Vector Delivery Methods Research Articles

A large-scale optogenetic neurophysiology platform for improving accessibility in NHP behavioral experiments.

Optogenetics has been a powerful scientific tool for two decades, yet its integration with non-human primate (NHP) electrophysiology has been limited due to several technical challenges. These include a lack of electrode arrays capable of supporting large-scale and long-term optical access, inaccessible viral vector delivery methods for transfection of large regions of cortex, a paucity of hardware designed for large-scale patterned cortical illumination, and inflexible designs for multi-modal experimentation. To address these gaps, we introduce a highly accessible platform integrating optogenetics and electrophysiology for behavioral and neural modulation with neurophysiological recording in NHPs. We employed this platform in two rhesus macaques and showcased its capability of optogenetically disrupting reaches, while simultaneously monitoring ongoing electrocorticography activity underlying the stimulation-induced behavioral changes. The platform exhibits long-term stability and functionality, thereby facilitating large-scale electrophysiology, optical imaging, and optogenetics over months, which is crucial for translationally relevant multi-modal studies of neurological and neuropsychiatric disorders.

Comparison of the efficiencies of intrathecal and intraganglionic injections in mouse dorsal root ganglion.

Dorsal root ganglia (DRG) are structures containing primary sensory neurons. Intraganglionic (IG) and intrathecal (IT) applications are the most common methods used for viral vector transfer to DRG. We aim to compare the efficiencies and pathological effects of IT and IG viral vector delivery methods to DRG, through in vivo imaging. Mice were divided into four groups of six each: IT, IG, IT-vehicle, and IG-vehicle. Adeno-associated virus (AAV) injection was performed for EGFP expression in IT/IG groups. DRGs were made visible through vertebral window surgery and visualized with multiphoton microscopy. After imaging, spinal cords and DRGs were removed and cleared, then imaged with light sheet microscopy. No neuronal death was observed after IT injection, while the death rate was 17% 24 h after IG injection. EGFP expression efficiencies were 90%-95% of neurons in both groups. EGFP expression was only observed in targeted L2 DRG after IG injection, while it was observed in DRGs located between L1-L5 levels after IT injection. IT injection is a more suitable method for labeling DRG neurons in neurodegenerative injury models. However, when the innervation of DRG needs to be specifically studied, IT injection reduces this specificity due to its spread. In these studies, IG injection is the most suitable method for labeling single DRG neurons.

State-of-the-art 2023 on gene therapy for phenylketonuria.

Phenylketonuria (PKU) or hyperphenylalaninemia is considered a paradigm for an inherited (metabolic) liver defect and is, based on murine models that replicate all human pathology, an exemplar model for experimental studies on liver gene therapy. Variants in the PAH gene that lead to hyperphenylalaninemia are never fatal (although devastating if untreated), newborn screening has been available for two generations, and dietary treatment has been considered for a long time as therapeutic and satisfactory. However, significant shortcomings of contemporary dietary treatment of PKU remain. A long list of various gene therapeutic experimental approaches using the classical model for human PKU, the homozygous enu2/2 mouse, witnesses the value of this model to develop treatment for a genetic liver defect. The list of experiments for proof of principle includes recombinant viral (AdV, AAV, and LV) and non-viral (naked DNA or LNP-mRNA) vector delivery methods, combined with gene addition, genome, gene or base editing, and gene insertion or replacement. In addition, a list of current and planned clinical trials for PKU gene therapy is included. This review summarizes, compares, and evaluates the various approaches for the sake of scientific understanding and efficacy testing that may eventually pave the way for safe and efficient human application.

