A review of the tortuous path of nonviral gene delivery and recent progress.

Gene therapy encompasses the transfer of exogenous genetic materials into the patient's target cells to treat or prevent diseases. Nevertheless, the transfer of genetic material into desired cells is challenging and often requires specialized tools or delivery systems. For the past 40 years, scientists are mainly pursuing various viruses as gene delivery vectors, and the overall progress has been slow and far from the expectation. As an alternative, nonviral vectors have gained substantial attention due to their several advantages, including superior safety profile, enhanced payload capacity, and stealth abilities. Since nonviral vectors encounter multiple extra- and intra-cellular barriers limiting the transfer of genetic payload into the target cell nucleus, we have discussed these barriers in detail for this review. A direct approach, utilizing physical methods like electroporation, sonoporation, gene gun, eliminate the requirement for a specific carrier for gene delivery. In contrast, chemical methods of gene transfer exploit natural or synthetic compounds as carriers to increase cellular targeting and gene therapy effectiveness. We have also emphasized the recent advancements aimed at enhancing the current nonviral approaches. Therefore, in this review, we have focused on discussing the current evolving state of nonviral gene delivery systems and their future perspectives.

Non-Viral Vector-Mediated Gene Therapy for ALS: Challenges and Future Perspectives.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease, for which no effective treatment is yet available to either slow or terminate it. Recent advances in gene therapy renew hope for developing an effective approach to control this disease. Non-viral vectors, such as lipid- and polymer-based nanoparticles, cationic polymers, and exosomes, can effectively transfer genes into primary neurons. The resulting gene expression can be long-term, stable, and without immunological complications, which is essential for the effective management of neurological disorders. This Review will first describe the current research and clinical stage of novel therapies for ALS. It will then touch on the journey of non-viral vector use in ALS, subsequently highlighting the application of non-viral vector-mediated gene therapy. The bottlenecks in the translation of non-viral vectors for ALS treatment are also discussed, including the biological barriers of systemic administration and the issues of "when, where, and how much?" for effective gene delivery. The prospect of employing emerging techniques, such as CRISPR-Cas9 gene editing, stem cell methodology, and low-intensity focused ultrasound for fueling the transport of non-viral vectors to the central nervous system for personalized gene therapy, is briefly discussed in the context of ALS. Despite the challenging road that lies ahead, with the current expansion in interest and technological advancement in non-viral vector-delivered gene therapy for ALS, we hold hope that the field is headed toward a positive future.

siRNA Design and GalNAc-Empowered Hepatic Targeted Delivery.

Small interfering RNA (siRNA) is a clinically approved therapeutic modality, which has attracted widespread attention not only from basic research but also from pharmaceutical industry. As siRNA can theoretically modulate any disease-related gene's expression, plenty of siRNA therapeutic pipelines have been established by tens of biotechnology companies. The drug performance of siRNA heavily depends on the sequence, the chemical modification, and the delivery of siRNA. Here, we describe the rational design protocol of siRNA, and provide some modification patterns that can enhance siRNA's stability and reduce its off-target effect. Also, the delivery method based on N-acetylgalactosamine (GalNAc)-siRNA conjugate that is widely employed to develop therapeutic regimens for liver-related diseases is also recapitulated.

Incorporating Gangliosides into PEGylated Cationic Liposomes that Complexed DNA Attenuates Anti-PEG Antibody Production but Not Anti-DNA Antibody Production in Mice.

Gangliosides (glycosphingolipids) reduce antibody production by inhibiting B-cell receptor (BCR) signaling. We have shown that a copresentation of gangliosides and polyethylene glycol (PEG) on the same liposomes suppresses anti-PEG IgM production in mice. In addition, we recently observed that pDNA incorporated in PEGylated cationic liposomes (PCLs) induces anti-DNA IgM, which could be a hurdle to the development of efficient gene delivery systems. Therefore, the focus of this study was to determine if the copresentation of gangliosides and DNA on the same PCL would suppress antibody production against DNA. PCLs including DNA induced both anti-PEG IgM production and anti-DNA IgM production. The extent of anti-PEG and anti-DNA IgM production was likely dependent on the immunogenicity of the complexed DNA. Treatment of clodronate-containing liposomes, which causes a depletion of phagocytic cells, suppressed anti-PEG IgM production from PCLs that did not include DNA but failed to suppress anti-PEG IgM production from PCLs that complexed DNA (PCLD). Both anti-PEG IgM and anti-DNA IgM was induced in T-cell-deficient nude mice as well as in normal mice following treatment with PCLs and PCLD, respectively. These results indicate that phagocytic cells contribute to anti-PEG IgM production but not to anti-DNA IgM production, while T-cells do not contribute to any form of antibody production. The copresentation of gangliosides and DNA significantly reduced anti-PEG IgM production but unfortunately did not reduce anti-DNA IgM production. It appears that the immunosuppressive effect of gangliosides, presumably via the CD22 signaling pathway, is limited only to anti-PEG immunity.

