Delivery Vectors Research Articles

Intracisternal vs intraventricular injection of AAV1 result in comparable, widespread transduction of the dog brain.

Widespread distribution of transduced brain cells following delivery of AAV vectors into the cerebrospinal fluid (CSF) of the cisterna magna (CM) has been demonstrated in large animal brains. In humans, intraventricular injection is preferred to intracisternal injection for CSF delivery due to the risk of brain stem injury. One study in the dog reported adverse reactions to AAV vectors expressing GFP injected into the lateral ventricle but not when injected into the CM. In contrast, AAV expressing mammalian genes in diseased animals have not triggered adverse responses since many genetic diseases also have compromised immune systems. Differences in circulation of CSF from each site could potentially affect vector spread within the brain, but a direct comparison has not been made using both a mammalian gene and immunologically normal animals. In this study we evaluated the dopamine-2-receptor (D2R) variant D2R80A, which is inactivated for intracellular signaling and has been used as a reporter gene in large animal brains. No adverse reactions to the D2R80A gene were observed from either injection route in normal dogs and both routes resulted in comparable distribution of D2R80A within the brain.

Enhancing non-viral DNA delivery systems: Recent advances in improving efficiency and target specificity.

DNA-based therapies are often limited by challenges such as stability, long-term integration, low transfection efficiency, and insufficient targeted DNA delivery. This review focuses on recent progress in the design of non-viral delivery systems for enhancing targeted DNA delivery and modulation of therapeutic efficiency. Cellular uptake and intracellular trafficking mechanisms play a crucial role in optimizing gene delivery efficiency. There are two main strategies employed to improve the efficiency of gene delivery vectors: (i) explore different administration routes (e.g., mucosal, intravenous, intramuscular, subcutaneous, intradermal, intratumoural, and intraocular) that best facilitates optimal uptake into the targeted cells and organs and (ii) modify the delivery vectors with cell-specific ligands (e.g., natural ligands, antibodies, peptides, carbohydrates, or aptamers) that enable targeted uptake to specific cells with higher specificity and improved biodistribution. We describe how recent progress in employing these DNA delivery strategies is advancing the field and increasing the clinical translation and ultimate clinical application of DNA therapies.

Enhancing the Deformability of the Adjuvant: Achieving Lymph Node Targeted Delivery to Elicit a Long-Lasting Immune Protection.

A key challenge in vaccine development is to inducean effective and durable immune response. Live virus vaccines induce lifelong antibody responses; however, the immune responses induced by inactivated or subunit vaccines decrease gradually. Activation of the germinal center (GC) reaction, which generates long-lived plasma cells (LLPCs), is a key mediator of long-term antibody responses. To enhance the activation of GC, lymph node-targeted delivery of the vaccine is promoted by enhancing the deformability of the delivery vector. In this study, a double emulsion is designed with strong deformability and containing chitosan nanoparticles (CSNP) in the internal aqueous phase (WNP) for efficient antigen loading, called WNP/O/W. The flexible oil layer and the internally loaded positively charged particles endow the emulsion with strong deformability, continuously enrich model antigen ovalbumin(OVA)in the lymph nodes, activate germinal center B (GC B) cells and T follicular helper (TFH) cells, induce LLPCs, and obtain high-level antibody persistence for more than 5 months, which is significantly better than the traditional oil emulsion adjuvant. Concurrently, it also improves the immune-protective effect in aged mice. Altogether, these results indicate that WNP/O/W achieves lymph node targeted delivery by strengthening deformability, generating high-intensity antibody responses, and long-lasting immune protection.

Metabolic engineering improves transduction efficiency and downstream vector isolation by altering the lipid composition of extracellular vesicle-enclosed AAV.

