Efficient Gene Delivery Research Articles

Gene therapy restores mitochondrial function and protects retinal ganglion cells in optic neuropathy induced by a mito-targeted mutant ND1 gene

Therapies for genetic disorders caused by mutated mitochondrial DNA are an unmet need, in large part due barriers in delivering DNA to the organelle and the absence of relevant animal models. We injected into mouse eyes a mitochondrially targeted Adeno-Associated-Virus (MTS-AAV) to deliver the mutant human NADH ubiquinone oxidoreductase subunit I (hND1/m.3460G>A) responsible for Leber’s hereditary optic neuropathy, the most common primary mitochondrial genetic disease. We show that the expression of the mutant hND1 delivered to retinal ganglion cells (RGC) layer colocalizes with the mitochondrial marker PORIN and the assembly of the expressed hND1 protein into host respiration complex I. The hND1 injected eyes exhibit hallmarks of the human disease with progressive loss of RGC function and number, as well as optic nerve degeneration. We also show that gene therapy in the hND1 eyes by means of an injection of a second MTS-AAV vector carrying wild type human ND1 restores mitochondrial respiratory complex I activity, the rate of ATP synthesis and protects RGCs and their axons from dysfunction and degeneration. These results prove that MTS-AAV is a highly efficient gene delivery approach with the ability to create mito-animal models and has the therapeutic potential to treat mitochondrial genetic diseases.

High-Throughput Optical Controlling and Recording Calcium Signal in iPSC-Derived Cardiomyocytes for Toxicity Testing and Phenotypic Drug Screening.

Understanding how excitable cells work in health and disease and how that behavior can be altered by small molecules or genetic manipulation is important. Genetically encoded calcium indicators (GECIs) with multiple emission windows can be combined (e.g., for simultaneous observation of distinct subcellular events) or used in extended applications with other light-dependent actuators in excitable cells (e.g., combining genetically encoded optogenetic control with spectrally compatible calcium indicators). Such approaches have been used in primary or stem cell-derived neurons, cardiomyocytes, and pancreatic beta-cells. However, it has been challenging to increase the throughput, or duration of observation, of such approaches due to limitations of the instruments, analysis software, indicator performance, and gene delivery efficiency. Here, a high-performance green GECI, mNeonGreen-GECO (mNG-GECO), and red-shifted GECI, K-GECO, is combined with optogenetic control to achieve all-optical control and visualization of cellular activity in a high-throughput imaging format using a High-Content Imaging System. Applications demonstrating cardiotoxicity testing and phenotypic drug screening with healthy and patient-derived iPSC-CMs are shown. In addition, multi-parametric assessments using combinations of spectral and calcium affinity indicator variants (NIR-GECO, LAR-GECO, and mtGCEPIA or Orai1-G-GECO) are restricted to different cellular compartments are also demonstrated in the iPSC-CM model.

High-Throughput Optical Controlling and Recording Calcium Signal in iPSC-Derived Cardiomyocytes for Toxicity Testing and Phenotypic Drug Screening

Understanding how excitable cells work in health and disease and how that behavior can be altered by small molecules or genetic manipulation is important. Genetically encoded calcium indicators (GECIs) with multiple emission windows can be combined (e.g., for simultaneous observation of distinct subcellular events) or used in extended applications with other light-dependent actuators in excitable cells (e.g., combining genetically encoded optogenetic control with spectrally compatible calcium indicators). Such approaches have been used in primary or stem cell-derived neurons, cardiomyocytes, and pancreatic beta-cells. However, it has been challenging to increase the throughput, or duration of observation, of such approaches due to limitations of the instruments, analysis software, indicator performance, and gene delivery efficiency. Here, a high-performance green GECI, mNeonGreen-GECO (mNG-GECO), and red-shifted GECI, K-GECO, is combined with optogenetic control to achieve all-optical control and visualization of cellular activity in a high-throughput imaging format using a High-Content Imaging System. Applications demonstrating cardiotoxicity testing and phenotypic drug screening with healthy and patient-derived iPSC-CMs are shown. In addition, multi-parametric assessments using combinations of spectral and calcium affinity indicator variants (NIR-GECO, LAR-GECO, and mtGCEPIA or Orai1-G-GECO) are restricted to different cellular compartments are also demonstrated in the iPSC-CM model.

