiv2025

A co-evolved peptide-GPCR system senses host entry to drive fungal infection

A successful infection requires pathogens to recognize the specific host environment in order to reprogram their physiology accordingly. One major way in which eukaryotic cells sense their surroundings is via G-Protein Coupled Receptors (GPCRs), which share a seven-transmembrane architecture and G-protein-mediated downstream signaling. While mammalian GPCRs are well-characterized and represent important drug targets, their fungal counterparts remain poorly understood. In the corn pathogen Ustilago maydis, we now uncover a GPCR-based mechanism that allows the fungus to scout the host environment to sense whether it has entered into the plant tissue. During infection, the fungus secretes the protein Pit2, which is cleaved by host apoplastic-cysteine proteases, releasing a peptide ligand hidden within Pit2. This ligand activates the fungal GPCR Gpe1 strongly promoting fungal proliferation after initial host penetration. Comparative analyses reveal conservation of the Gpe1/Pit2 system, with co-evolutionary signatures preserving receptor-ligand specificity. Furthermore, this GPCR system recognizing hidden peptide ligands shows conceptual similarities to the fungal pheromone mating system, without sharing sequence similarity. Our findings reveal a co-evolved mechanism between fungus and host that encodes environmental context into a protein scaffold, establishing a novel paradigm for host-dependent signaling with implications for inter-organismic communication.

microbiology↗

bioRxiv2025

Spend today or build for tomorrow? Kinship dynamics and the evolution of alternative helping strategies in cooperative breeders.

Cooperatively breeding animal groups are characterised by the presence of helpers, which assist breeders in raising offspring through a variety of behaviours, such as provisioning young or defence against predators. While these systems have been studied to investigate how costly forms of help can evolve, there is little theory explaining the considerable variation in helping strategies observed across such societies. Here, we use mathematical models to investigate the evolution of alternative helping strategies. Helpers in our models can allocate effort to immediate, short-term benefits or durable, long-term benefits. We find that allocation depends on the kinship dynamics within the group. Specifically, where helpers are more related to future breeders than current breeders, often because they themselves become the future breeder, our model typically predicts greater allocation to durable help. Conversely, immediate help is favoured when helpers are less related to future breeders than to current breeders. We use our model to explore how demographic features impact kinship dynamics within the group, identify which factors select for immediate versus durable help, and which amplify conflict between breeders and helpers over helping strategies. The model is a first step towards explaining variation in types of help observed among cooperatively breeding societies.

evolutionary biology↗

bioRxiv2025

Mapping and reprogramming microenvironment-induced cell states in human disease using generative AI

Tissue microenvironments reprogram local cellular states in disease, yet current computational spatial methods remain descriptive and do not simulate tissue perturbation. We present MintFlow, a generative AI algorithm that learns how the tissue microenvironment influences cell states and predicts how tissue perturbations can reprogram them. Applied to three human diseases, MintFlow uncovered distinct pathogenic spatial reprogramming in inflammatory and tumor microenvironments. In atopic dermatitis, MintFlow identified a novel, spatially-imprinted, type 2 (IL13+ITGAE+) epidermal T resident memory cell population (type 2 TRM), and decoded signaling pathways within the perivascular lymphoid niche. In melanoma, MintFlow identified fibrotic stroma resembling keloid scar tissue. In kidney cancer, MintFlow resolved immunosuppressed CD8+ T cell states within tertiary lymphoid structures. Furthermore, MintFlow enabled in silico perturbations of disease-relevant cell states and tissue environments. Regulatory T cell modulation in atopic dermatitis was predicted to suppress the pro-inflammatory tissue environment, supporting manipulation of these cells as a therapeutic target. In kidney cancer, in silico T cell replacement recapitulated immune checkpoint blockade, while spatially targeted macrophage depletion reverted immunosuppressed T cell states. The corresponding gene programs correlated with survival in large kidney cancer patient cohorts. Together, these findings position MintFlow as a tool for unbiased disease mechanism prediction and in silico perturbation, accelerating translational hypothesis generation and guiding therapeutic strategies.

genomics↗

bioRxiv2025

Non-canonical enhancers control gene expression and cell fate in human pluripotent stem cells