Adeno-associated viruses (AAV) are promising vectors for gene therapy due to their efficacy in vivo. However, there is room for improvement to address key limitations such as the pre-existing immunity to AAV in patients, high-dose toxicity, and relatively low efficiency for some cell types. This study introduces a metabolic engineering approach, using knockout of the enzyme phosphatidylserine synthase 1 (PTDSS1) to increase the abundance of extracellular vesicle-enclosed AAV (EV-AAV) relative to free AAV in the supernatant of producer cells, simplifying downstream purification processes. The lipid-engineered HEK293T-ΔPTDSS1 cell line achieved a 42.7-fold enrichment of EV-AAV9 compared to free AAV9 in the supernatant. The rational genetic strategy also led to a 300-fold decrease of free AAV in supernatant compared to wild-type HEK293T. The membrane-engineered EV-AAV9 (mEV-AAV9) showed unique envelope composition alterations, including cholesterol enrichment and improved transduction efficiency in human AC16 cardiomyocytes by 1.5-fold compared to conventional EV-AAV9 and by 11-fold compared to non-enveloped AAV9. Robust in-vivo transduction four weeks after intraparenchymal administration of mEV-AAV9 was observed in the murine brain. This study shows promise in the potential of lipid metabolic engineering strategies to improve the efficiency and process development of enveloped gene delivery vectors.

Advances in the study of LNPs for mRNA delivery and clinical applications.

Messenger ribonucleic acid (mRNA) was discovered in 1961 as an intermediary for transferring genetic information from DNA to ribosomes for protein synthesis. The COVID-19 pandemic brought worldwide attention to mRNA vaccines. The emergency use authorization of two COVID-19 mRNA vaccines, BNT162b2 and mRNA-1273, were major achievements in the history of vaccine development. Lipid nanoparticles (LNPs), one of the most superior non-viral delivery vectors available, have made many exciting advances in clinical translation as part of the COVID-19 vaccine and therefore has the potential to accelerate the clinical translation of many gene drugs. In addition, due to these small size, biocompatibility and excellent biodegradability, LNPs can efficiently deliver nucleic acids into cells, which is particularly important for current mRNA therapeutic regimens. LNPs are composed cationic or pH-dependent ionizable lipid bilayer, polyethylene glycol (PEG), phospholipids, and cholesterol, represents an advanced system for the delivery of mRNA vaccines. Furthermore, optimization of these four components constituting the LNPs have demonstrated enhanced vaccine efficacy and diminished adverse effects. The incorporation of biodegradable lipids enhance the biocompatibility of LNPs, thereby improving its potential as an efficacious therapeutic approach for a wide range of challenging and intricate diseases, encompassing infectious diseases, liver disorders, cancer, cardiovascular diseases, cerebrovascular conditions, among others. Consequently, this review aims to furnish the scientific community with the most up-to-date information regarding mRNA vaccines and LNP delivery systems.

Expression and delivery of HA1-M2e antigen using an innovative attenuated Salmonella–mediated delivery system confers promising protection against H9N2 avian influenza challenge

This study explores a dual expression vector system for delivering prokaryotic and eukaryotic antigens to improve conventional vaccination strategies. To enhance immune protection against H9N2 avian influenza virus (AIV), which threatens poultry and humans, we used the previously constructed pJHL270 and pJHL305 plasmids with the Ptrc and CMV promoters to stimulate MHC class II and I responses through exogenous and endogenous antigenic presentation. Salmonella Gallinarum (SG), a delivery vector, was engineered to have defective lipopolysaccharide structures through lon, pagL, and rfaL deletion. It demonstrated a safety profile with lower induction of inflammatory cytokines than the wild-type strain. Bioinformatics tools predicted that the HA1 and M2e sequences, which were designed as consensus sequences of South Korean strains (2000–2021), would have high antigenicity and favorable structures. In vitro expression of the vaccine constructs was validated by western blotting. Birds immunized with attenuated SG harboring pJHL270 (JOL3025) or pJHL305 (JOL3027) containing HA-M2e showed significant increases in serum IgY and mucosal IgA antibodies, indicating strong humoral and mucosal immune responses, comparable with inactivated commercial vaccine. Post-immunization, we found a substantial rise in the hemagglutination inhibition titer, suggesting effective prevention of viral attachment and robust cell-mediated immunity, with a 1.96-fold and 2.80-fold increase in CD4+ and CD8+ T cells, respectively, for JOL3025 and a 1.75-fold and 2.49-fold increase for JOL3027. Furthermore, MHC class I and II expression increased 1.35-fold and 1.63-fold, for JOL3025, and 1.61-fold and 1.68-fold, respectively, for JOL3027. The IL-4 and IFN-γ levels were elevated, indicating a balanced Th-1 and Th-2 response. Post-challenge, birds immunized with vaccine candidates or the commercial vaccine exhibited minimal to no clinical signs, reduced lesions, lower lung viral titers, and negligible impacts on egg production compared to controls. In conclusion, both plasmids successfully delivered HA1-M2e immunogens through the engineered SG strains, eliciting strong humoral, mucosal, and cell-mediated immune responses and co-stimulating MHC class I and II antigen presentation pathways to provide effective protection against H9N2 AIV with minimal adverse effects.

