Supramolecular assembly of polycation/mRNA nanoparticles and in vivo monocyte programming.

Size-dependent phagocytosis is a well-characterized phenomenon in monocytes and macrophages. However, this size effect for preferential gene delivery to these important cell targets has not been fully exploited because commonly adopted stabilization methods for electrostatically complexed nucleic acid nanoparticles, such as PEGylation and charge repulsion, typically arrest the vehicle size below 200 nm. Here, we bridge the technical gap in scalable synthesis of larger submicron gene delivery vehicles by electrostatic self-assembly of charged nanoparticles, facilitated by a polymer structurally designed to modulate internanoparticle Coulombic and van der Waals forces. Specifically, our strategy permits controlled assembly of small poly(ß-amino ester)/messenger ribonucleic acid (mRNA) nanoparticles into particles with a size that is kinetically tunable between 200 and 1,000 nm with high colloidal stability in physiological media. We found that assembled particles with an average size of 400 nm safely and most efficiently transfect monocytes following intravenous administration and mediate their differentiation into macrophages in the periphery. When a CpG adjuvant is co-loaded into the particles with an antigen mRNA, the monocytes differentiate into inflammatory dendritic cells and prime adaptive anticancer immunity in the tumor-draining lymph node. This platform technology offers a unique ligand-independent, particle-size-mediated strategy for preferential mRNA delivery and enables therapeutic paradigms via monocyte programming.

Systematic multi-trait AAV capsid engineering for efficient gene delivery.

Broadening gene therapy applications requires manufacturable vectors that efficiently transduce target cells in humans and preclinical models. Conventional selections of adeno-associated virus (AAV) capsid libraries are inefficient at searching the vast sequence space for the small fraction of vectors possessing multiple traits essential for clinical translation. Here, we present Fit4Function, a generalizable machine learning (ML) approach for systematically engineering multi-trait AAV capsids. By leveraging a capsid library that uniformly samples the manufacturable sequence space, reproducible screening data are generated to train accurate sequence-to-function models. Combining six models, we designed a multi-trait (liver-targeted, manufacturable) capsid library and validated 88% of library variants on all six predetermined criteria. Furthermore, the models, trained only on mouse in vivo and human in vitro Fit4Function data, accurately predicted AAV capsid variant biodistribution in macaque. Top candidates exhibited production yields comparable to AAV9, efficient murine liver transduction, up to 1000-fold greater human hepatocyte transduction, and increased enrichment relative to AAV9 in a screen for liver transduction in macaques. The Fit4Function strategy ultimately makes it possible to predict cross-species traits of peptide-modified AAV capsids and is a critical step toward assembling an ML atlas that predicts AAV capsid performance across dozens of traits.

Biodegradable silica nanoparticles for efficient linear DNA gene delivery.

Targeting, safety, scalability, and storage stability of vectors are still challenges in the field of nucleic acid delivery for gene therapy. Silica-based nanoparticles have been widely studied as gene carriers, exhibiting key features such as biocompatibility, simplistic synthesis, and enabling easy surface modifications for targeting. However, the ability of the formulation to incorporate DNA is limited, which restricts the number of DNA molecules that can be incorporated into the particle, thereby reducing gene expression. Here we use polymerase chain reaction (PCR)-generated linear DNA molecules to augment the coding sequences of gene-carrying nanoparticles, thereby maximizing nucleic acid loading and minimizing the size of these nanocarriers. This approach results in a remarkable 16-fold increase in protein expression six days post-transfection in cells transfected with particles carrying the linear DNA compared with particles bearing circular plasmid DNA. The study also showed that the use of linear DNA entrapped in DNA@SiO2 resulted in a much more efficient level of gene expression compared to standard transfection reagents. The system developed in this study features simplicity, scalability, and increased transfection efficiency and gene expression over existing approaches, enabled by improved embedment capabilities for linear DNA, compared to conventional methods such as lipids or polymers, which generally show greater transfection efficiency with plasmid DNA. Therefore, this novel methodology can find applications not only in gene therapy but also in research settings for high-throughput gene expression screenings.

Acoustically targeted measurement of transgene expression in the brain.