Challenges in adeno-associated virus-based treatment of central nervous system diseases through systemic injection.

Adeno-associated virus (AAV) vector, an excellent gene therapy vector, has been widely used in the treatment of various central nervous system (CNS) diseases. Due to the presence of the blood-brain barrier (BBB), early attempts at AAV-based CNS diseases treatment were mainly performed through intracranial injections. Subsequently, systemic injections of AAV9, the first AAV that was shown to have BBB-crossing ability in newborn and adult mice, were assessed in clinical trials for multiple CNS diseases. However, the development of systemic AAV injections to treat CNS diseases is still associated with many challenges, such as the efficiency of AAV in crossing the BBB, the peripheral toxicity caused by the expression of AAV-delivered genes, and the immune barrier against AAV in the blood. In this review, we will introduce the biology of the AAV vector and the advantages of systemic AAV injections to treat CNS diseases. Most importantly, we will introduce the challenges associated with systemic injection of therapeutic AAV in treating CNS diseases and suggest feasible solutions.

Advantages and challenges in nanomedicines for chronic liver diseases: A hepatologist's perspectives.

Chronic liver diseases (CLD) are responsible for significant morbidity and mortality worldwide. CLD patients are at a high risk of developing progressive liver fibrosis, cirrhosis, hepatocellular carcinoma (HCC), and subsequent liver failure. To date, there is no specific and effective therapies exist for patients with various forms of CLD. The application of nanotechnology has emerged as a rapidly developing area of interest for the safe and target-specific delivery of poorly aqueous soluble hepatoprotective agents and nucleic acids (siRNA/miRNAs) in CLD. The nanoparticle combination improves bioavailability and plasma stability of drugs with poor aqueous solubility. However, the extent of successful functional delivery of nanoparticles into hepatocytes is often surprisingly low. High Kupffer cells interaction reduces the nanomedicine efficacy. During fibrosis, the extracellular matrix accumulation in the perisinusoidal space restricts nanoparticle delivery to hepatocytes. The availability and uptake of nanoparticles exposure to different cells in the liver microenvironment is as Kupffer cells > sinusoidal endothelial cells > HSCs > hepatocytes. The most widely used strategy to reduce nanoparticles and macrophages interaction is to coat the particle surface with polyethylene glycol. The cationic charged nanoparticles have increased hepatocyte delivery by increased cellular interaction by disrupting the endosomal system via their pH buffering capacity. The immune clearance and toxicity of nanoparticles are mainly unpredictable. Therefore, more elaborate knowledge on exact cellular uptake and intracellular accumulation, trafficking, and endosomal sorting of nanoparticle is the need of the hour to improve the rational carrier design.

Capillary electroporation affects the expression of miRNA-122-5p from bull sperm cells.

Sperm-mediated gene transfer (SMGT) has a potential application in the generation of transgenic animals. Capillary electroporation consists of the application of electrical pulses, resulting in an increased transfection rate. Little is known about the impacts of the transfection of exogenous DNA on sperm epigenetics. MicroRNAs are epigenetic factors that are related to sperm motility. MiRNA-122-5p regulates genes that influence motility, and consequently, the fertilizing potential of sperm. Therefore, we aimed at identifying whether epigenetic factors such as microRNAs could be altered after DNA transfection, using the capillary electroporation technique. In this study, bull sperm was electroporated using voltages of 600 V, 1500 V, and 0 V (control group), with or without exogenous DNA. Parameters of sperm quality were analyzed using CASA and flow cytometry, and expression of the miRNA-122-5p was analyzed using RT-qPCR. It was observed that electroporation increased the internalization of exogenous DNA (P < 0.05), but did not impair the mitochondrial activity (P > 0.05). It reduced sperm motility (P < 0.05). The expression of miRNA-122-5p was upregulated in sperm electroporated at 1500 V, and the presence of exogenous DNA did not affect its expression. Thus, we can conclude that electroporation influences the expression of miRNA-122-5p from bull sperm cells.

Ultrasound-assisted magnetic nanoparticle-based gene delivery.