Enhancers are key gene regulatory elements that ensure the precise spatiotemporal execution of developmental gene expression programmes. However recent findings indicate that approaches to identify enhancers may not capture the full repertoire of active enhancers in mammalian genomes. Here, we combine massively parallel enhancer assays with chromatin structure and transcriptome profiling to functionally annotate enhancers genome-wide in human induced pluripotent stem cells. We find that a substantial fraction ([~]40%) of accessible chromatin regions with enhancer function lack key features associated with active enhancers, including the active enhancer mark histone H3 lysine 27 acetylation and enhancer-associated RNAs. Perturbation of this class of non-canonical enhancers by CRISPR-mediated epigenome editing results in decreased levels of target gene expression and, in one instance, loss of pluripotent stem cell characteristics. Collectively, our data demonstrate enhancer activity for a class of gene regulatory elements that had until now only been associated with a neutral or inactive status, challenging current models of enhancer function.

genomics↗

bioRxiv2025

Evolutionary responses to historic drought across the range of scarlet monkeyflower

Adaptive evolution is a key means for populations to persist under environmental change, yet whether populations across a species range can adapt quickly enough to keep pace with climate change remains unknown. The breeders equation predicts the evolutionary change in a trait from one generation to the next as the product of the selection differential and the narrow-sense heritability in that trait. Incorporating these aspects of the breeders equation, we performed a resurrection study with the scarlet monkeyflower (Mimulus cardinalis) to evaluate whether traits associated with drought adaptation have evolved in populations across a species range in response to extreme drought. We compared trait and fitness differences of pre-drought ancestors and post-drought descendants from six populations transplanted into three latitudinally-arrayed common gardens and quantified phenotypic selection and trait heritabilities. The strength, direction, and mode of selection varied among traits and gardens. Trait heritabilities were relatively low, and did not differ dramatically among populations or gardens. Overall, instances of evolutionary responses between ancestors and descendants were few and small in magnitude, but the magnitude of these evolutionary differences varied among gardens. Together, these results suggest that the expression of genetic variation, and thus traits, depend on the environment, and that environmental variability in field settings may mask the genetic variation that is often detected in greenhouse environments.

evolutionary biology↗

bioRxiv2025

A spatiotemporal cancer cell trajectory underlies glioblastoma heterogeneity

Cancer cells display highly heterogeneous and plastic states in glioblastoma, an incurable brain tumour. However, how these malignant states arise and whether they follow defined cellular trajectories across tumours is poorly understood. Here, we generated a deep single cell and spatial multi-omic atlas of human glioblastoma that pairs transcriptomic, epigenomic and genomic profiling of 12 tumours across multiple regions. We identify that glioblastoma heterogeneity is driven by spatially-patterned transitions of cancer cells from developmental-like states towards those defined by a glial injury response and hypoxia. This cellular trajectory regionalises tumours into distinct tissue niches and manifests in a molecularly conserved manner across tumours as well as genetically distinct tumour subclones. Moreover, using a new deep learning framework to map cancer cell states jointly with clones in situ, we show that tumour subclones are finely spatially intermixed through glioblastoma tissue niches. Finally, we show that this cancer cell trajectory is intimately linked to myeloid heterogeneity and unfolds across regionalised myeloid signalling environments. Our findings define a stereotyped trajectory of cancer cells in glioblastoma and unify glioblastoma tumour heterogeneity into a tractable cellular and tissue framework.

cancer biology↗

bioRxiv2025

Alleviation of Drought Stress and Plant Growth Promotion in Mungbean through Rhizospheric Actinobacteria

