Gene Delivery Research Articles

Silencing the glycerol-3-phosphate acyltransferase-1 gene in the liver of mice fed a high-fat diet, enhances insulin sensitivity and glucose metabolism by promoting fatty acid beta-oxidation

BackgroundLiver plays a central role in systemic glucose and lipid metabolism. High-fat diet (HFD) and obesity are related to hepatic lipid accumulation and insulin resistance (InsR). Diacylglycerols (DAG) play a key role in the induction of InsR, however their involvement in hepatic InsR remains debated. This study aimed to clarify and confirm the role of glycero-3-phosphate acyltransferase 1 (GPAT1), a rate-limiting enzyme in DAG synthesis, in the progression of hepatic InsR in the context of HFD-induced lipid accumulation and insulin resistance in the liver. MethodsLiver-targeted GPAT1 silencing was performed using shRNA-mediated hydrodynamic gene delivery. Lipid species including LCA-CoA, sphingolipids, DAG and acyl-carnitines were quantified using UHPLC/MS/MS while insulin signalling was assessed at protein level by Western Blot. Hepatic glucose metabolism, including glucose-6-pasphate content and gluconeogenesis rate was evaluated using GC/MS. ResultsHFD-fed animals developed InsR, evidenced by increased HOMA-IR, enhanced gluconeogenesis and reduced glycogen content compared to controls. Hepatic GPAT1 silencing in HFD-fed animals resulted in a significant reduction of DAG and TAG levels, increased acyl-carnitines content and upregulated mitochondrial β-oxidation protein expression. These changes were accompanied by improved insulin signalling, enhanced glycogen storage, and reduced gluconeogenesis. ConclusionsSilencing GPAT1, and thereby reducing glycerolipid synthesis, promotes β-oxidation and ameliorates HFD-induced hepatic insulin resistance, confirming the enzyme's pivotal role in liver metabolic dysfunction associated with increased lipid supply.

PH-Responsive Micelles Containing Quinine Functionalities Enhance Intracellular Gene Delivery and Expression.

Quinine is a promising building block for creating polymer carriers for intracellular nucleic acid delivery. This is due to its ability to bind to genetic material through intercalation and electrostatic interactions and the balance of hydrophobicity and hydrophilicity dependent on the pH/charge state. Yet, studies utilizing cinchona alkaloid natural products in gene delivery are limited. Herein, we present the incorporation of a quinine functionalized monomer (Q) into block polymer architectures to form self-assembled micelles for highly efficient gene delivery. Q was incorporated into the core and/or the shell of the micelles to introduce the unique advantages of quinine to the system. We found that incorporation of Q into the core of the micelle resulted in acid-induced disassembly of the micelle and a boost in transfection efficiency by promoting endosomal escape. This effect was especially evident in the cancerous cell line, A549, which has a more acidic intracellular environment. Incorporation of Q into the shell of the micelles resulted in intercalative binding to the genetic payload as well as larger micelle-DNA complexes (micelleplexes) from the hydrophobicity of Q in the shell. These factors enable the micelleplexes to be more resistant to serum and have more persistent protein expression post-transfection. Overall, this study is the first to demonstrate the benefits of including quinine functionalities into self-assembled micelles for highly efficient gene delivery and presents a platform for inclusion of other natural products with similar properties into micellar systems.

An optimized protocol for quality control of gene therapy vectors using nanopore direct RNA sequencing.

Despite recent advances made toward improving the efficacy of lentiviral gene therapies, a sizeable proportion of produced vector contains an incomplete and thus potentially nonfunctional RNA genome. This can undermine gene delivery by the lentivirus as well as increase manufacturing costs and must be improved to facilitate the widespread clinical implementation of lentiviral gene therapies. Here, we compare three long-read sequencing technologies for their ability to detect issues in vector design and determine nanopore direct RNA sequencing to be the most powerful. We show how this approach identifies and quantifies incomplete RNA caused by cryptic splicing and polyadenylation sites, including a potential cryptic polyadenylation site in the widely used Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE). Using artificial polyadenylation of the lentiviral RNA, we also identify multiple hairpin-associated truncations in the analyzed lentiviral vectors (LVs), which account for most of the detected RNA fragments. Finally, we show that these insights can be used for the optimization of LV design. In summary, nanopore direct RNA sequencing is a powerful tool for the quality control and optimization of LVs, which may help to improve lentivirus manufacturing and thus the development of higher quality lentiviral gene therapies.