Delivery of dCas9 Activator System Using Magnetic Nanoparticles Technology as a Vector Delivery Method for Human Skin Fibroblast

The overexpression of stem cell-related genes such as octamer-binding transcription factor 4 (OCT4) and (sex determining region Y)-box 2 (SOX2) has been indicated to play several critical roles in stem cell self-renewal; moreover, the elevation of the self-renewal of cancer cells with stem cell-like properties has been suggested. The clustered and regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) protein fused to transactivation domains can be used to activate gene expression in human cells. CRISPR-mediated activation (CRISPRa) systems represent an effective genome editing tool for highly specific gene activation in which a nuclease-deficient Cas9 (dCas9) is utilized to target a transcriptional activator to the gene’s regulatory element, such as a promoter and enhancer. The main drawback of typical delivery methods for CRISPR/Cas9 components is their low transfection efficiency or toxic effects on cells; thus, we generated superparamagnetic iron oxide nanoparticles (SPIONs) coated with polyethylenimine (PEI) to improve the delivery of CRISPR/Cas9 constructs into human foreskin fibroblast cells. The delivery system with magnetic PEI-coated nanoparticles complex was applied to constitute plasmid DNA lipoplexes. CRISPRa systems were used to overexpress the endogenous OCT4 and SOX2 in fibroblast cells. The quantitative polymerase chain reaction (QPCR) assessment exhibited a three-times higher expression of OCT4 and SOX2 transfected by CRISPRa using MNPs. Moreover, no additional cytotoxicity was observed with the application of magnetic nanoparticles (MNPs) compared to lipofectamine. Our results demonstrate that MNPs enable the effective delivery of the CRISPR/Cas9 construct into human foreskin fibroblasts with low cell toxicity and a consequential overexpression of endogenous OCT4 and SOX2.

CRISPR/Cas9 technology and its application in horticultural crops

Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated 9 (CRISPR/Cas9) system has recently become one popular technology due to its efficiency, precision, and simplicity compared with other genome editing tools such as Zinc Finger Nucleases (ZFNs) and Transcription Activator Like Effector Nucleases (TALENs). Horticultural crops provide energy and health-keeping nutrients to humankind. Genome-editing technology has become widely adopted in horticultural breeding with the increasing demand for high yield and better-quality horticultural crops. Here, we describe the CRISPR/Cas9 system construction, its optimization, including sgRNA promoter, sgRNA design, Cas9 protein promoter, SpCas9 variants and orthologs, and vector delivery methods. We also summarized the application of this technology in horticultural plants for stress responses enhancement, fruit quality improvement, and cultivation traits modification. This detailed review was compiled to help establish comprehensive understanding of the CRISPR/Cas9 systems and provide a reference for further developing this technology to manipulate horticultural plant traits effectively.

Recent Advances and Challenges in Gene Delivery Mediated by Polyester-Based Nanoparticles.

Gene therapy is a promising approach for the treatment of several diseases, such as chronic or viral infections, inherited disorders, and cancer. The cellular internalization of exogenous nucleic acids (NA) requires efficient delivery vehicles to overcome their inherent pharmacokinetic drawbacks, e.g. electrostatic repulsions, enzymatic degradation, limited cellular uptake, fast clearance, etc. Nanotechnological advancements have enabled the use of polymer-based nanostructured biomaterials as safe and effective gene delivery systems, in addition to viral vector delivery methods. Among the plethora of polymeric nanoparticles (NPs), this review will provide a comprehensive and in-depth summary of the polyester-based nanovehicles, including poly(lactic-co-glycolic acid) (PLGA) and polylactic acid (PLA) NPs, used to deliver a variety of foreign NA, e.g. short interfering RNA (siRNA), messenger RNA (mRNA), and plasmid DNA (pDNA). The article will review the versatility of polyester-based nanocarriers including their recent application in the delivery of the clustered, regularly‐interspaced, short palindromic repeats/Cas (CRISPR/Cas) genome editing system for treating gene-related diseases. The remaining challenges and future trend of the targeted delivery of this revolutionary genome-editing system will be discussed. Special attention will be given to the pivotal role of nanotechnology in tackling emerging infections such as coronavirus disease 2019 (COVID-19): ground-breaking mRNA vaccines delivered by NPs are currently used worldwide to fight the pandemic, pushing the boundaries of gene therapy.

Viral Vector Delivery of DREADDs for CNS Therapy.

Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) are genetically modified G-protein-coupled receptors (GPCRs), that can be activated by a synthetic ligand which is otherwise inert at endogenous receptors. DREADDs can be expressed in cells in the central nervous system (CNS) and subsequently offer the opportunity for remote and reversible silencing or activation of the target cells when the synthetic ligand is systemically administered. In neuroscience, DREADDs have thus far shown to be useful tools for several areas of research and offer considerable potential for the development of gene therapy strategies for neurological disorders. However, in order to design a DREADD-based gene therapy, it is necessary to first evaluate the viral vector delivery methods utilised in the literature to deliver these chemogenetic tools. This review evaluates each of the prominent strategies currently utilised for DREADD delivery, discussing their respective advantages and limitations. We focus on adeno-associated virus (AAV)-based and lentivirus-based systems, and the manipulation of these through cell-type specific promoters and pseudotyping. Furthermore, we address how virally mediated DREADD delivery could be improved in order to make it a viable gene therapy strategy and thus expand its translational potential.

Macrophage Modification Strategies for Efficient Cell Therapy.

Macrophages, important cells of innate immunity, are known for their phagocytic activity, capability for antigen presentation, and flexible phenotypes. Macrophages are found in all tissues and therefore represent an attractive therapeutic target for the treatment of diseases of various etiology. Genetic programming of macrophages is an important issue of modern molecular and cellular medicine. The controllable activation of macrophages towards desirable phenotypes in vivo and in vitro will provide effective treatments for a number of inflammatory and proliferative diseases. This review is focused on the methods for specific alteration of gene expression in macrophages, including the controllable promotion of the desired M1 (pro-inflammatory) or M2 (anti-inflammatory) phenotypes in certain pathologies or model systems. Here we review the strategies of target selection, the methods of vector delivery, and the gene editing approaches used for modification of macrophages.

Human Organic Anion Transporting Polypeptide 1B3 Applied as an MRI-Based Reporter Gene.

ObjectiveRecent innovations in biology are boosting gene and cell therapy, but monitoring the response to these treatments is difficult. The purpose of this study was to find an MRI-reporter gene that can be used to monitor gene or cell therapy and that can be delivered without a viral vector, as viral vector delivery methods can result in long-term complications.Materials and MethodsCMV promoter-human organic anion transporting polypeptide 1B3 (CMV-hOATP1B3) cDNA or CMV-blank DNA (control) was transfected into HEK293 cells using Lipofectamine. OATP1B3 expression was confirmed by western blotting and confocal microscopy. In vitro cell phantoms were made using transfected HEK293 cells cultured in various concentrations of gadoxetic acid for 24 hours, and images of the phantoms were made with a 9.4T micro-MRI. In vivo xenograft tumors were made by implanting HEK293 cells transfected with CMV-hOATP1B3 (n = 4) or CMV-blank (n = 4) in 8-week-old male nude mice, and MRI was performed before and after intravenous injection of gadoxetic acid (1.2 µL/g).ResultsWestern blot and confocal microscopy after immunofluorescence staining revealed that only CMV-hOATP1B3-transfected HEK293 cells produced abundant OATP1B3, which localized at the cell membrane. OATP1B3 expression levels remained high through the 25th subculture cycle, but decreased substantially by the 50th subculture cycle. MRI of cell phantoms showed that only the CMV-hOATP1B3-transfected cells produced a significant contrast enhancement effect. In vivo MRI of xenograft tumors revealed that only CMV-hOATP1B3-transfected HEK293 tumors demonstrated a T1 contrast effect, which lasted for at least 5 hours.ConclusionThe human endogenous OATP1B3 gene can be non-virally delivered into cells to induce transient OATP1B3 expression, leading to gadoxetic acid-mediated enhancement on MRI. These results indicate that hOATP1B3 can serve as an MRI-reporter gene while minimizing the risk of long-term complications.

Localized Intra-Arterial Gene Delivery Using AAV.

In vivo gene therapy is a tremendous tool for a wide variety of genetic modifications. However, often a specific and precise local administration of the viral vector is necessary to deliver the genetic payload in vivo. For many animal studies using viral vectors, such as those investigating neurological disorders, the vector is targeted directly into the tissue/organ of interest. On the other hand, in vascular disease research, viral vectors are administered systemically, either via a tail vein injection or through catheter-mediated infusion, which results in off-target transduction of cells and tissues. Targeting cells in the vascular wall without off-target activity, however, requires localized delivery in order to efficiently target cells of the internal vasculature. Here we describe a novel murine in vivo targeted intra-arterial viral vector delivery method, which has been developed in order to be able to perform more intricate studies in cardiovascular disease.