Drought is a major cause for decreasing in crop yield and agricultural productivity around the Globe. The application of drought tolerance Actinobacteria to agroecosystem improves the growth and protect from water scarcity and they can be better alternative for natural farming and sustainable agriculture for aride and semi-aride regions. Herein, we isolated ten morphologically different actinobacteria from the rhizospheric soil of Datura and Khejri plants which are surviving in the nature without any special treatment provided. All the isolated bacteria were screened in-vitro for their drought tolerance capability for different time intervals against polyethylene glycol 8000 (PEG8000) with 5%, 10% and 10% concentration to media which produce moisture stress in which medium bacteria were growing. Among them best five bacterial isolates were selected and named RADM1 to RADM5 for further experimentation. Physiological characterizations were done through their ability to grow in different temperature, pH ranges and NaCl concentration supplemented with the 5% PEG8000. Morphology of the bacteria was Grams positive and mycelium producing same as typical Actinobacterial species and for biochemical diversity stains were able to IMVIC test and carbohydrate utilization through IMVIC test. Enzymatic observations of isolates were resulted positive for nitrate reduction, catalase production and negative for starch hydrolysis. PCR amplification and 16S rRNA gene sequencing in BlastN found maximum similarity of three species with Streptomyces clavuligerus and two species with Rhodococcus erythropolis and submitted to NCBI gene bank database with accession number PQ114139 (RADM1), PQ120343 (RADM2), PQ114109 (RADM3), PQ120390 (RADM4), PQ039766 (RADM5). In pot trial of mungbean, all the isolates showed better performance in bacterial treatments with an average increase in physiological parameters like, seedlings shoot length (52.70% and 9.62%), root length (35.63% and 17.25%), fresh weight (42.8% and 13.76%) dry weight (45.25% and 21.24%) in compared to uninoculated induced drought and watered treatments. Stress features of seedlings revealed significant increase in proline and chlorophyll contents whereas decrease in MDA contents than the untreated plants which indicates actinobacteria reduced water scarcity and damage of plant membrane. IMPORTANCERhizosphere of plant contains diversity of microbes with different abilities to support the plant growth. The role of plant growth promoting bacteria in soil presented and their association with plant can be the one reason for survive of the plant against the abiotic stress conditions like drought, salinity, temperature or pH. The actinobacteria found in the rhizosphere have evolved multiple strategies to assist plants in managing the adverse effects of drought. These strategies may include alterations in phytohormones, which play a vital role in enabling plants to withstand environmental stresses, modifications to root structures, accumulation of osmolytes, enhancements in the plants antioxidant defense mechanisms, synthesis of exopolysaccharides, and the identification of particular genes that support plant growth and improve drought resilience. These actinobacteria as bioinoculant can be the better alternative for the chemical fertilizers use and protect from the water scarcity.

microbiology↗

bioRxiv2025

Intracellular Trafficking SNARE Protein, Syntaxin-6, is a Modifier of Prion and Tau Pathogenesis in vivo and in Cellular Models

Syntaxin-6, a SNARE protein involved in intracellular protein trafficking, is a proposed risk factor for sporadic prion disease, progressive supranuclear palsy and Alzheimers disease. However, no study has validated its functional role in these diseases, explored the disease stage at which it is acting nor its mechanism of action. Here, we show that syntaxin-6 acts at early stages of prion disease in experimental mice by increasing disease transmission risk following inoculation with low prion doses. Conversely, syntaxin-6 does not affect prion propagation kinetics or toxicity during established disease. Syntaxin-6 manipulation in cellular models profoundly alters the subcellular distribution and morphologies of disease-related PrP and modifies prion export. Furthermore, syntaxin-6 knockout in a transgenic tauopathy mouse model exerts protective effects on numerous physiological, behavioural and neuropathological outcome measures. Therefore, our studies firmly establish syntaxin-6 as a modifier of prion and tau pathogenesis, providing key insights into a fundamental mechanism of neurodegeneration.

neuroscience↗

bioRxiv2025

How do egocentric boundary cells depend upon the coordinate system of environmental features?

Neurons in the retrosplenial (RSC) (Alexander et al 2020a, LaChance & Hasselmo 2024) and postrhinal cortex (POR) respond to environmental boundaries and configurations using egocentric coordinates relative to an animals current position. Neurons in these structures and adjacent structures also respond to spatial dimensions of self-motion such as running velocity (Carstensen et al 2021, Robinson et al 2023). Experimental and modeling data suggest that these responses could be essential for guiding behaviors such as obstacle avoidance and goal-directed navigation (Erdem & Hasselmo 2012, Erdem & Hasselmo 2014). However, these findings still leave the unanswered question: What specific features, and in what coordinate frames, drive these egocentric neural responses? Here we present models of the potential circuit mechanisms generating egocentric responses in RSC. One model posits that neurons encode internal representations of barriers in head-centered coordinates, defined by distance and angle, which are modulated by running velocity to enable trajectory planning and obstacle avoidance. We contrast this with a complementary hypothesis in which neurons respond to retinotopic features--such as the top, bottom, or edges of walls, which may serve as precursors to head-centered representations. Additional hypotheses include trajectory-based forward scanning (e.g., ray tracing) for barrier detection or comparing optic flow across the visual field. These hypotheses generate complementary modeling predictions about how changes in environmental parameters could alter the neural responses of egocentric boundary cells that are presented here.

neuroscience↗

bioRxiv2024

R-Spondin Mimetic, SZN-043, Induced Proliferation and Wnt Activity, Two Features Deficient in Human Alcohol-Associated Liver Disease