Comparative analysis of six adeno-associated viral vector serotypes in mouse inferior colliculus and cerebellum.

Adeno-associated viral vector (AAV) serotypes vary in how effectively they express genes across different cell types and brain regions. Here we report a systematic comparison of the AAV serotypes 1, 2, 5, 8, 9, and the directed evolution derived AAVrg, in the inferior colliculus and cerebellum. The AAVs were identical apart from their different serotypes, each having a synapsin promotor and expressing GFP (AAV-hSyn-GFP). Identical titers and volumes were injected into the inferior colliculus and cerebellum of adult male and female mice and brains were sectioned and imaged 2 weeks later. Transduction efficacy, anterograde labeling of axonal projections, and retrograde labeling of somata, were characterized and compared across serotypes. Cell-type tropism was assessed by analyzing the morphology of the GFP-labeled neurons in the cerebellar cortex. In both the cerebellum and inferior colliculus, AAV1 expressed GFP in more cells, labeled a larger volume, and produced significantly brighter labeling than all other serotypes, indicating superior transgene expression. AAV1 labeled more Purkinje cells, unipolar brush cells, and molecular layer interneurons than the other serotypes, while AAV2 labeled a greater number of granule cells. These results provide guidelines for the use of AAVs as gene delivery tools in these regions.Significance statement AAVs have become ubiquitous gene expression tools in neuroscience research and are becoming more common in clinical settings. Naturally occurring and engineered serotypes have varying abilities to infect neurons and cause them to produce proteins of interest. The efficacy of AAV transduction in specific cell types depends on many factors and remains difficult to predict, so an empirical approach is often required to determine the best performing serotype in each population of cells. In the present study we show that AAV1 produces the highest expression in these two regions, labels the most axonal projections, and labels Purkinje cells and unipolar brush cells better than the other serotypes tested, while AAV2 labels granule cells most effectively.

Efficient Polymeric Nanoparticle Gene Delivery Enabled Via Tri- and Tetrafunctional Branching.

Poly(β-amino ester) (PBAE) nanoparticles (NPs) show great promise for nonviral gene delivery. Recent studies suggest branched PBAEs (BPBAEs) offer advantages over linear counterparts, but the effect of polymer structure has not been well investigated across many chemical constituents. Here, a library of BPBAEs was synthesized with tri- and tetrafunctional branching. These polymers self-assemble with DNA to form highly cationic, monodisperse NPs with notably small size (∼50 nm). Optimal transfection occurred with polymer structures that featured moderate PBAE branching, enabling complete DNA encapsulation, rapid NP uptake, and robust expression at low DNA doses and polymer amounts. Optimized NPs enabled efficient DNA delivery to diverse cell types in vitro while maintaining high cellular viability, demonstrating significant improvements over a well-performing linear PBAE counterpart. BPBAEs also facilitated efficient mRNA and siRNA delivery, highlighting the versatility of these structures and demonstrating the broad utility of BPBAE NPs as vectors for nucleic acid delivery.

Cationized extracellular vesicles for gene delivery

Last decade, extracellular vesicles (EVs) attracted a lot of attention as potent versatile drug delivery vehicles. We reported earlier the development of EV-based delivery systems for therapeutic proteins and small molecule chemotherapeutics. In this work, we first time engineered EVs with multivalent cationic lipids for the delivery of nucleic acids. Stable, small size cationized EVs were loaded with plasmid DNA (pDNA), or mRNA, or siRNA. Nucleic acid loaded EVs were efficiently taken up by target cells as demonstrated by confocal microscopy and delivered their cargo to the nuclei in triple negative breast cancer (TNBC) cells and macrophages. Efficient transfection was achieved by engineered cationized EVs formulations of pDNA- and mRNA in vitro. Furthermore, siRNA loaded into cationized EVs showed significant knockdown of the reporter gene in Luc-expressing cells. Overall, multivalent cationized EVs represent a promising strategy for gene delivery.