Influence of Linker Orientation and Regulative Factor(s) in Liposomal Gene Delivery: A Molecular Level Investigation.

Molecular level understanding of liposome-gene interaction is immensely important for the research progress and technological advancement of gene delivery, which is highly significant due to a wide range of applications of gene therapy. The liposomal gene delivery method is one of the most promising techniques due to its efficacy to easily fuse with the cell membrane and its lower toxicity. In vivo gene delivery using liposomes is reported to be extremely successful. However, the success of gene delivery depends on various factors including the chemical nature of the structural unit of the liposome. To explore the regulative factor(s) for liposomal gene delivery, we systematically analyze the linker orientation effect on the gene delivery efficiency of liposomes through a density functional theory (DFT) study. Interestingly, it is observed that the liposome-gene interaction is not the regulating factor for successful gene delivery. The success depends on the gel to liquid melting temperature of the liposome.

Electroporation: An Effective Method For In Vivo Gene Delivery

Background: Gene therapy is a promising approach for the treatment of various diseases, including cancer, hereditary disorders, and some viral infections. The development of efficient and safe gene delivery systems is essential for facilitating gene trans-fer to various organs and tissues in vivo. Objective: In this review, we briefly describe the principal mechanisms of gene delivery systems, particularly electroporation, and discuss the latest advancements in the application of electro-poration for in vivo gene transfer. Methods: A narrative review of all the relevant publication known to the authors was conducted. Results: In recent years, electroporation-based strategies have emerged as an auspicious and versa-tile platform for efficient and controlled delivery of various biomolecules, including nucleic acids. Applying electric pulses of enough magnitude leads to the formation of hydrophilic pores in the cell membrane and allows the entry of otherwise membrane-impermeant molecules, such as DNA. Alt-hough electroporation has been initially developed for in vitro transfection of cells, it has recently advanced to preclinical in vivo applications and finally to clinical trials. Conclusion: Electroporation has already entered the clinical practice for antitumor therapy and may be an essential part of future personalized treatments. Given the ability of electroporation to deliver multiple genes in a single event, it will also certainly be further developed both as a stand-alone de-livery approach and when coupled with other technologies.

Fluorination Promotes the Cytosolic Delivery of Genes, Proteins, and Peptides.

The cytosolic delivery of biomolecules such as genes, proteins, and peptides is of great importance for biotherapy but usually limited by multiple barriers during the process. Cell membrane with high hydrophobic character is one of the representative biological barriers for cytosolic delivery. The introduction of hydrophobic ligands such as aliphatic lipids onto materials or biomolecules could improve their membrane permeability. However, these ligands are lipophilic and tend to interact with the phospholipids in the membrane as well as serum proteins, which may hinder efficient intracellular delivery. To solve this issue, our research group proposed the use of fluorous ligands with both hydrophobicity and lipophobicity as ideal alternatives to aliphatic lipids to promote cytosolic delivery.In our first attempt, fluorous ligands were conjugated onto cationic polymers to increase their gene delivery efficacy. The fluorination dramatically increased the gene delivery performance at low polymer doses. In addition, the strategy greatly improved the serum tolerance of cationic polymers, which is critical for efficient gene delivery in vivo. Besides serum tolerance, mechanism studies revealed that fluorination increases multiple steps such as cellular uptake and endosomal escape. Fluorination also allowed the assembly of low-molecular-weight polymers and achieved highly efficient gene delivery with minimal material toxicity. The method showed robust efficiency for polymers, including linear polymers, branched polymers, dendrimers, bola amphiphilies, and dendronized polymers.Besides gene delivery, fluorinated polymers were also used for intracellular protein delivery via a coassembly strategy. For this purpose, two lead fluoropolymers were screened from a library of amphiphilic materials. The fluoropolymers are greatly superior to their nonfluorinated analogues conjugated with aliphatic lipids. The fluorous lipids are beneficial for polymer assembly and protein encapsulation, reduced protein denaturation, facilitated endocytosis, and decreased polymer toxicity compared to nonfluorinated lipids. The materials exhibited potent efficacy in therapeutic protein and peptide delivery to achieve cancer therapy and were able to fabricate a personalized nanovaccine for cancer immunotherapy. Finally, the fluorous lipids were directly conjugated to peptides via a disulfide bond for cytosolic peptide delivery. Fluorous lipids drive the assembly of cargo peptides into uniform nanoparticles with much improved proteolytic stability and promote their delivery into various types of cells. The delivery efficacy of this strategy is greatly superior to traditional techniques such as cell-penetrating peptides both in vitro and in vivo. Overall, the fluorination techniques provide efficient and promising strategies for the cytosolic delivery of biomolecules.