Liver regeneration is impaired in patients suffering from alcohol-associated liver (ALD) diseases. Wnt ligands and their FZD receptors are dysregulated in diseased livers. R-spondin and their receptors are known to regulate Wnt activity via the stabilization of FZD receptors. Here, we investigated the components of the Wnt and R-Spondin-signaling pathways and their activity in patients with ALD. We found that while hepatocytes retained high levels of differentiation markers such as ASGR1 and ASGR2, the expression of two R-spondin co-receptors, LGR4 and LGR5, and of Wnt target genes, CYP1A2 and others, were strongly reduced. SZN-043, a hepatocyte-targeted R-Spondin mimetic, is a new investigational drug that stimulates the physiological Wnt repair pathway and proliferation of hepatocytes. Here, we show that SZN-043 induced hepatocyte proliferation in all models tested, including humanized mouse livers, a chronic-binge alcohol-induced liver injury, and a CCl4-induced fibrosis mouse model. Altogether, SZN-043 could be beneficial for the treatment of ALD.

pathology↗

bioRxiv2024

Haploinsufficiency of lysosomal enzyme genes in Alzheimers disease

There is growing evidence suggesting that the lysosome or lysosome dysfunction is associated with Alzheimers disease (AD). Pathway analysis of post mortem brain-derived proteomic data from AD patients shows that the lysosomal system is perturbed relative to similarly aged unaffected controls. However, it is unclear if these changes contributed to the pathogenesis or are a response to the disease. Consistent with the hypothesis that lysosome dysfunction contributes to AD pathogenesis, whole genome sequencing data indicate that heterozygous pathogenic mutations and predicted protein-damaging variants in multiple lysosomal enzyme genes are enriched in AD patients compared to matched controls. Heterozygous loss-of-function mutations in the palmitoyl protein thioesterase-1 (PPT1), -L-iduronidase (IDUA), {beta}-glucuronidase (GUSB), N-acetylglucosaminidase (NAGLU), and galactocerebrosidase (GALC) genes have a gene-dosage effect on A{beta}40 levels in brain interstitial fluid in C57BL/6 mice and significantly increase A{beta} plaque formation in the 5xFAD mouse model of AD, thus providing in vivo validation of the human genetic data. A more detailed analysis of PPT1 heterozygosity in 18-month-old mice revealed changes in -, {beta}-, and {gamma}-secretases that favor an amyloidogenic pathway. Proteomic changes in brain tissue from aged PPT1 heterozygous sheep are consistent with both the mouse data and the potential activation of AD pathways. Finally, CNS-directed, AAV-mediated gene therapy significantly decreased A{beta} plaques, increased life span, and improved behavioral performance in 5xFAD/PPT1+/- mice. Collectively, these data strongly suggest that heterozygosity of multiple lysosomal enzyme genes represent risk factors for AD and may identify precise therapeutic targets for a subset of genetically-defined AD patients. Significance StatementLysosomes play a role in the degradation of aggregation-prone proteins such as amyloid {beta} (A{beta}). Homozygous lysosomal enzyme gene defects result in fatal pediatric lysosomal storage diseases and, historically, carriers were considered normal. However, a human genetic analysis identified deleterious heterozygous variants in multiple lysosomal enzyme genes that are enriched in Alzheimers disease (AD) patients. Those findings were validated in vivo by demonstrating that heterozygous loss-of-function (LoF) mutations in five different lysosomal enzyme genes affect A{beta} processing and exacerbate A{beta} plaque formation. CNS-directed gene therapy ameliorated the effects of a heterozygous LoF mutation in one of those genes in a mouse model of AD. These findings provide insights into the role of lysosomes in AD and have important therapeutic implications.

neuroscience↗

bioRxiv2024

Multi-stage-mixing to create a core-then-shell structure improves DNA-loaded lipid nanoparticles transfection by orders of magnitude

As they became the dominant gene therapy platform, lipid nanoparticles (LNPs) experienced nearly all their innovation in varying the structure of individual molecules in LNPs. This ignored control of the spatial arrangement of molecules, which is suboptimal because supramolecular structure determines function in biology. To control LNPs supramolecular structure, we introduce multi-stage-mixing (MSM) to successively add different molecules to LNPs. We first utilize MSM to create a core-then-shell (CTS) synthesis. CTS-LNPs display a clear core-shell structure, vastly lower frequency of LNPs containing no detectable mRNA, and improved mRNA-LNP expression. With DNA-loaded LNPs, which for decades lagged behind mRNA-LNPs due to low expression, CTS improved DNA-LNPs protein expression by 2-3 orders of magnitude, bringing it within range of mRNA-LNPs. These results show that supramolecular arrangement is critical to LNP performance and can be controlled by mixing methodology. Further, MSM/CTS have finally made DNA-LNPs into a practical platform for long-term gene expression.

bioengineering↗