Gene expression is a critical component of brain physiology, but monitoring this expression in the living brain represents a major challenge. Here, we introduce a new paradigm called recovery of markers through insonation (REMIS) for noninvasive measurement of gene expression in the brain with cell type, spatial, and temporal specificity. Our approach relies on engineered protein markers that are produced in neurons but exit into the brain's interstitium. When ultrasound is applied to targeted brain regions, it opens the blood-brain barrier and releases these markers into the bloodstream. Once in blood, the markers can be readily detected using biochemical techniques. REMIS can noninvasively confirm gene delivery and measure endogenous signaling in specific brain sites through a simple insonation and a subsequent blood test. REMIS is reliable and demonstrated consistent improvement in recovery of markers from the brain into the blood. Overall, this work establishes a noninvasive, spatially specific method of monitoring gene delivery and endogenous signaling in the brain.

Stable transduction of the neonatal mouse liver using a hybrid rAAV/sleeping beauty transposon gene delivery system.

BACKGROUND: Conventional adeno-associated viral (AAV) vectors, while highly effective in quiescent cells such as hepatocytes in the adult liver, confer less durable transgene expression in proliferating cells owing to episome loss. Sustained therapeutic success is therefore less likely in liver disorders requiring early intervention. We have previously developed a hybrid, dual virion approach, recombinant AAV (rAAV)/piggyBac transposon system capable of achieving stable gene transfer in proliferating hepatocytes at levels many fold above conventional AAV vectors. An alternative transposon system, Sleeping Beauty, has been widely used for ex vivo gene delivery; however liver-targeted delivery using a hybrid rAAV/Sleeping Beauty approach remains relatively unexplored. METHODS: We investigated the capacity of a Sleeping Beauty (SB)-based dual rAAV virion approach to achieve stable and efficient gene transfer to the newborn murine liver using transposable therapeutic cassettes encoding coagulation factor IX or ornithine transcarbamylase (OTC). RESULTS: At equivalent doses, rAAV/SB100X transduced hepatocytes with high efficiency, achieving stable expression into adulthood. Compared with conventional AAV, the proportion of hepatocytes transduced, and factor IX and OTC activity levels, were both markedly increased. The proportion of hepatocytes stably transduced increased 4- to 8-fold from <5%, and activity levels increased correspondingly, with markedly increased survival and stable urinary orotate levels in the OTC-deficient Spfash mouse following elimination of residual endogenous murine OTC. CONCLUSIONS: The present study demonstrates the first in vivo utility of a hybrid rAAV/SB100X transposon system to achieve stable long-term therapeutic gene expression following delivery to the highly proliferative newborn mouse liver. These results have relevance to the treatment of genetic metabolic liver diseases with neonatal onset.

Analyses of Neurite and Spine Formation by In Utero Electroporation in Mice.

Dendrite morphology and dendritic spines are key features of the neuronal networks in the brain. Abnormalities in these features have been observed in patients with psychiatric disorders and mouse models of these diseases. In utero electroporation is an easy and efficient gene transfer system for developing mouse embryos in the uterus. By combining with the Cre-loxP system, the morphology of individual neurons can be clearly and sparsely visualized. Here, we describe how this labeling system can be applied to visualize and evaluate the dendrites and dendritic spines of cortical neurons.

PAX4 gene delivery improves ß-cell function in human islets of Type II diabetes.

Aim: Type II diabetes (T2D) stems from insulin resistance, with ß-cell dysfunction as a hallmark in its progression. Studies reveal that ß cells undergo apoptosis or dedifferentiation during T2D development. The transcription factor PAX4 is vital for ß differentiation and survival, thus may be a potential enhancer of ß-cell function in T2D islets. Materials & methods: Human PAX4 cDNA was delivered into T2D human islets with an adenoviral vector, and its effects on ß cells were examined. Results: PAX4 gene delivery significantly improved ß-cell survival, and increased ß-cell composition in the T2D human islets. Basal insulin and glucose-stimulated insulin secretion in PAX4-expressing islets were substantially higher than untreated or control-treated T2D human islets. Conclusion: Introduced PAX4 expression in T2D human islets improves ß-cell function, thus could provide therapeutic benefits for T2D treatment.