Structure–activity relationships of pH-responsive and ionizable lipids for gene delivery

Ionizable lipids are the leading vectors for gene therapy. Understanding the effects of molecular structure on efficient gene delivery is one of the most important challenges for maximizing the utility of such lipid vectors. We synthesized an array of pH-responsive and ionizable lipids to investigate the relationship between lipid structure and activity. The optimized lipid (EDM) has double tertiary amines in the headgroup and an ester linker. EDM exhibited efficient DNA and siRNA delivery to, and gene silencing of, A549 cells. EDM has a pKa value of 6.67, which enabled it to quickly escape from the endosome after entering the cell; the ester linkages rapidly degraded and enabled gene release into the cytoplasm. EDM also delivered IGF-1R siRNA to inhibit tumor growth and induce cancer cell apoptosis by efficient inhibition of IGF-1R expression in mice. Our study on the structure–activity relationships of ionizable lipids will facilitate clinical applications.

Cancer gene therapy goes viral: viral vector platforms come of age.

BackgroundSince the advent of viral vector gene therapy in 1990s, cancer treatment with viral vectors promised to revolutionize the field of oncology. Notably, viral vectors offer a unique combination of efficient gene delivery and engagement of the immune system for anti-tumour response. Despite the early potential, viral vector-based cancer treatments are only recently making a big impact, most prominently as gene delivery devices in approved CAR-T cell therapies, cancer vaccines and targeted oncolytic therapeutics. To reach this broad spectrum of applications, a number of challenges have been overcome – from our understanding of cancer biology to vector design, manufacture and engineering. Here, we take an overview of viral vector usage in cancer therapy and discuss the latest advancements. We also consider production platforms that enable mainstream adoption of viral vectors for cancer gene therapy.ConclusionsViral vectors offer numerous opportunities in cancer therapy. Recent advances in vector production platforms open new avenues in safe and efficient viral therapeutic strategies, streamlining the transition from lab bench to bedside. As viral vectors come of age, they could become a standard tool in the cancer treatment arsenal.

Chemically Tuned Intracellular Gene Delivery by Core-Shell Nanoparticles: Effects of Proton Buffering, Acid Degradability, and Membrane Disruption.

Nanoparticles consisting of a condensed nucleic acid core surrounded by protective layers which aid to overcome extracellular and intracellular hurdles to gene delivery (i. e., core-shell nanoparticles, CSNPs) synthetically mimic viruses. The outer shells shield the core and are particularly designed to enable facilitated release of the gene payload into the cytoplasm, the major limiting step in intracellular gene delivery. The hypothetical proton sponge effect and degradability in response to a stimulus (i. e., mildly acidic pH in the endosome) are two prevailing, although contested, principles in designing effective carriers for intracellular gene delivery via endosomal escape. Utilizing the highly flexible chemical-tuning of the polymeric shell via surface-initiated photo-polymerization of the various monomers at different molecular ratios, the effects of proton buffering capacity, acid-degradability, and endosomal membrane-lysis property on intracellular delivery of plasmid DNA by CSNPs were investigated. This study demonstrated the equivalently critical roles of proton buffering and acid-degradability in achieving efficient intracellular gene delivery, independent of cellular uptake. Extended proton buffering resulted in further improved transfection as long as the core structure was not compromised. The results of the study present a promising synthetic strategy to the development of an efficient, chemically-tunable gene delivery carrier.