A doxycycline inducible, adenoviral bone morphogenetic protein-2 gene delivery system to bone.

We report the novel use of a tuneable, non-integrating viral gene delivery system to bone that can be combined with clinically approved biomaterials in an 'off-the-shelf' manner. Specifically, a doxycycline inducible Tet-on adenoviral vector (AdTetBMP-2) in combination with mesenchymal stromal cells (MSCs), fibrin and a biphasic calcium phosphate ceramic (MBCP®) was used to repair large bone defects in nude rats. Bone morphogenetic protein-2 (BMP-2) transgene expression could be effectively tuned by modification of the doxycycline concentration. The effect of adenoviral BMP-2 gene delivery upon bone healing was investigated in vivo in 4mm critically sized, internally fixated, femoral defects. MSCs were transduced either by direct application of AdTetBMP-2 or by pre-coating MBCP granules with the virus. Radiological assessment scores post-mortem were significantly improved upon delivery of AdTetBMP-2. In AdTetBMP-2 groups, histological analysis revealed significantly more newly formed bone at the defect site compared with controls. Newly formed bone was vascularized and fully integrated with nascent tissue and implanted biomaterial. Improvement in healing outcome was achieved using both methods of vector delivery (direct application vs. pre-coating MCBP). Adenoviral delivery of BMP-2 enhanced bone regeneration achieved by the transplantation of MSCs, fibrin and MBCP in vivo. Importantly, our in vitro and in vivo data suggest that this can be achieved with relatively low (ng/ml) levels of the growth factor. Our model and novel gene delivery system may provide a powerful standardized tool for the optimization of growth factor delivery and release for the healing of large bone defects. Copyright © 2016 John Wiley & Sons, Ltd.

AAV vector distribution in the mouse respiratory tract following four different methods of administration

BackgroundTargeted delivery of gene therapy vectors to the mouse respiratory tract is often performed via intranasal or intratracheal administration; however, there can be a great deal of variability between these methods, which could potentially influence experimental results. Improving the accuracy and precision of lung delivery will not only reduce the number of animals required to detect statistically significant differences, but may reduce the variability of studies from different laboratories.ResultsHere we evaluated three different methods of adeno-associated virus (AAV) vector administration to the respiratory tract in mice (intranasal, intubation, and intratracheal injection) and discuss the advantages, challenges, and shortcomings of each. We also present a modified-intranasal delivery technique that is superior to passive administration of vector into the nares of anesthetized supine animals. Transgene expression was consistently visible in the nasal cavity, trachea, and proximal to middle aspect of all lung lobes for all four methods, whereas transgene expression was consistently observed in the most distal aspect of lung lobes only with the intubation and intratracheal injection techniques. AAV vector genome copy numbers in the lung were approximately four-fold lower in mice that received vector via intranasal administration in comparison to the other three methods of vector delivery. The modified intranasal, intubation and intratracheal injection methods of vector administration did not yield statistical differences in AAV vector genome copy numbers in the lung. With regard to reproducibility of vector distribution within and between animals, the modified-intranasal technique was superior.ConclusionOur results show that mode of AAV vector administration to the murine respiratory tract should be selected based on desired target site and skill of the researcher, and that appropriate technique selection may greatly influence experimental outcomes.

Gene Transfer to Rodent Hearts In Vivo.

Altering expression of specific genes in rodent models allows the dissection of various pathways involved in various disease states. Advances in gene transfer technologies produced vectors with tissue specificity, various time courses, increasing or knocking down genes in an externally controlled fashion. To achieve this goal, appropriate vector delivery method is essential for successfully conducting experiments. In this chapter, we focus on cardiac gene transfer in rodents that can be employed for delivering both viral and nonviral vectors.