Remote-Controlled Gene Delivery in Coaxial 3D-Bioprinted Constructs using Ultrasound-Responsive Bioinks

Abstract Introduction Coaxial 3D bioprinting has advanced the formation of tissue constructs that recapitulate key architectures and biophysical parameters for in-vitro disease modeling and tissue-engineered therapies. Controlling gene expression within these structures is critical for modulating cell signaling and probing cell behavior. However, current transfection strategies are limited in spatiotemporal control because dense 3D scaffolds hinder diffusion of traditional vectors. To address this, we developed a coaxial extrusion 3D bioprinting technique using ultrasound-responsive gene delivery bioinks. These bioink materials incorporate echogenic microbubble gene delivery particles that upon ultrasound exposure can sonoporate cells within the construct, facilitating controllable transfection. Methods Phospholipid-coated gas-core microbubbles were electrostatically coupled to reporter transgene plasmid payloads and incorporated into cell-laden alginate bioinks at varying particle concentrations. These bioinks were loaded into the coaxial nozzle core for extrusion bioprinting with CaCl2 crosslinker in the outer sheath. Resulting bioprints were exposed to 2.25 MHz focused ultrasound and evaluated for microbubble activation and subsequent DNA delivery and transgene expression. Results Coaxial printing parameters were established that preserved the stability of ultrasound-responsive gene delivery particles for at least 48 h in bioprinted alginate filaments while maintaining high cell viability. Successful sonoporation of embedded cells resulted in DNA delivery and robust ultrasound-controlled transgene expression. The number of transfected cells was modulated by varying the number of focused ultrasound pulses applied. The size region over which DNA was delivered was modulated by varying the concentration of microbubbles in the printed filaments. Conclusions Our results present a successful coaxial 3D bioprinting technique designed to facilitate ultrasound-controlled gene delivery. This platform enables remote, spatiotemporally-defined genetic manipulation in coaxially bioprinted tissue constructs with important applications for disease modeling and regenerative medicine.

Gene Augmentation Techniques to Stimulate Wound Healing Process: Progress and Prospects.

Gene therapy has traditionally been used to treat individuals with late-stage cancers or congenital abnormalities. Numerous prospects for therapeutic genetic modifications have emerged with the discovery that gene therapy applications are far more extensive, particularly in skin and exterior wounds. Cutaneous wound healing is a complex, multistep process involving multiple steps and mediators that operate in a network of activation and inhibition processes. This setting presents a unique obstacle for gene delivery. Many gene delivery strategies have been developed, including liposomal administration, high-pressure injection, viral transfection, and the application of bare DNA. Among several gene transfer techniques, categorical polymers, nanoparticles, and liposomalbased constructs show great promise for non-viral gene transfer in wounds. Clinical experiments have shown that efficient transportation of certain polypeptides to the intended wound location is a crucial factor in wound healing. Genetically engineered cells can be used to produce and control the delivery of specific growth factors, thereby addressing the drawbacks of mechanically administered recombinant growth factors. We have discussed how repair mechanisms are based on molecules and cells, as well as their breakdown, and provided an overview of the methods and research conducted on gene transmission in tissue regeneration.

Structuring lipid nanoparticles, DNA, and protein corona into stealth bionanoarchitectures for in vivo gene delivery

Lipid nanoparticles (LNPs) play a crucial role in addressing genetic disorders, and cancer, and combating pandemics such as COVID-19 and its variants. Yet, the ability of LNPs to effectively encapsulate large-size DNA molecules remains elusive. This is a significant limitation, as the successful delivery of large-size DNA holds immense potential for gene therapy. To address this gap, the present study focuses on the design of PEGylated LNPs, incorporating large-sized DNA, departing from traditional RNA and ionizable lipids. The resultant LNPs demonstrate a unique particle morphology. These particles were further engineered with a DNA coating and plasma proteins. This multicomponent bionanoconstruct exhibits enhanced transfection efficiency and safety in controlled laboratory settings and improved immune system evasion in in vivo tests. These findings provide valuable insights for the design and development of bionanoarchitectures for large-size DNA delivery, opening new avenues for transformative gene therapies.