A Purely Biomanufactured System for Delivering Nanoparticles and STING Agonists.

Nanovaccines have significantly contributed in the prevention and treatment of diseases. However, most of these technologies rely on chemical or hybrid semibiological synthesis methods, which limit the manufacturing performance advantages and improved inoculation outcomes compared with traditional vaccines. Herein, a universal and purely biological nanovaccine system is reported. This system integrates three modules: (1) self-assembling nanoparticles, (2) self-catalyzed synthesis of small-molecule stimulator of interferon gene (STING) agonists, and (3) delivery vectors that target the cytosolic surveillance system. Various nanoparticles are efficiently self-assembled using this system. After confirming the excellent immunostimulatory and lymph node targeting of this system, its broad-spectrum antiviral efficacy is further demonstrated. By leveraging the comprehensive biosynthetic capabilities of bacterial cells, this system can efficiently combine various adjuvant-active modular components and antigenic cargo, thereby providing a highly diversified and potent vaccine platform.

Focused Ultrasounds as an Adeno-Associated Virus Gene Therapy-Empowering Tool in Juvenile Mice via Intracerebroventricular Administration.

Systemic delivery of adeno-associated virus (AAV) vectors targeting the central nervous system has the potential to solve many neurodevelopmental disorders, yet it is made difficult by the filtering effect of the blood-brain barrier and systemic complications. To overcome this limitation, we attempted to inject a Venus-expressing, oligodendrocyte-selective AAV9 viral vector in the ventricles together with lipid microbubbles and subjected them to focused ultrasound (FUS); the resulting mechanical stimulation on the brain ventricles is able to open small, temporary gaps from which vector particles can leak and spread. Our findings indicate that FUS can increase viral vector diffusion across both the anteroposterior and left-right axes without influencing cell tropism; significant effects were found with 60 and 90 s exposure time, but no effects were observed with longer intervals. Taken together, these results highlight a new strategy for the safe and effective delivery of viral vectors and offer new perspectives for the development and application of gene therapies for central nervous system diseases.

Polyfunctional T cells and unique cytokine clusters imprint the anti rAAV2/rAAV9 vector immune response.

Polyfunctional T cells programmed to perform activities such as degranulation of lytic enzymes and simultaneous production of multiple cytokines are associated with more effective control of viral infections. Immune responses to recombinant adeno-associated virus (rAAV) vector delivery systems can critically influence therapeutic efficacy and safety of gene therapy. However, knowledge of polyfunctional T cells in anti-AAV immune responses is scarce. To bridge this knowledge gap, we have investigated the polyfunctionality of primary human CD4 T cells from healthy donors after in-vitro exposure to rAAV2 or rAAV9 vectors. By performing proliferation assays of co-cultured T cells and rAAV pulsed monocyte-derived dendritic cells from healthy donors we demonstrate T cell reactivity of 43% and 50% to rAAV2 and rAAV9 vectors, respectively. We validated this frequency in a second screen using another set of healthy donors measuring CD25 and CD71 T cell activation. Single T cell secretome analysis of reactive donors uncovered a Th1 pro-inflammatory, cytolytic and chemoattractive cytokine release profile after stimulation with rAAV2 or rAAV9 vectors. 12.4% and 9.6% of the stimulated T cells displayed a polyfunctional cytokine response, respectively, including elevated polyfunctional inflammatory indices. These responses were characterized by cytokine clusters such as Granzyme B, MIP1-α and TNF-α released in combination by single T cells. Overall, our results provide insights into adaptive immunity with rAAV vector serotypes which will be important in advancing gene therapy safety, vector selection, immunogenicity assessment and better patient selection for AAV gene therapy.