Targeted gene delivery is important in biomedical research and applications. In this paper, we synergistically combine non-viral chemical materials, magnetic nanoparticles (MNPs), and a physical technique, low-intensity pulsed ultrasound (LIPUS), to achieve efficient and targeted gene delivery. The MNPs are iron oxide super-paramagnetic nanoparticles, coated with polyethyleneimine (PEI), which makes a high positive surface charge and is favorable for the binding of genetic materials. Due to the paramagnetic properties of the MNPs, the application of an external magnetic field increases transfection efficiency while LIPUS stimulation enhances cell viability and permeability. We found that stimulation at the intensity of 30 mW/cm2 for 10 minutes yields optimal results with a minimal adverse effect on the cells. By combining the effect of the external magnetic field and LIPUS, the genetic material (GFP or Cherry Red plasmid) can enter the cells. The flow cytometry results showed that by using just a magnetic field to direct the genetic material, the transfection efficiency on HEK 293 cells that were treated by our MNPs was 56.1%. Coupled with LIPUS stimulation, it increased to 61.5% or 19% higher than the positive control (Lipofectamine 2000). Besides, compared with the positive control, our method showed less toxicity. Cell viability after transfection was 63.61%, which is 19% higher than the standard transfection technique. In conclusion, we designed a new gene-delivery method that is affordable, targeted, shows low-toxicity, yet high transfection efficiency, compared to other conventional approaches.

Long-Term Follow-Up of the First in Human Intravascular Delivery of AAV for Gene Transfer: AAV2-hFIX16 for Severe Hemophilia B.

Adeno-associated virus (AAV) vectors are a leading platform for gene-based therapies for both monogenic and complex acquired disorders. The success of AAV gene transfer highlights the need to answer outstanding clinical questions of safety, durability, and the nature of the human immune response to AAV vectors. Here, we present longitudinal follow-up data of subjects who participated in the first trial of a systemically delivered AAV vector. Adult males (n = 7) with severe hemophilia B received an AAV2 vector at doses ranging from 8 × 1010 to 2 × 1012 vg/kg to target hepatocyte-specific expression of coagulation factor IX; a subset (n = 4) was followed for 12-15 years post-vector administration. No major safety concerns were observed. There was no evidence of sustained hepatic toxicity or development of hepatocellular carcinoma as assessed by liver transaminase values, serum α-fetoprotein, and liver ultrasound. Subjects demonstrated persistent, increased AAV neutralizing antibodies (NAbs) to the infused AAV serotype 2 (AAV2) as well as all other AAV serotypes tested (AAV5 and AAV8) for the duration of follow-up. These data represent the longest available longitudinal follow-up data of subjects who received intravascular AAV and support the preliminary safety of intravascular AAV administration at the doses tested in adults. Data demonstrate, for the first time, the persistence of high-titer, multi-serotype cross-reactive AAV NAbs for up to 15 years post- AAV vector administration. Our observations are broadly applicable to the development of AAV-mediated gene therapy.

Effect of Transgenesis on mRNA and miRNA Profiles in Cucumber Fruits Expressing Thaumatin II.

Transgenic plants are commonly used in breeding programs because of the various features that can be introduced. However, unintended effects caused by genetic transformation are still a topic of concern. This makes research on the nutritional safety of transgenic crop plants extremely interesting. Cucumber (Cucumis sativus L.) is a crop that is grown worldwide. The aim of this study was to identify and characterize differentially expressed genes and regulatory miRNAs in transgenic cucumber fruits that contain the thaumatin II gene, which encodes the sweet-tasting protein thaumatin II, by NGS sequencing. We compared the fruit transcriptomes and miRNomes of three transgenic cucumber lines with wild-type cucumber. In total, we found 47 differentially expressed genes between control and all three transgenic lines. We performed the bioinformatic functional analysis and gene ontology classification. We also identified 12 differentially regulated miRNAs, from which three can influence the two targets (assigned as DEGs) in one of the studied transgenic lines (line 224). We found that the transformation of cucumber with thaumatin II and expression of the transgene had minimal impact on gene expression and epigenetic regulation by miRNA, in the cucumber fruits.

Engineering NK Cells for CAR Therapy-Recent Advances in Gene Transfer Methodology.

The development of chimeric antigen receptor (CAR) T cell therapy has introduced a new and effective strategy to guide and promote the immune response against tumors in the clinic. More recently, in an attempt to enhance its utility, this method has been expanded to novel cell types. One of the more successful variants has proven to be the expression of CARs in Natural Killer (NK) cells (CAR-NK). Gene engineering NK cells to express an exogenous CAR receptor allows the innate anti-tumor ability of NK cells to be harnessed and directed against a target tumor antigen. In addition, the biology of NK cells allows the development of an allogeneic cell therapeutic product useable with most or all patient haplotypes. NK cells cause little or no graft versus host disease (GvHD) and are therefore suitable for development of an "off the shelf" therapeutic product. Initial trials have also shown that CAR-NK cells rarely cause cytokine release syndrome. However, despite their potential NK cells have proven to be difficult to engineer, with high sensitivity to apoptosis and low levels of gene expression. The creation of optimized methods to introduce genes into NK cells will promote the widespread application of CAR-NK in research laboratories and the clinics.