Optimizing lipid nanoparticles for fetal gene delivery in vitro, ex vivo, and aided with machine learning

There is a clinical need to develop lipid nanoparticles (LNPs) to deliver congenital therapies to the fetus during pregnancy. The aim of these therapies is to restore normal fetal development and prevent irreversible conditions after birth. As a first step, LNPs need to be optimized for transplacental transport, safety on the placental barrier and fetal organs and transfection efficiency. We developed and characterized a library of LNPs of varying compositions and used machine learning (ML) models to delineate the determinants of LNP size and zeta potential. Utilizing different in vitro placental models with the help of a Random Forest algorithm, we could identify the top features driving percentage LNP transport and kinetics at 24 h, out of a total of 18 input features represented by 41 LNP formulations and 48 different transport experiments. We further evaluated the LNPs for safety, placental cell uptake, transfection efficiency in placental trophoblasts and fetal lung fibroblasts. To ensure the integrity of the LNPs following transplacental transport, we screened LNPs for transport and transfection using a high-throughput integrated transport-transfection in vitro model. Finally, we assessed toxicity of the LNPs in a tracheal occlusion fetal lung explant model. LNPs showed little to no toxicity to fetal and placental cells. Immunoglobin G (IgG) orientation on the surface of LNPs, PEGylated lipids, and ionizable lipids had significant effects on placental transport. The Random Forest algorithm identified the top features driving LNPs placental transport percentage and kinetics. Zeta potential emerged in the top driving features. Building on the ML model results, we developed new LNP formulations to further optimize the transport leading to 622 % increase in transport at 24 h versus control LNP formulation. To induce preferential siRNA transfection of fetal lung, we further optimized cationic lipid percentage and the lipid-to-siRNA ratio. Studying LNPs in an integrated placental and fetal lung fibroblasts model showed a strong correlation between zeta potential and fetal lung transfection. Finally, we assessed the toxicity of LNPs in a tracheal occlusion lung explant model. The optimized formulations appeared to be safe on ex vivo fetal lungs as indicated by insignificant changes in apoptosis (Caspase-3) and proliferation (Ki67) markers. In conclusion, we have optimized an LNP formulation that is safe, with high transplacental transport and preferential transfection in fetal lung cells. Our research findings represent an important step toward establishing the safety and effectiveness of LNPs for gene delivery to the fetal organs.

Recent Updates on Diverse Nanoparticles and Nanostructures in Therapeutic and Diagnostic Applications with Special Focus on Smart Protein Nanoparticles: A Review.

Nanomedicine enables advanced therapeutics, diagnostics, and predictive analysis, enhancing treatment outcomes and patient care. The choices and development of high-quality organic nanoparticles with relatively lower toxicity are important for achieving advanced medical goals. Among organic molecules, proteins have been prospected as smart candidates to revolutionize nanomedicine due to their inherent fascinating features. The advent of protein nanoarchitectures, which explore the biomolecular corona, offers new insights into their efficient tissue penetration and therapeutic potential. This review examines various animal- and plant-based protein nanoparticles, highlighting their source, activity, products, and unique biomedical applications in regenerative medicine, targeted therapies, gene and drug delivery, antimicrobial activity, bioimaging, immunological adjuvants, etc. It provides an extensive discussion on recent applications of protein nanoparticles across diverse biomedical fields as well as the evolving landscape of other nanoproducts and nanodevices for sensitive medical procedures. Furthermore, this review introduces different preparation technologies of protein nanoparticles, emphasizing how their design and construction significantly influence loading capacity, stability, and targeting effects. Additionally, we delve into the construction of different user-friendly multifunctional modular bioarchitectures by the assembly of protein nanoparticles (PNPs), marking a significant breakthrough in therapies. This review also considers the challenges of synthetic nanomaterials and the emergence of natural alternatives, which provides insights into protein nanoparticle research.