Noninvasive Imaging of Transgene Expression in Neurons Using Chemical Exchange Saturation Transfer MRI.

Advances in gene therapy, especially for brain diseases, have created new imaging demands for noninvasive monitoring of gene expression. While reporter gene imaging using co-expression of fluorescent protein-encoding gene has been widely developed, these conventional methods face significant limitations in longitudinal invivo applications. Magnetic resonance imaging (MRI), specifically chemical exchange saturation transfer (CEST) MRI, provides a robust noninvasive alternative that offers unlimited depth penetration, reliable spatial resolution, and specificity toward particular molecules. In this study, we explore the potential of CEST-MRI for monitoring gene expression in neurons. We designed a CEST polypeptide reporter expressing 150 arginine residues and evaluated its expression in the living brain after viral vector delivery. A longitudinal study performed at one and 2 months postinjection showed that specific CEST signal was observable. In particular, the CEST contrast exhibited distinct peaks at 0.75 and 1.75 ppm, consistent with the expected hydroxyl and guanidyl protons resonance frequencies. Histological study confirmed the specific neuronal expression of the transgene evidenced by the fluorescence signal from the td-Tomato fluorophore fused to the polypeptide. The ability to image noninvasively a neuron-specific CEST-MRI reporter gene could offer valuable insights for further developments of gene therapy for neurological disorders.

Comparison of Cell-Penetrating and Fusogenic TAT-HA2 Peptide Performance in Peptideplex, Multicomponent, and Conjugate siRNA Delivery Systems.

In this study, the performance of the cell-penetrating and fusogenic peptide, TAT-HA2, which consists of a cell-permeable HIV trans-activator of transcription (TAT) protein transduction domain and a pH-responsive influenza A virus hemagglutinin protein (HA2) domain, was comparatively evaluated for the first time in peptideplex, multicomponent, and conjugate siRNA delivery systems. TAT-HA2 in all three systems protected siRNA from degradation, except in the conjugate system with a low Peptide/siRNA ratio. The synergistic effect of different peptide domains enhanced the transfection efficiency of multicomponent and conjugate systems compared to that of peptideplexes, which was attributed to the surface configuration of TAT-HA2 peptides depending on the nature of attachment. Particularly, the multicomponent system showed better cellular uptake and endosomal escape than the peptideplexes, resulting in enhanced siRNA delivery in the cytoplasm. In addition, the presence of cleavable disulfide bonds in multicomponent and conjugate systems promoted the effective siRNA delivery in the cytoplasm, resulting in improved gene silencing activity. The multicomponent system reduced the level of luciferase expression in SKOV3 cells to 45% (±4). In contrast, the conjugate system and the commercially available siRNA transfection agent, Lipofectamine RNAiMax, caused luciferase suppression down to 55% (±2) at a siRNA dose of 100 nM. For the same dose, the peptideplex system could only reduce the luciferase expression to 65% (±5). None of the developed systems showed significant toxicity at any dose. Overall, the TAT-HA2 peptide is promising as a siRNA delivery vector; however, its performance depends on the nature of attachment and, as a result, its surface configuration on the developed delivery system.

Is ASCT2 a Suitable Vector for the Selective Delivery of Anticancer Drugs? Modification of Glutamine at Either the Carboxylate or the Side Chain Hinders Binding and Transport.