Molecular Therapies for Inherited Retinal Diseases-Current Standing, Opportunities and Challenges.

Inherited retinal diseases (IRDs) are both genetically and clinically highly heterogeneous and have long been considered incurable. Following the successful development of a gene augmentation therapy for biallelic RPE65-associated IRD, this view has changed. As a result, many different therapeutic approaches are currently being developed, in particular a large variety of molecular therapies. These are depending on the severity of the retinal degeneration, knowledge of the pathophysiological mechanism underlying each subtype of IRD, and the therapeutic target molecule. DNA therapies include approaches such as gene augmentation therapy, genome editing and optogenetics. For some genetic subtypes of IRD, RNA therapies and compound therapies have also shown considerable therapeutic potential. In this review, we summarize the current state-of-the-art of various therapeutic approaches, including the pros and cons of each strategy, and outline the future challenges that lie ahead in the combat against IRDs.

Delivery of CRISPR/Cas9 for therapeutic genome editing.

The clustered, regularly-interspaced, short palindromic repeat (CRISPR)-associated nuclease 9 (CRISPR/Cas9) is emerging as a promising genome-editing tool for treating diseases in a precise way, and has been applied to a wide range of research in the areas of biology, genetics, and medicine. Delivery of therapeutic genome-editing agents provides a promising platform for the treatment of genetic disorders. Although viral vectors are widely used to deliver CRISPR/Cas9 elements with high efficiency, they suffer from several drawbacks, such as mutagenesis, immunogenicity, and off-target effects. Recently, non-viral vectors have emerged as another class of delivery carriers in terms of their safety, simplicity, and flexibility. In this review, we discuss the modes of CRISPR/Cas9 delivery, the barriers to the delivery process and the application of CRISPR/Cas9 system for the treatment of genetic disorders. We also highlight several representative types of non-viral vectors, including polymers, liposomes, cell-penetrating peptides, and other synthetic vectors, for the therapeutic delivery of CRISPR/Cas9 system. The applications of CRISPR/Cas9 in treating genetic disorders mediated by the non-viral vectors are also discussed.

A Critical Review of Electroporation as A Plasmid Delivery System in Mouse Skeletal Muscle.

The gene delivery to skeletal muscles is a promising strategy for the treatment of both muscular disorders (by silencing or overexpression of specific gene) and systemic secretion of therapeutic proteins. The use of a physical method like electroporation with plate or needle electrodes facilitates long-lasting gene silencing in situ. It has been reported that electroporation enhances the expression of the naked DNA gene in the skeletal muscle up to 100 times and decreases the changeability of the intramuscular expression. Coelectransfer of reporter genes such as green fluorescent protein (GFP), luciferase or beta-galactosidase allows the observation of correctly performed silencing in the muscles. Appropriate selection of plasmid injection volume and concentration, as well as electrotransfer parameters, such as the voltage, the length and the number of electrical pulses do not cause long-term damage to myocytes. In this review, we summarized the electroporation methodology as well as the procedure of electrotransfer to the gastrocnemius, tibialis, soleus and foot muscles and compare their advantages and disadvantages.

Clinical translation of gene medicine.

Gene therapy has recently witnessed accelerated progress as a new therapeutic strategy with the potential to treat a range of inherited and acquired diseases. Billions of dollars have been invested in basic and clinical research on gene medicine, with ongoing clinical trials focused on cancer, monogenic diseases, cardiovascular diseases and other refractory diseases. Advances addressing the inherent challenges of gene therapy, particularly those related to retaining the delivery efficacy and minimizing unwanted immune responses, provide the basis for the widespread clinical application of gene medicine. Several types of genes delivered by viral or non-viral delivery vectors have demonstrated encouraging results in both animals and humans. As augmented by clinical indications, gene medicine techniques have rapidly become a promising alternative to conventional therapeutic strategies because of their better clinical benefit and lower toxicities. Their application in the clinic has been extensive as a result of the approval of many gene therapy drugs in recent years. In this review, we provide a comprehensive overview of the clinical translation of gene medicine, focusing on the key events and latest progress made regarding clinical gene therapy products. We also discuss the gene types and non-viral materials with respect to developing gene therapeutics in clinical trials.