Type II diabetes (T2D) results from insulin resistance, with ß-cell dysfunction playing a pivotal role in its progression. Deficits in ß-cell mass and function have been attributed primarily to ß-cell death through apoptosis; however, recent studies suggest ß-cell failure can also arise from ß-cell dedifferentiation ­ that is, ß cells undergo a loss of mature identity, adopting either progenitor-like or glucagon-producing α cell states during T2D development. Therefore, a strategy preventing ß-cell dedifferentiation while promoting its survival is beneficial for T2D treatment. In this study, we explored whether PAX4, a critical transcription factor for ß differentiation and survival, could alleviate ß-cell dysfunction in human islets derived from T2D patients. To accomplish that, human PAX4 cDNA was delivered into human islets isolated from T2D donors by an adenoviral vector-based vector, Ad5.Pax4 and its effects on ß-cell function were evaluated. The results showed PAX4 expression significantly improved ß-cell survival and increased ß-cell composition in the T2D islets. Notably, PAX4-treated T2D islets exhibited significantly higher basal insulin secretion and glucose-stimulated insulin secretion than control-treated islets. The data demonstrate that PAX4 gene delivery into T2D human islets enhances ß-cell mass and function, and thus may offer therapeutic benefits in the treatment of T2D.

Tea polyphenol nanoparticles enable targeted siRNA delivery and multi-bioactive therapy for abdominal aortic aneurysms.

Abdominal aortic aneurysm (AAA) is a life-threatening vascular disease, while there is a lack of pharmaceutical interventions to halt AAA progression presently. To address the multifaceted pathology of AAA, this work develops a novel multifunctional gene delivery system to simultaneously deliver two siRNAs targeting MMP-2 and MMP-9. The system (TPNs-siRNA), formed through the oxidative polymerization and self-assembly of epigallocatechin gallate (EGCG), efficiently encapsulates siRNAs during self-assembly. TPNs-siRNA safeguards siRNAs from biological degradation, facilitates intracellular siRNA transfection, promotes lysosomal escape, and releases siRNAs to silence MMP-2 and MMP-9. Additionally, TPNs, serving as a multi-bioactive material, mitigates oxidative stress and inflammation, fosters M1-to-M2 repolarization of macrophages, and inhibits cell calcification and apoptosis. In experiments with AAA mice, TPNs-siRNA accumulated and persisted in aneurysmal tissue after intravenous delivery, demonstrating that TPNs-siRNA can be significantly distributed in macrophages and VSMCs relevant to AAA pathogenesis. Leveraging the carrier's intrinsic multi-bioactive properties, the targeted siRNA delivery by TPNs exhibits a synergistic effect for enhanced AAA therapy. Furthermore, TPNs-siRNA is gradually metabolized and excreted from the body, resulting in excellent biocompatibility. Consequently, TPNs emerges as a promising multi-bioactive nanotherapy and a targeted delivery nanocarrier for effective AAA therapy.

Nucleic acid-based nanotherapeutics for treating sepsis and associated organ injuries.

In recent years, gene therapy has been made possible with the success of nucleic acid drugs against sepsis and its related organ dysfunction. Therapeutics based on nucleic acids such as small interfering RNAs (siRNAs), microRNAs (miRNAs), messenger RNAs (mRNAs), and plasmid DNAs (pDNAs) guarantee to treat previously undruggable diseases. The advantage of nucleic acid-based therapy against sepsis lies in the development of nanocarriers, achieving targeted and controlled gene delivery for improved efficacy with minimal adverse effects. Entrapment into nanocarriers also ameliorates the poor cellular uptake of naked nucleic acids. In this study, we discuss the current state of the art in nanoparticles for nucleic acid delivery to treat hyperinflammation and apoptosis associated with sepsis. The optimized design of the nanoparticles through physicochemical property modification and ligand conjugation can target specific organs-such as lung, heart, kidney, and liver-to mitigate multiple sepsis-associated organ injuries. This review highlights the nanomaterials designed for fabricating the anti-sepsis nanosystems, their physicochemical characterization, the mechanisms of nucleic acid-based therapy in working against sepsis, and the potential for promoting the therapeutic efficiency of the nucleic acids. The current investigations associated with nanoparticulate nucleic acid application in sepsis management are summarized in this paper. Noteworthily, the potential application of nanotherapeutic nucleic acids allows for a novel strategy to treat sepsis. Further clinical studies are required to confirm the findings in cell- and animal-based experiments. The capability of large-scale production and reproducibility of nanoparticle products are also critical for commercialization. It is expected that numerous anti-sepsis possibilities will be investigated for nucleic acid-based nanotherapeutics in the future.