The Alanine, Serine, and Cysteine Transporter 2 (ASCT2) transports glutamine into cells and is upregulated in many cancers. Attachment to glutamine to enable ASCT2 to transport anticancer agents into cells has been proposed, but the impact of such modifications is a critical determinant of the potential of this strategy. Transport via ASCT2 of two glutamine analogues modified in ways that reflect possible mechanisms for attaching anticancer agents was studied. The aim was to determine if the modification of glutamine interferes with its transport via ASCT2 and thereby establish whether the conjugation of drugs to glutamine can facilitate the accumulation of anticancer drugs in cancer cells. L-theanine and a glutamine derivative modified at the carboxylate (7) were applied to Xenopus laevis oocytes expressing ASCT2. Two-electrode voltage clamp electrophysiology was used to measure substrate-elicited currents over a range of membrane potentials. Compound 7 was identified as neither a substrate nor an inhibitor while L-theanine was identified as an inhibitor of ASCT2. Thus, modification of glutamine in these ways prevents it from acting as a substrate and suggests that ASCT2 may not be a suitable target for delivery of anticancer drugs attached via either the carboxylate or side chain positions.

Spatially Resolved in vivo CRISPR Screen Sequencing via Perturb-DBiT.

Perturb-seq enabled the profiling of transcriptional effects of genetic perturbations in single cells but lacks the ability to examine the impact on tissue environments. We present Perturb-DBiT for simultaneous co- sequencing of spatial transcriptome and guide RNAs (gRNAs) on the same tissue section for in vivo CRISPR screen with genome-scale gRNA libraries, offering a comprehensive understanding of how genetic modifications affect cellular behavior and tissue architecture. This platform supports a variety of delivery vectors, gRNA library sizes, and tissue preparations, along with two distinct gRNA capture methods, making it adaptable to a wide range of experimental setups. In applying Perturb-DBiT, we conducted un-biased knockouts of tens of genes or at genome-wide scale across three cancer models. We mapped all gRNAs in individual colonies and corresponding transcriptomes in a human cancer metastatic colonization model, revealing clonal dynamics and cooperation. We also examined the effect of genetic perturbation on the tumor immune microenvironment in an immune-competent syngeneic model, uncovering differential and synergistic perturbations in promoting immune infiltration or suppression in tumors. Perturb-DBiT allows for simultaneously evaluating the impact of each knockout on tumor initiation, development, metastasis, histopathology, and immune landscape. Ultimately, it not only broadens the scope of genetic inquiry, but also lays the groundwork for developing targeted therapeutic strategies.

The potential of long non-coding RNAs for motor function recovery after spinal cord injury in rodents: A systematic review and meta-analysis

ObjectiveLong non-coding RNAs (LncRNAs) have garnered significant attention in preclinical studies for their potential in treating spinal cord injury (SCI). This meta-analysis aimed to assess the overall efficacy of lncRNA treatments in improving motor function in rodent models of SCI. MethodsThe Embase, PubMed, Web of Science, and Scopus databases were searched. Meta-analysis was performed using STATA 14.0. The standardized mean difference (SMD) was employed to combine various motor function scores. ResultsA total of 33 studies were included in this review. Key findings indicated that lncRNA treatments could markedly enhance locomotor function in rodents with SCI compared to control groups (SMD = 4.20, 95% CI: 3.35 to 5.05, I2 = 80.0%, P < 0.0001). Furthermore, in male rats with contusion/compression injuries, targeting specific cytosol-enriched lncRNAs to downregulate their expression may significantly improve motor function recovery. Specifically, intrathecal injection of non-viral vectors for lncRNA delivery proved to be the most effective method in this study. ConclusionsLncRNA treatments have demonstrated the potential to improve motor function in rodent models with SCI. However, the therapeutic efficacy may be overestimated. Future research should rigorously assess the clinical translational efficacy and safety of lncRNA treatments.