Preferred transduction with AAV8 and AAV9 via thalamic administration in the MPS IIIB model: A comparison of four rAAV serotypes

Sanfilippo syndrome type B (MPS IIIB) is a lysosomal storage disease caused by a deficiency of N-acetyl-glucosaminidase (NAGLU) activity. Since early therapeutic intervention is likely to yield the most efficacious results, we sought to determine the possible therapeutic utility of rAAV in early gene therapy based interventions. Currently, the application of recombinant adeno-associated virus (AAV) vectors is one of the most widely used gene transfer systems, and represents a promising approach in the treatment of MPS IIIB. From a translational standpoint, a minimally invasive, yet highly efficient method of vector administration is ideal. The thalamus is thought to be the switchboard for signal relay in the central nervous system (CNS) and therefore represents an attractive target. To identify an optimal AAV vector for early therapeutic intervention, and establish whether thalamic administration represents a feasible therapeutic approach, we performed a comprehensive assessment of transduction and biodistribution profiles of four green fluorescent protein (GFP) bearing rAAV serotypes, -5, -8, -9 and -rh10, administered bilaterally into the thalamus. Of the four serotypes compared, AAV8 and -9 proved superior to AAV5 and -rh10 both in biodistribution and transduction efficiency profiles. Genotype differences in transduction efficiency and biodistribution patterns were also observed. Importantly, we conclude that AAV8 and to a lesser extent, AAV9 represent preferable candidates for early gene therapy based intervention in the treatment of MPS IIIB. We also highlight the feasibility of thalamic rAAV administration, and conclude that this method results in moderate rAAV biodistribution with limited treatment capacity, thus suggesting a need for alternate methods of vector delivery.

Correction of Hyperbilirubinemia in Gunn Rats by Surgical Delivery of Low Doses of Helper-Dependent Adenoviral Vectors

Helper-dependent adenoviral (HDAd) vectors are attractive for liver-directed gene therapy because they can drive sustained high levels of transgene expression without chronic toxicity. However, high vector doses are required to achieve efficient hepatic transduction by systemic delivery because of a nonlinear dose response. Unfortunately, such high doses result in systemic vector dissemination and dose-dependent acute toxicity with potential lethal consequences. We have previously shown in nonhuman primates that delivery of HDAd in surgically isolated livers resulted in a significantly higher hepatic transduction with reduced systemic vector dissemination compared with intravenous delivery and multiyear transgene expression. Encouraged by these data, we have now employed a surgical vector delivery method in the Gunn rat, an animal model for Crigler-Najjar syndrome. After vector delivery into the surgically isolated liver, we show phenotypic correction at the low and clinically relevant vector dose of 1 × 10(11) vp/kg. Correction of hyperbilirubinemia and increased glucuronidation of bilirubin in bile was achieved for up to 1 year after vector administration. Surgical delivery of the vector was well tolerated without signs of acute or chronic toxicity. This method of delivery could thereby be a safer alternative to liver transplantation for long-term treatment of Crigler-Najjar syndrome type I.

Virus-induced gene silencing in two novel functional plants, Lycium barbarum L. and Lycium ruthenicum Murr.

Two species of Goji, Lycium barbarum L. and Lycium ruthenicum Murr., are novel functional vegetables and functional fruits widely used in China and other Asian countries. Both species possess complex secondary metabolic pathways and show high tolerance and adaptability to saline-alkali stress, making them novel targets for functional genetic analysis of the biochemical pathways involved. Although stable transgenic Goji lines have been produced, the process is very labor-intensive and time-consuming. Virus-induced gene silencing (VIGS) presents an effective and rapid alternative for creating targeted gene knock-outs to study gene function in plants. In this study, the first application of VIGS in Lycium species is presented, using the Tobacco rattle virus (TRV) vectors. A number of vector delivery methods were trialed, including leaf syringe-infiltration, agrodrench, seedling vacuum-infiltration and sprout vacuum-infiltration (SVI). Vacuum-infiltration was the most effective method and was used to successfully silence two reporter genes, phytoene desaturase (PDS) and Mg-chelatase H subunit (Chl H), concomitant with photobleaching and yellow leaf phenotypes, respectively. The proven application of VIGS to these Lycium sp. will expedite the functional characterization of novel genes involved in the biosynthesis of functional components both in leaves and fruits, as well as the abiotic stress tolerances.