Adeno-Associated Virus Engineering and Load Strategy for Tropism Modification, Immune Evasion and Enhanced Transgene Expression.

Gene therapy aims to add, replace or turn off genes to help treat disease. To date, the US Food and Drug Administration (FDA) has approved 14 gene therapy products. With the increasing interest in gene therapy, feasible gene delivery vectors are necessary for inserting new genes into cells. There are different kinds of gene delivery vectors including viral vectors like lentivirus, adenovirus, retrovirus, adeno-associated virus et al, and non-viral vectors like naked DNA, lipid vectors, polymer nanoparticles, exosomes et al, with viruses being the most commonly used. Among them, the most concerned vector is adeno-associated virus (AAV) because of its safety, natural ability to efficiently deliver gene into cells and sustained transgene expression in multiple tissues. In addition, the AAV genome can be engineered to generate recombinant AAV (rAAV) containing transgene sequences of interest and has been proven to be a safe gene vector. Recently, rAAV vectors have been approved for the treatment of various rare diseases. Despite these approvals, some major limitations of rAAV remain, namely nonspecific tissue targeting and host immune response. Additional problems include neutralizing antibodies that block transgene delivery, a finite transgene packaging capacity, high viral titer used for per dose and high cost. To deal with these challenges, several techniques have been developed. Based on differences in engineering methods, this review proposes three strategies: gene engineering-based capsid modification (capsid modification), capsid surface tethering through chemical conjugation (surface tethering), and other formulations loaded with AAV (virus load). In addition, the major advantages and limitations encountered in rAAV engineering strategies are summarized.

Recent advances in gene delivery nanoplatforms based on spherical nucleic acids.

Gene therapy is a therapeutic option for mitigating diseases that do not respond well to pharmacological therapy. This type of therapy allows for correcting altered and defective genes by transferring nucleic acids to target cells. Notably, achieving a desirable outcome is possible by successfully delivering genetic materials into the cell. In-vivo gene transfer strategies use two major classes of vectors, namely viral and nonviral. Both of these systems have distinct pros and cons, and the choice of a delivery system depends on therapeutic objectives and other considerations. Safe and efficient gene transfer is the main feature of any delivery system. Spherical nucleic acids (SNAs) are nanotechnology-based gene delivery systems (i.e., non-viral vectors). They are three-dimensional structures consisting of a hollow or solid spherical core nanoparticle that is functionalized with a dense and highly organized layer of oligonucleotides. The unique structural features of SNAs confer them a high potency in internalization into various types of tissue and cells, a high stability against nucleases, and efficay in penetrating through various biological barriers (such as the skin, blood-brain barrier, and blood-tumor barrier). SNAs also show negligible toxicity and trigger minimal immune response reactions. During the last two decades, all these favorable physicochemical and biological attributes have made them attractive vehicles for drug and nucleic acid delivery. This article discusses the unique structural properties, types of SNAs, and also optimization mechanisms of SNAs. We also focus on recent advances in the synthesis of gene delivery nanoplatforms based on the SNAs.

New Perspectives and Prospects of microRNA Delivery in Diabetic Wound Healing.