A Non-Invasive and DNA-free Approach to Upregulate Mammalian Voltage-Gated Calcium Channels and Neuronal Calcium Signaling via Terahertz Stimulation.

Mammalian voltage-gated calcium channels (CaV) play critical roles in cardiac excitability, synaptic transmission, and gene transcription. Dysfunctions in CaV are implicated in a variety of cardiac and neurodevelopmental disorders. Current pharmacological approaches to enhance CaV activity are limited by off-target effects, drug metabolism issues, cytotoxicity, and imprecise modulation. Additionally, genetically-encoded channel activators and optogenetic tools are restricted by gene delivery challenges and biosafety concerns. Here a novel terahertz (THz) wave-based method to upregulate CaV1.2, a key subtype of CaV, and boost CaV1-mediated Ca2+ signaling in neurons without introducing exogenous DNA is presented. Using molecular dynamics simulations, it is shown that 42.5 THz (7.05µm, 1418cm-1) waves enhance Ca2+ conductance in CaV1.2 by resonating with the stretching mode of the -COO- group in the selectivity filter. Electrophysiological recordings and Ca2+ imaging confirm that these waves rapidly, reversibly, and non-thermally increase calcium influx of CaV1.2 in HEK293 cells and induce acute Ca2+ signals in neurons. Furthermore, this irradiation upregulates critical CaV1 signals, including CREB phosphorylation and c-Fos expression, in vitro and in vivo, without raising significant biosafety risks. This DNA-free, non-invasive approach offers a promising approach for modulating CaV gating and Ca2+ signaling and treating diseases characterized by deficits in CaV functions.

Enhancing gene delivery efficiency with amphiphilic chitosan modified by myristic acid and tertiary amino groups

The aim of this study is to synthesize new amphiphilic chitosan containing myristic acid as the hydrophobic tail and tertiary amine groups as the hydrophilic head and to evaluate the gene delivery efficiency. In this context, the primary amine groups of chitosan were first modified with myristic acid (Chi-M), followed by the modification of the methylol groups with 3-dimethylamino-1-propyl chloride hydrochloride. The chemical characterization of this chitosan formulation (Chi-MA) was determined using nuclear magnetic resonance (NMR), Fourier-transform infrared spectroscopy (FTIR) analysis and gel permeation chromatography-size exclusion chromatography. Chi-MA nanoparticles were prepared via ionic gelation, and particle size, polydispersity and zeta potential were determined. The nanoparticles were evaluated for their proton buffering capacity and gene complexing capacity. Additionally, the cytotoxicity of Chi-MA on HEK293T cells was determined via MTT assay, and the transfection efficiency of Chi-MA was analyzed by a flow cytometer. The results indicate a significant increase in gene complexing capacity (8-fold) and nanoparticle formation ability of Chi-MA compared to other chitosan formulations. Chi-MA nanoparticles showed no toxicity against HEK293T cells and exhibited the highest transfection efficiency with significantly lower nanoparticle: gene ratios compared to previous studies. These findings demonstrate the effective use of amphiphilic Chi-MA as a gene carrier.

Metal Ion-Condensed DNA Nanoparticle Library: Phase Separation and Transition and Antisense Therapy Applications.