Linear-branched poly(β-amino esters)/DNA nano-polyplexes for effective gene transfection and neural stem cell differentiation

Controllable regulation of stem cell differentiation is a critical concern in stem cell-based regenerative medicine. In particular, there are still great challenges in controlling the directional differentiation of neural stem cells (NSCs) into neurons. Herein, we developed a novel linear-branched poly(β-amino esters) (S4-TMPTA-BDA-DT, STBD) through a two-step reaction. The synthesized linear-branched polymers possess multiple positively charged amine terminus and degradable intermolecular ester bonds, thus endowing them with excellent properties such as high gene load, efficient gene delivery, and effective gene release and transcription in cells. In the mCherry transfection test, a high transfection efficiency of approximately 70% was achieved in primary NSCs after a single transfection. Moreover, STBD also showed high biocompatibility to NSCs without disturbing their viability and neural differentiation. With the high gene delivery property, STBD is capable of delivering siRNA (shSOX9) expression plasmid into NSCs to significantly interfere with the expression of SOX9, thus enhancing the neuronal differentiation and maturation of NSCs. The STBD/DNA nano-polyplex represents a powerful non-viral approach of gene delivery for manipulating the differentiation of stem cells, showing broad application prospects in NSC-based regenerative therapy for treating neurodegenerative diseases.

Efficient polycation non-viral gene delivery system with high buffering capacity and low molecular weight for primary cells: Branched poly(β-aminoester) containing primary, secondary and tertiary amine groups

Gene transfer studies, preferred in many biotechnology studies such as in vitro transgenic cell modeling systems, gene therapy, transgenic animal production by cloning, etc., are quite limited due to transfection difficulties on primary cell lines. The main purpose of this study is on the production of biocompatible gene carrier systems with high transfection efficiency for Primary Ovine Fibroblast cell lines (POF). To achieve this goal, the synthesis of the branched poly(β-aminoester) (PBAE) containing primary, secondary and tertiary amino groups in their molecular structures was carried out via step-growth polymerization by a typical Michael addition reaction between bisphenol A-ethoxylate diacrylate (BPAEDA) and ethylenediamine (EDA) for the first time. PBAEs were characterized using FTIR and NMR techniques. GPC-SEC system was used to determine the molecular weight of these polymers. Proton buffering capacity of the PBAE was also determined. In order to optimize the preparation of the PBAE nanoparticles (nPBAE), two different methods, which are solvent evaporation and solvent displacement techniques, were used. Their physicochemical properties such as particle size, polydispersity index (PDI), zeta potential and stability were identified. nPBAE-gene complexes (gnPBAE) were obtained according to the adsorption or encapsulation mechanisms and their properties were evaluated by comparative surface charge and gel electrophoresis. In vitro studies including gene binding capacity, cytotoxicity and the transfection efficiency for primary ovine fibroblast (POF) cell lines were also realized. Consequently, (egnPBAE)dw formulation, which is obtained from PBAEEDA with the lowest Mw by encapsulation method in this study, have very high in vitro transfection efficiency up to 78.3% in POF cells and it was also exhibited almost 1.5 times more transfection efficiency against commercial product Lipofectamine because of its low Mw and the presence of the secondary and tertiary amine structures as well as primary amine end groups in its backbone.

One-pot synthesis of functional peptide-modified gold nanoparticles for gene delivery

A facile and mild method for construction of peptide-modified gold nanoparticles (AuNPs) for gene delivery has been developed. A peptide with sequence of (CRGDKGPDC)GPLGLAGIIIGRRRRRRRR-NH2 (CPIR28) has been de novo designed to have four functional segments, that is, the cyclic iRGD segment of c(CRGDKGPDC) with Au binding cystine group, the enzyme responsive segment of PLGLA, the hydrophobic segment of III and the positive transmembrane segment of R8. By a simple one-pot reaction between CPIR28, HAuCl4 and NaBH4 in aqueous solution, the AuNPs with sizes < 10 nm are successfully produced, which have CPIR28 molecules grafted on their surface. The AuNPs have a high density of surface positive charges attributing to R8 segment. They can interact with negatively charged DNA electrostatically to form condensed complexes, which show low level of cytotoxicity. In this system, CPIR28 not only serves as a modifier to regulate the nucleation and growth of AuNPs for controlling their morphology and size but also acts as an excellent stabilizer by grafting on the AuNPs surface to prevent their aggregation and precipitation. Moreover, the surface grafted peptide molecules provide positive charges to facilitate interactions with cells, which benefit the efficient gene delivery. The study paves a new way for synthesizing peptide-functionalized AuNPs for gene delivery applications.