The remarkable potential of microRNAs (miRNAs) as a class of biotherapeutic agents in the treatment of diverse pathological conditions has garnered significant interest in recent years. To heal both acute and chronic wounds, miRNAs work by post-transcriptionally controlling various proteins and the pathways that are linked to them. Diabetes mellitus predisposes to several macro- and microvascular defects of end organs such as atherosclerosis, peripheral artery disease, retinopathy, nephropathy, neuropathy, and impaired wound healing. Here, miRNAs emerge as a beacon of hope, with the capacity to heal diabetic wounds by precisely modulating the expression of genes involved in the healing process. Despite the therapeutic promise, the journey to realizing the full potential of miRNAs is fraught with challenges. Their intrinsic instability and the inefficient delivery into target cells pose significant barriers to their clinical application. Consequently, a major focus of current research is the discovery of novel miRNAs and the development of innovative delivery systems that can effectively transport these nucleic acids into the cells where they are needed most. This review delves into the intricate roles that miRNAs play at various stages of diabetic wound healing, providing a comprehensive overview of the latest research findings. The review also addresses the obstacles and opportunities that come with translating miRNA-based strategies into clinical practice, offering a critical assessment of the field's advancements and the hurdles that remain to be overcome. SIGNIFICANCE STATEMENT: The potential of microRNA delivery using new biological or nonbiological carriers may create a revolutionary treatment method for chronic wounds of diabetes.

In utero adeno-associated virus (AAV)-mediated gene delivery targeting sensory and supporting cells in the embryonic mouse inner ear.

In vivo gene delivery to tissues using adeno-associated vector (AAVs) has revolutionized the field of gene therapy. Yet, while sensorineural hearing loss is one of the most common sensory disorders worldwide, gene therapy applied to the human inner ear is still in its infancy. Recent advances in the development recombinant AAVs have significantly improved their cell tropism and transduction efficiency across diverse inner ear cell types to a level that renders this tool valuable for conditionally manipulating gene expression in the context of developmental biology studies of the mouse inner ear. Here, we describe a protocol for in utero micro-injection of AAVs into the embryonic inner ear, using the AAV-PHP.eB and AAV-DJ serotypes that respectively target the sensory hair cells and the supporting cells of the auditory sensory epithelium. We also aimed to standardize procedures for imaging acquisition and image analysis to foster research reproducibility and allow accurate comparisons between studies. We find that AAV-PHP.eB and AAV-DJ provide efficient and reliable tools for conditional gene expression targeting cochlear sensory and supporting cells in the mouse inner ear, from late embryonic stages on.

A black phosphorus nanosheet-based RNA delivery system for prostate cancer therapy by increasing the expression level of tumor suppressor gene PTEN via CeRNA mechanism.

BACKGROUND: Prostate cancer (PCa) has a high incidence in men worldwide, and almost all PCa patients progress to the androgen-independent stage which lacks effective treatment measures. PTENP1, a long non-coding RNA, has been shown to suppress tumor growth through the rescuing of PTEN expression via a competitive endogenous RNA (ceRNA) mechanism. However, PTENP1 was limited to be applied in the treatment of PCa for the reason of rapid enzymatic degradation, poor intracellular uptake, and excessively long base sequence to be synthesized. Considering the unique advantages of artificial nanomaterials in drug loading and transport, black phosphorus (BP) nanosheet was employed as a gene-drug carrier in this study. RESULTS: The sequence of PTENP1 was adopted as a template which was randomly divided into four segments with a length of about 1000 nucleotide bases to synthesize four different RNA fragments as gene drugs, and loaded onto polyethyleneimine (PEI)-modified BP nanosheets to construct BP-PEI@RNA delivery platforms. The RNAs could be effectively delivered into PC3 cells by BP-PEI nanosheets and elevating PTEN expression by competitive binding microRNAs (miRNAs) which target PTEN mRNA, ultimately exerting anti-tumor effects. CONCLUSIONS: Therefore, this study demonstrated that BP-PEI@RNAs is a promising gene therapeutic platform for PCa treatment.

Exosome for mRNA delivery: strategies and therapeutic applications.

Messenger RNA (mRNA) has emerged as a promising therapeutic molecule with numerous clinical applications in treating central nervous system disorders, tumors, COVID-19, and other diseases. mRNA therapies must be encapsulated into safe, stable, and effective delivery vehicles to preserve the cargo from degradation and prevent immunogenicity. Exosomes have gained growing attention in mRNA delivery because of their good biocompatibility, low immunogenicity, small size, unique capacity to traverse physiological barriers, and cell-specific tropism. Moreover, these exosomes can be engineered to utilize the natural carriers to target specific cells or tissues. This targeted approach will enhance the efficacy and reduce the side effects of mRNAs. However, difficulties such as a lack of consistent and reliable methods for exosome purification and the efficient encapsulation of large mRNAs into exosomes must be addressed. This article outlines current breakthroughs in cell-derived vesicle-mediated mRNA delivery and its biomedical applications.