Mechanistic, Technical, and Clinical Perspectives in Therapeutic Stimulation of Coronary Collateral Development by Angiogenic Growth Factors

Stimulation of collateral vessel development in the heart by angiogenic growth factor therapy has been tested in animals and humans for almost two decades. Discordance between the outcome of preclinical studies and clinical trials pointed to the difficulties of translation from animal models to patients. Lessons learned in this process identified specific mechanistic, technical, and clinical hurdles, which need to be overcome. This review summarizes current understanding of the mechanisms leading to the establishment of a functional coronary collateral network and the biological processes growth factor therapies should stimulate even under conditions of impaired natural adaptive vascular response. Vector delivery methods are recommended to maximize angiogenic gene therapy efficiency and reduce side effects. Optimization of clinical trial design should include the choice of clinical end points which provide mechanistic proof-of-concept and also reflect clinical benefits (e.g., surrogates to assess increased collateral flow reserve, such as myocardial perfusion imaging). Guidelines are proposed to select patients who may respond to the therapy with high(er) probability. Both short and longer term strategies are outlined which may help to make therapeutic angiogenesis (TA) work in the future.

Percutaneous methods of vector delivery in preclinical models

Cardiovascular disease remains a leading cause of hospitalization and mortality worldwide. Conventional heart failure treatment is making steady and substantial progress to reduce the burden of disease. Nevertheless novel therapies and especially cardiac gene therapy have been emerging in the past and successfully made their way into first clinical trials. Gene therapy was initially a visionary treatment strategy for inherited, monogenetic diseases but has now developed to have potential for polygenic diseases as atherosclerosis, arrhythmias and heart failure. These novel therapeutic strategies require testing in clinically relevant animal models to transition from 'bench to bedside'. One of the major hurdles for effective cardiovascular gene therapy is the delivery of the viral vectors to the heart. In this review we present the currently available vector-mediated cardiac gene delivery methods in vivo considering the specific merits and deficiencies.

Cardiac Gene Therapy: From Concept to Reality

Heart failure is increasing in incidence throughout the world, especially in industrialized countries. Although the current therapeutic modalities have been successful in stabilizing the course of heart failure, morbidity and mortality remain quite high and there remains a great need for innovative breakthroughs that will offer new treatment strategies for patients with advanced forms of the disease. The past few years have witnessed a greater understanding of the molecular underpinnings of the failing heart, paving the way for novel strategies in modulating the cellular environment. As such, gene therapy has recently emerged as a powerful tool offering the promise of a new paradigm for alleviating heart failure. Current gene therapy research for heart failure is focused on exploring potential cellular targets and preclinical and clinical studies are ongoing toward the realization of this goal. Efforts also include the development of sophisticated viral vectors and vector delivery methods for efficient transduction of cardiomyocytes.

Efficient transgene expression from naked DNA delivered into an arterio‐venous fistula model for kidney dialysis

Patients with kidney failure frequently require the formation of an arterio-venous fistula (AVF) in which a vein is connected to an artery resulting in arterialization of the vein to allow adequate blood flow into an external 'artificial kidney'. In most patients, neo-intimal hyperplasia (NIH) ensues, causing narrowing and subsequent occlusion of the vein, leading to significant morbidity. The cellular events causing venous NIH may serve as ideal targets for molecular-based therapies. However, therapeutic gene delivery into the vascular system is seriously impeded by problems related to the low efficacy and toxicity of targeted viral vector delivery. To explore the feasibility of a plasmid-based vascular gene delivery system, we established a rat AVF model that develops NIH. Plasmids encoding for reporter or therapeutic genes were delivered into the blood vessels at the time or after AVF formation. Intra-luminal injection of plasmid into the AVF resulted in extensive and long-term reporter gene expression at the venous limb mainly at the site of NIH formation. Transgene expression was confined to endothelial cells and myofibroblasts that migrate inwards from the adventitia and form the NIH lesion. There was no detrimental tissue reaction to plasmid delivery, contrasting with the severe inflammatory response observed after adenovirus infection. Intra-vascular delivery of a plasmid carrying the endothelial nitric oxide synthase gene resulted in sustained production of nitric oxide, previously shown to mitigate NIH formation. These findings open the possibility of vascular transduction with naked DNA bearing therapeutic genes in areas prone to NIH to ameliorate vein graft pathologies using simple and clinically applicable vector delivery methods.