DNA condensation has long been investigated as a fundamental cellular activity and is known to be driven by the mediation of diverse condensing agents. The phase behaviors of DNA during condensation are particularly interesting because the complicated molecular structure of natural nucleotides fundamentally allows electrostatic, coordinate covalent, and various other secondary interactions with the condensing agents. Recently, metal ion (Mn+)-induced DNA condensation has emerged as a powerful approach to synthesizing nanoparticulate DNA structures suitable for therapeutic gene delivery. However, how the DNA phase changes during Mn+-induced DNA condensation has rarely been observed and is not understood yet. In this study, a library of Mn+-condensed DNA nanoparticles (Mn+-CDNPs) was established using 30 different types of Mn+s, and their phase behaviors during condensation were elucidated using spherical nucleic acids (SNAs) as electron microscopic labels. Importantly, the phase transition and separation of DNA were demonstrated to be driven by the Mn+s into either the growth of individual DNA particles or the fission of bulky DNA aggregates. Pt2+ and Eu3+ were chosen as model systems for the demonstration. The hard and soft acid nature of Mn+ is presumably the underlying driving force of these phase transitions. In addition, the Mn+-controlled anticancer therapeutic efficiency of the Mn+-CDNP library as a state-of-the-art gene delivery platform was demonstrated even for unmodified antisense oligonucleotides in association with the potential toxicity of the Mn+s released from the Mn+-CDNPs. This comprehensive study of the Mn+-dependent condensation of nucleic acids provides profound insights into the chemistry of the nucleic acid-Mn+ interactions and the reliable theragnostic applications of Mn+-CDNPs as functional nucleic acid nanostructures.

Development of an RNA virus-based episomal vector with artificial aptazyme for gene silencing

RNA virus-based episomal vector (REVec), engineered from Borna disease virus, is an innovative gene delivery tool that enables sustained gene expression in transduced cells. However, the difficulty in controlling gene expression and eliminating vectors has limited the practical use of REVec. In this study, we overcome these shortcomings by inserting artificial aptazymes into the untranslated regions of foreign genes carried in vectors or downstream of the viral phosphoprotein gene, which is essential for vector replication. Non-transmissive REVec carrying GuaM8HDV or the P1-F5 aptazyme showed immediate suppression of gene expression in a guanine or theophylline concentration-dependent manner. Continuous compound administration also markedly reduced the percentage of vector-transduced cells and eventually led to the complete elimination of the vectors from the transduced cells. This new REVec is a safe gene delivery technology that allows fine-tuning of gene expression and could be a useful platform for gene therapy and gene-cell therapy, potentially contributing to the cure of many genetic disorders.Key points• We developed a bornavirus vector capable of silencing transgene expression by insertion of aptazyme• Transgene expression was markedly suppressed in a compound concentration-dependent manner• Artificial aptazyme systems allowed complete elimination of the vector from transduced cells

Precise Preparation of Supramolecular Spherical Nucleic Acids for Nucleolin‐Targeted Gene Delivery

AbstractMolecular spherical nucleic acids (m‐SNAs) are a second generation of spherical nucleic acids (SNAs), which are of significance in potential application of targeted delivery of nucleic acids or gene regulation due to their defined molecular structures. Nevertheless, m‐SNAs typically involve a single DNA sequence which greatly limits its functions as either targeting purpose or gene regulation. In response, we proposed here a third generation, supramolecular spherical nucleic acids (Supra‐SNAs) with two different sequences to achieve both above‐mentioned functions. Specifically, we constructed a series of supramolecular self‐assembly structures by coupling a cell membrane receptor (i.e., nucleolin)‐recognizing aptamer (AS1411)‐modified adamantine as targeting probe and human epithelial growth factor receptor 2 (HER2) antisense‐functionalized β‐cyclodextrin to specifically inhibit the overexpression of HER2 proteins for gene regulations. In comparison to the m‐SNA precursors, such Supra‐SNA structures exhibited enhanced levels of resistance to nuclease degradation, cellular uptake, gene regulation capabilities and tumor retention capacity. We demonstrated that Supra‐SNAs exhibited optimal cell suppression rates and cell apoptosis via a phosphatidylinositol 3‐kinase/protein kinase B signaling pathway. The well‐defined molecular structures provide an attractive platform for investigating interrelationship between structure and property at the molecular level.

Growth Factor Gene Therapy for Alzheimer's Disease.