Zoledronic acid-loaded cationic methylcellulose polyplex nanoparticles for enhanced gene delivery efficiency and breast cancer cell killing effect

Zoledronic acid-loaded cationic methylcellulose polyplex nanoparticles for enhanced gene delivery efficiency and breast cancer cell killing effect

Transient depletion of pre-existing antibodies for efficient AAV gene delivery

Transient depletion of pre-existing antibodies for efficient AAV gene delivery

The inhibitory role of si-UBB delivered by degradable dendrimers-based lipid nanoparticles in ovarian cancer

ObjectiveRNA interference holds tremendous potential in the treatment of malignant tumors. However, efficient and biocompatible delivery methods are needed for systemic delivery of small interfering RNA (siRNA). In this study, we explored the delivery efficiency and therapy effect of si-UBB-5A2SC8 in ovarian cancer.Methods and materialsSi-UBB-5A2SC8 nanoparticles were successfully prepared according to the established procedure and the characteristic of nanoparticles was determined by digital laser scanning. Flow cytometry and confocal analysis demonstrated si-UBB was efficiently transfected to cell by the delivery of 5A2SC8 complexes. The in vitro gene knockdown efficiency of ubiquitin B was demonstrated by RT-qPCR and Western blot analysis, which was further verified by the inhibition of proliferation and migration of ovarian cancer cells. Accumulative efficiency of si-UBB-5A2SC8 nanoparticles was investigated in BALB/c mice bearing SKOV3-GFP tumor xenograft. In vivo imaging was adopted to test the accurate location of the nanoparticle in the tumor. The feature of the tumor and pivotal organ was determined. TUNEL and caspase-3 expression was used to analyze the underling mechanism of the inhibition effect.ResultsThe average size and the zeta potential of the si-UBB-5A2SC8 was (150 ± 11) nm and − (20 ± 4) mV, respectively. Transmission electronic microscopy showed the nanoparticle was near-spherical with the mean size of (100 ± 15) nm. Flow cytometry and confocal microscopic images demonstrated 5A2SC8 complex could successfully deliver Cy5.5-siRNA to the cytoplasm of ovarian cancer cells. qRT-PCR and western blot demonstrated the mRNA and protein expression of ubiquitin B was decreased to 62.5% and 36.5% of the control group, which was accompanied with the decreased proliferation and migration ability in si-UBB-5A2SC8-transfected cells. Ex vivo fluorescence imaging demonstrated 5A2SC8 complex could successfully carry siRNA to the tumor lesion and exert the inhibition effect, which was verified by the decreased tumor weight and increased apoptosis and caspase-3 expression in mice treated with si-UBB-5A2SC8.Conclusions5A2SC8 complex was a safe and efficient gene delivery vehicle and ubiquitin B was a potential target for the ovarian cancer targeted therapy.

Metallohelix vectors for efficient gene delivery via cationic DNA nanoparticles.

The design of efficient and safe gene delivery vehicles remains a major challenge for the application of gene therapy. Of the many reported gene delivery systems, metal complexes with high affinity for nucleic acids are emerging as an attractive option. We have discovered that certain metallohelices—optically pure, self-assembling triple-stranded arrays of fully encapsulated Fe—act as nonviral DNA delivery vectors capable of mediating efficient gene transfection. They induce formation of globular DNA particles which protect the DNA from degradation by various restriction endonucleases, are of suitable size and electrostatic potential for efficient membrane transport and are successfully processed by cells. The activity is highly structure-dependent—compact and shorter metallohelix enantiomers are far less efficient than less compact and longer enantiomers.

Ultrasound-Enhanced Reactive Oxygen Species Responsive Charge-Reversal Polymeric Nanocarriers for Efficient Pancreatic Cancer Gene Delivery.