Bat adeno-associated viruses as gene therapy vectors with the potential to evade human neutralizing antibodies.

The prevalence of adeno-associated virus (AAV) has been investigated in bat populations, but little is known about the biological properties of this virus. In this study, four full-length bat AAV capsid genes were isolated in China, with their amino acid sequences sharing 61% identity with those of AAV2 on average. These capsid genes could package AAV particles in combination with AAV2 rep and ITRs, albeit at a lower efficiency. Bat AAVs could only slightly infect mouse liver but could transduce mouse muscle to some extent after systemic administration with a higher muscle/liver ratio than that of primate AAVs. Bat AAV 10HB showed moderate muscle transduction, similar to that of AAV2, during direct intramuscular injection and, compared with other AAV serotypes, was also relatively efficient in resisting human antibody neutralization after intramuscular injection. Evolutionary analysis revealed a number of codons in bat AAV capsid genes subject to positive selection, with sites corresponding to V259 and N691 in 10HB capsids being localized on the surface of the AAV2 capsid. Mutagenesis studies indicated that the positive selection in bat AAV capsids is driven by their tropism evolution in host species. Taken together, the results of this study indicate that bat AAV 10HB vector has the possible applications for muscular gene therapy, especially in the presence of human AAV neutralizing antibodies.

Bile-duct proliferation as an unexpected side-effect after AAV2-LDLR gene transfer to rabbit liver.

Familial hypercholesterolemia (FH) is an inherited disease of lipoprotein metabolism caused by a defect in the LDL receptor (LDLR) leading to severe hypercholesterolemia, and associated with an increased risk of coronary heart disease and myocardial infarction. We have developed a gene therapy protocol for FH using AAV2, AAV9 and lentiviral vectors and tested safety and efficacy in LDL receptor deficient Watanabe Heritable Hyperlipidemic rabbits. We show that LV-LDLR produced a significant long-lasting decrease in total serum cholesterol whereas AAV9-LDLR resulted only in a transient decrease and AAV2-LDLR failed to reduce serum cholesterol levels. A significant pathological side effect, bile-duct proliferation, was seen in the liver of AAV2-LDLR rabbits associated with an increased expression of Cyr61 matricellular protein. Special attention should be given to liver changes in gene therapy applications when genes affecting cholesterol and lipoprotein metabolism are used for therapy.

Adeno-associated virus neutralising antibodies in type 1 diabetes mellitus.

Recombinant Adeno-associated viruses (AAVs) are an attractive vector for gene therapy delivery which may be blocked by AAV neutralising antibodies (NAbs). As Type 1 Diabetes (T1DM) is an endocrine disease of immunological origin, it is likely that NAb profiles are altered in the disease. In this study NAb to AAV2, AAV5, AAV6, and AAV8 in 72 subjects with T1DM and 45 non-diabetic patients were measured over a 4-year follow-up period. AAV2 NAb titres were significantly lower in non-diabetic subjects (P = 0.036). The T1DM group had more AAV8 NAb activity at baseline (P = 0.019), whilst after 4 years follow-up the T1DM group displayed developed increased AAV 5 (P = 0.03), 6 (P = 0.03) and 8 (P = 0.002) activity relative to the control group, however, overall AAV5 and 8 NAb levels were very low in patients <40. AAV NAb titre activity and prevalence generally appears higher in T1DM, however, low levels of AAV 5 and 8, particular in younger adult age groups at which T1DM can be targeted, could make these attractive vectors to target the disease.

Non-viral gene delivery for cancer immunotherapy.

Recent decades have witnessed the revolutionary development of cancer immunotherapies, which boost cancer-specific immune responses for long-term tumor regression. However, immunotherapy still has limitations, including off-target side effects, long processing times and limited patient responses. These disadvantages of current immunotherapy are being addressed by improving our understanding of the immune system, as well as by establishing combinational approaches. Advanced biomaterials and gene delivery systems overcome some of these delivery issues, harnessing adverse effects and amplifying immunomodulatory effects, and are superior to standard formulations with respect to eliciting antitumor immunity. Nucleic acid-based nanostructures have diverse functions, ranging from gene expression and gene regulation to pro-inflammatory effects, as well as the ability to specifically bind different molecules. A brief overview is provided of the recent advances in the non-viral gene delivery methods that are being used to activate cancer-specific immune responses. Furthermore, the tumor microenvironment-responsive synergistic strategies that modulate the immune response by targeting various signaling pathways are discussed. Nanoparticle-based non-viral gene delivery strategies have great potential to be implemented in the clinic for cancer immunotherapy.