Heterologous protein exposure and secretion optimization in Mycoplasma pneumoniae

The non-pathogenic Mycoplasma pneumoniae engineered chassis (Mycochassis) has demonstrated the ability to express therapeutic molecules in vitro and to be effective for treatment of lung infectious diseases in in vivo mouse models. However, the expression of heterologous molecules, whether secreted or exposed on the bacterial membrane has not been optimized to ensure sufficient secretion and/or exposure levels to exert a maximum in vivo biological effect. Here, we have improved the currently used secretion signal from MPN142 protein. We found that mutations at P1’ position of the signal peptide cleavage site do not abrogate secretion but affect it. Increasing hydrophobicity and mutations at the C-terminal of the signal peptide increases secretion. We tested different lipoprotein signal peptides as possible N-terminal protein anchoring motifs on the Mpn cell surface. Unexpectedly we found that these peptides exhibit variable retention and secretion rates of the protein, with some sequences behaving as full secretion motifs. This raises the question of the biological role of the lipobox motif traditionally thought to anchor membrane proteins without a helical transmembrane domain. These results altogether represent a step forward in chassis optimization, offering different sequences for secretion or membrane retention, which could be used to improve Mycochassis as a delivery vector, and broadening its therapeutic possibilities.

Fine-Tuned "Click" Functionalization of PAMAM Dendrimers with a Linear Fluorinated Guanidino Linker: Synthesis, Characterization, and Applications.

This study presents the synthesis, characterization, and application of multifunctional PAMAM G2 and G4 dendrimers decorated with a linear fluorinated guanidino linker designed to improve gene delivery efficiency while minimizing cytotoxicity. For the first time, we were able to fine-tune the degree of grafting (DG) during the functionalization process through efficient "click" Michael addition, achieving the synthesis of a collection of six PAMAM conjugates that showed a significant enhancement in transfection efficiency (TE), surpassing the performance of traditional nonviral vectors. The incorporation of fluorinated moieties not only facilitated better deoxyribonucleic acid (DNA) condensation and TE but also introduced potential applications in 19F magnetic resonance imaging thanks to the sharp and intense fluorine nuclear magnetic resonance signals and favorable relaxation parameters. The new dendrimer conjugates demonstrated a promising balance between low cytotoxicity and high TE, with the low-generation PAMAM G2 with lower DG being the best-performing conjugate, making them strong candidates for further development in gene therapy. These findings highlight the potential of these multifunctional PAMAM dendrimers as efficient, nontoxic, and trackable gene delivery vectors.

Brief Comparison of the Efficacy of Cationic and Anionic Liposomes as Nonviral Delivery Systems.

In recent decades, the development and application of nonviral vectors, such as liposomes and lipidic nanoparticles, for gene therapy and drug delivery have seen substantial progress. The interest in the physicochemical properties and structures of the complexes liposome/DNA and liposome/RNA is due to their potential to substitute viruses as carriers of drugs or genetic material into cells with minimal cytotoxicity, which could lead to their use in gene therapy. Initially, cationic liposomes were utilized as nonviral DNA delivery vectors; subsequently, different molecules, such as polymers, were incorporated to enhance transfection efficiency. Additionally, liposome/protein complexes have been developed as nonviral vectors for the treatment of diseases. The most relevant internalization pathways of these vectors and the few transfection results obtained using targeted and nontargeted liposomes are discussed below. The high cytotoxicity of cationic liposomes represents a significant challenge for the development of gene therapy and drug delivery. Anionic liposomes offer a promising alternative to address the limitations of conventional cationic liposomes, including immune response, short circulation time, and low toxicity. This review will discuss the advantages of cationic liposomes and the novel anionic liposome-based systems that have emerged as a result. The advent of novel designs and manufacturing techniques has facilitated the development of innovative systems, designated as lipid nanoparticles (LNPs), which serve as highly efficacious regulators of the immune system.

Engineering an Ultrasound-Responsive Glycopolymersome for Hepatocyte-Specific Gene Delivery.