Nervous system growth factors are natural proteins of the brain that influence neuronal survival and function throughout life, from embryonic development to old age. In animal models of Alzheimer's disease (AD), the growth factor brain derived neurotrophic factor (BDNF) prevents neuronal death, activates neuronal function, builds new synapses and improves learning and memory. Accordingly, we are determining whether gene delivery of BDNF in patients with AD will slow disease progression and improve memory. In a previous clinical trial of nerve growth factor (NGF) gene therapy in AD patients (NCT00017940, June 2001), we learned that growth factors can unequivocally elicit classic trophic responses from degenerating neurons in AD. Experience gained from the earlier NGF gene therapy trial is guiding our effort to optimize gene delivery of BDNF in our present clinical program (NCT05040217, June 2021).

Precise Preparation of Supramolecular Spherical Nucleic Acids for Nucleolin-Targeted Gene Delivery.

Molecular spherical nucleic acids (m-SNAs) are a second generation of spherical nucleic acids (SNAs), which are of significance in potential application of targeted delivery of nucleic acids or gene regulation due to their defined molecular structures. Nevertheless, m-SNAs typically involve a single DNA sequence which greatly limits its functions as either targeting purpose or gene regulation. In response, we proposed here a third generation, supramolecular spherical nucleic acids (Supra-SNAs) with two different sequences to achieve both above-mentioned functions. Specifically, we constructed a series of supramolecular self-assembly structures by coupling a cell membrane receptor (i.e., nucleolin)-recognizing aptamer (AS1411)-modified adamantine as targeting probe and human epithelial growth factor receptor 2 (HER2) antisense-functionalized β-cyclodextrin to specifically inhibit the overexpression of HER2 proteins for gene regulations. In comparison to the m-SNA precursors, such Supra-SNA structures exhibited enhanced levels of resistance to nuclease degradation, cellular uptake, gene regulation capabilities and tumor retention capacity. We demonstrated that Supra-SNAs exhibited optimal cell suppression rates and cell apoptosis via a phosphatidylinositol 3-kinase/protein kinase B signaling pathway. The well-defined molecular structures provide an attractive platform for investigating interrelationship between structure and property at the molecular level.

Spatially-Resolved Organoid Transfection by Porous Silicon-Mediated Optoporation.

Engineering the spatial organisation of organotypic cultures is pivotal for refining tissue models that are useful for gaining deeper insights into complex, non-cell autonomous processes. These advanced models are key to improving the understanding of fundamental biological mechanisms and therapeutic strategies. Controlling gene regulation through spatially-resolved delivery of nucleic acids provides an attractive approach to produce such tissue models. An emerging strategy for spatially-resolved transfection uses photosensitizing nanoparticles coupled with laser pulses to optoporate cells in culture and locally mediate gene delivery. However, localized optoporation in 3D systems remains challenging. Here we propose a solution to this longstanding hurdle, demonstrating that porous silicon nanoparticles are a safe and bioresorbable photosensitising nanomaterial capable of spatially-resolved transfection of mRNA in MCF-7 organoids by near-infrared two-photon optoporation. Functionalization with an azobenzene-lysine photo-switchable moiety enhances the transfection efficiency of the nanoparticles up to 84% in a 2D cell system. Moreover, the nanoparticles enable spatially selective mRNA transfection to MCF-7 spheroids, demonstrating targeted gene delivery in complex 3D cellular environments. The approach for spatially-resolved 3D optoporation offers a way forward for the design of tailored spheroids and organoids by spatially selective nucleic acids delivery.

Identification of SLC35A1 as an essential host factor for the transduction of multi-serotype recombinant adeno-associated virus (AAV) vectors.

rAAV is an essential tool for gene delivery in the treatment of genetic disorders, yet the mechanisms of rAAV transduction remain partially understood. GPR108 is vital for the transduction of most rAAV vectors, but not for rAAV5. We aimed to identify host factors that impact AAV5 transduction akin to GPR108. Using a genome-wide CRISPR/Cas9 screen in 293-F cells, we identified SLC35A1, a Golgi apparatus-localized CMP-sialic acid transporter that transports CMP-sialic acid from cytoplasm into the Golgi apparatus for sialylation, is essential to rAAV transduction. Further studies across various AAV serotypes showed SLC35A1 significantly affects vector nuclear import post-internalization. These results underscore the crucial role of SLC35A1 in intracellular trafficking beyond the initial cell attachment of rAAV.