Inefficient intracellular gene release and transfection limit nonviral gene delivery applications in cancer therapy. Reactive oxygen species (ROS) responsive nonviral gene delivery is the most widely explored strategy for such applications, yet the development of fast and safe ROS responsive nanocarriers proves to be a challenge because of the intracellular chemical equilibrium of high ROS and glutathione levels. Here, we report an ultrasound-enhanced ROS responsive charge-reversal polymeric nanocarrier (BTIL) for fast and efficient pancreatic cancer gene delivery. The BTIL is composed of B-PDEAEA/DNA polyplex-based cores and IR780-loaded liposome coatings. The IR780 is able to produce an excess of ROS under low intensity ultrasound irradiation, thus disequilibrating the chemical equilibrium of ROS and glutathione, and promoting the ROS-responsive positive-to-negative charge-reversal of the B-PDEAEA polymer. This charge conversion results in fast polyplex dissociation and intracellular gene release, inducing efficient gene transfection and cancer cell apoptosis. Moreover, following the intravenous administration, BTIL maintains a stable and long circulation in the bloodstream, achieves orthotopic pancreatic ductal adenocarcinoma distribution, and exhibits potent antitumor activity with negligible side effects. Our results reveal the proposed strategy to be both promising and universal for the development of fast and safe ROS responsive nonviral gene delivery in cancer therapy.

Tailoring head-tail mesoporous silica nanoparticles for enhanced gene transfection.

Plasmid DNA (pDNA) delivery has attracted extensive research interest due to its great potential in gene therapy. The design of efficient nano-vectors to promote cellular delivery and transfection of gene molecules is the key to success. Compared to conventional nanocarriers with spherical geometry, asymmetric nanoparticles have been well documented showing enhanced cellular uptake and drug delivery capability. However, the impact of asymmetric nanostructures on pDNA binding and following intracellular delivery performance has been less reported. Herein, asymmetric head-tail mesoporous silica nanoparticles (HTMSNs) with tailored tail lengths were synthesized and employed as nano-vectors for pDNA delivery. The nanostructures of HTMSNs were carefully characterized by electron tomography. The pDNA binding, cellular uptake and gene transfection capabilities of engineered asymmetric nanoparticles were compared with symmetric dendritic mesoporous silica nanoparticles (DMSNs). The results showed that the asymmetric morphology of nanoparticles promoted pDNA binding and cell internalization, where HTMSNs-66 with a specific tail length of 66 nm achieved the highest transfection efficiency. This study reveals the impact of asymmetric nanostructure on DNA interaction, and provides guidance in future designs of non-viral nano-vectors for efficient gene delivery.

Nicotinic acid-based cationic vectors for efficient gene delivery to glioblastoma cells

A tocopherol-conjugated nicotinic acid-based lipid (NGT) was used for liposomal formation with the co-lipid DOPE and exhibited enhanced transfection of glioblastoma cells for eGFP and β-galactosidase protein expression.

Preparation and Characterization of a Liver Targeted, Poly(amidoamine) Based, Gene Delivery System.

Nonalcoholic steatohepatitis (NASH) is an aggressive liver disease that is considered a major cause of liver cirrhosis and hepatocellular carcinoma. NASH is characterized by multiple underlying genetic mutations, with no approved cure to date. Gene therapies that target those genetic mutations may play a major role in treating this disease, once delivered specifically to the hepatocytes. In this chapter we present, in detail, the synthesis and the characterization of an efficient gene delivery system capable of targeting hepatocytes by exploiting the overexpression of asialoglycoprotein receptors on their cell surface. The targeting ligand, galactose derivative, lactobionic acid (Gal), is first conjugated to bifunctional poly(ethylene glycol) (PEG), and then the formed PEG-Gal is further conjugated to the positively charged polymer, poly(amidoamine) (PAMAM) to form a PAMAM-PEG-Gal construct that can complex and deliver genetic material (e.g., pDNA, siRNA, mRNA) specifically to hepatocytes. We first synthesize PAMAM-PEG-Gal using carbodiimide click chemistry. The synthesized conjugate is characterized using 1H NMR spectroscopy and mass spectrometry. Next, nanoplexes are prepared by combining the positively charged conjugate and the negatively charged genetic material at different nitrogen to phosphate (N/P) ratios; then the size, charge, electrophoretic mobility, and surface morphology of those nanoplexes are estimated. The simplicity of complexing our conjugate with any type of genetic material, the ability of our delivery system to overcome the current limitations of delivering naked genetic material, and the efficiency of delivering its payload specifically to hepatocytes, makes our formulation a promising tool to treat any type of genetic abnormality that arises in hepatocytes, and specifically NASH.