The ability to design liver-targeted gene delivery vectors is plagued with difficulties ranging from carrier-mediated cellular toxicity to challenges in encapsulating sensitive nucleic acids. Herein, we present an ultrasound-responsive glycopolymersome strategy for in situ loading of nucleic acids and achieving hepatocyte-specific gene delivery. This glycopolymersome is self-assembled from a block copolymer, N-acetylgalactosamine-grafted poly(glutamic acid)-block-poly(ε-caprolactone) (PGAGalNAc-b-PCL). GalNAc is introduced to afford liver targeting through the selective binding to the asialoglycoprotein receptor overexpressed on hepatocytes. External ultrasound is utilized to assist in encapsulating nucleic acids within the hydrophilic lumen of glycopolymersomes by exploiting their ultrasound responsiveness nature. Biological studies confirmed the successful encapsulation of plasmid DNA (pDNA) and small interfering RNA (siRNA), rapid nuclear internalization, and efficient gene transfection. These findings collectively demonstrated that this ultrasound-responsive glycopolymersome could be exploited as a novel safe and efficient gene vector targeting hepatocytes.

Modified PEG-Lipids Enhance the Nasal Mucosal Immune Capacity of Lipid Nanoparticle mRNA Vaccines.

Omicron, the predominant variant of SARS-CoV-2, exhibits strong immune-evasive properties, leading to the reduced efficacy of existing vaccines. Consequently, the development of versatile vaccines is imperative. Intranasal mRNA vaccines offer convenient administration and have the potential to enhance mucosal immunity. However, delivering vaccines via the nasal mucosa requires overcoming complex physiological barriers. The aim of this study is to modify PEGylated lipids to enhance the mucosal immune efficacy of the vaccine. The PEGylated lipid component of lipid nanoparticle (LNP) delivery vectors was modified with chitosan or mannose to generate novel LNPs that enhance vaccine adhesion or targeting on mucosal surfaces. The impact of the mRNA encoding the receptor-binding domain of Omicron BA.4/BA.5 on the immune response was examined. Compared to the unmodified LNP group, the IgG and IgA titers in the chitosan or mannose-modified LNP groups showed an increasing trend. The chitosan-modified group showed better effects. Notably, the PEGylated lipid with 1.5 mol% of chitosan modification produced high levels of IgG1 and IgG2a antibodies, promoting Th1/Th2 responses while also generating high levels of IgA, which can induce stronger cellular immunity, humoral immunity, and mucosal immunity. The 1.5 mol% of chitosan-modified LNPs (mRNA-LNP-1.5CS) can serve as a safe and effective carrier for intranasal mRNA vaccines, offering a promising strategy for combating the Omicron variant.

Rational Design of Enhanced Nme2Cas9 and Nme2SmuCas9 Nucleases and Base Editors.

CRISPR-Cas genome editing tools enable precise, RNA-guided modification of genomes within living cells. The most clinically advanced genome editors are Cas9 nucleases, but many nuclease technologies provide only limited control over genome editing outcomes. Adenine base editors (ABEs) and cytosine base editors (CBEs) enable precise and efficient nucleotide conversions of A:T-to-G:C and C:G-to-T:A base pairs, respectively. Therapeutic use of base editors (BEs) provides an avenue to correct approximately 30% of human pathogenic variants. Nonetheless, factors such as protospacer adjacent motif (PAM) availability, accuracy, product purity, and delivery limit the full therapeutic potential of BEs. We previously developed Nme2Cas9 and its BE derivatives, including ABEs compatible with single adeno-associated virus (AAV) vector delivery, in part to enable editing near N4CC PAMs. Further engineering yielded domain-inlaid BEs with enhanced activity, as well as Nme2Cas9/SmuCas9 chimeras that target single-cytidine (N4C) PAMs. Here we further enhance Nme2Cas9 and Nme2SmuCas9 editing effectors for improved efficiency and vector compatibility through site-directed mutagenesis and deaminase linker optimization. Finally, we define the editing and specificity profiles of the resulting variants by using paired guide-target libraries.