Viral and Non-Viral Systems to Deliver Gene Therapeutics to Clinical Targets.

Clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has revolutionized the field of gene therapy as it has enabled precise genome editing with unprecedented accuracy and efficiency, paving the way for clinical applications to treat otherwise incurable genetic disorders. Typically, precise genome editing requires the delivery of multiple components to the target cells that, depending on the editing platform used, may include messenger RNA (mRNA), protein complexes, and DNA fragments. For clinical purposes, these have to be efficiently delivered into transplantable cells, such as primary T lymphocytes or hematopoietic stem and progenitor cells that are typically sensitive to exogenous substances. This challenge has limited the broad applicability of precise gene therapy applications to those strategies for which efficient delivery methods are available. Electroporation-based methodologies have been generally applied for gene editing applications, but procedure-associated toxicity has represented a major burden. With the advent of novel and less disruptive methodologies to deliver genetic cargo to transplantable cells, it is now possible to safely and efficiently deliver multiple components for precise genome editing, thus expanding the applicability of these strategies. In this review, we describe the different delivery systems available for genome editing components, including viral and non-viral systems, highlighting their advantages, limitations, and recent clinical applications. Recent improvements to these delivery methods to achieve cell specificity represent a critical development that may enable in vivo targeting in the future and will certainly play a pivotal role in the gene therapy field.

Stereotaxic Surgery as a Method to Deliver Epigenetic Editing Constructs in Rodent Brain.

Modern neuroscience research is increasingly discovering that alterations in epigenetic states within key brain cells is correlated with brain diseases. These epigenetic alterations may include changes in histone post-translational modifications and/or DNA modifications, all of which affect transcription and other gene expression programs within the brain cells that comprise central brain regions. However, the exact causal contribution of these epigenome changes to brain disease cannot be elucidated in the absence of direct in vivo manipulations in the implicated brain areas. Combining the design and creation of epigenetic editing constructs, gene delivery strategies, and stereotaxic surgery enables neuroscience researchers to target and manipulate the epigenetic state of the brain cells of laboratory rodents in a locus-specific manner and test its causal contribution to disease-related pathology and behaviors. Here, we describe the surgical protocol utilized by our group and others, which is optimized for herpes simplex virus delivery into the mouse brain, although the protocol outlined herein could be applied for delivery of adeno-associated viruses, lentiviruses, or nonviral gene-delivery methods in both mice and rats. The method allows for the overexpression of engineered DNA-binding proteins for direct and targeted epigenome editing in rodent brain with excellent spatiotemporal control. Nearly any brain region of interest can be targeted in rodents at every stage of postnatal life. Owing to the versatility, reproducibility, and utility of this technique, it is an important method for any laboratory interested in studying the cellular, circuit, and behavioral consequences of manipulating the brain epigenome in laboratory rodents.

Stable germline transgenesis using the Minos Tc1/mariner element in the sea urchin Lytechinus pictus.

Stable transgenesis is a transformative tool in model organism biology. Although the sea urchin is one of the oldest animal models in cell and developmental biology, studies in this animal have largely relied on transient manipulation of wild animals, without a strategy for stable transgenesis. Here, we build on recent progress to develop a more genetically tractable sea urchin species, Lytechinus pictus, and establish a robust transgene integration method. Three commonly used transposons (Minos, Tol2 and piggyBac) were tested for non-autonomous transposition, using plasmids containing a polyubiquitin promoter upstream of a H2B-mCerulean nuclear marker. Minos was the only transposable element that resulted in significant expression beyond metamorphosis. F0 animals were raised to sexual maturity, and spawned to determine germline integration and transgene inheritance frequency, and to characterize expression patterns of the transgene in F1 progeny. The results demonstrate transgene transmission through the germline, the first example of a germline transgenic sea urchin and, indeed, of any echinoderm. This milestone paves the way for the generation of diverse transgenic resources that will dramatically enhance the utility, reproducibility and efficiency of sea urchin research.