Ultrasound-mediated Gene Delivery Research Articles

Development of a Nonviral Gene Therapy for X-linked Alport Syndrome by Targeted Transcutaneous Ultrasound-Mediated Gene Delivery

Development of a Nonviral Gene Therapy for X-linked Alport Syndrome by Targeted Transcutaneous Ultrasound-Mediated Gene Delivery

50-nm Gas-Filled Protein Nanostructures to Enable the Access of Lymphatic Cells by Ultrasound Technologies.

Ultrasound imaging and ultrasound-mediated gene and drug delivery are rapidly advancing diagnostic and therapeutic methods; however, their use is often limited by the need for microbubbles, which cannot transverse many biological barriers due to their large size. Here, the authors introduce 50-nm gas-filled protein nanostructures derived from genetically engineered gas vesicles(GVs)that are referred to as 50 nmGVs. These diamond-shaped nanostructures have hydrodynamic diameters smaller than commercially available 50-nm gold nanoparticles and are, to the authors' knowledge, the smallest stable, free-floating bubbles made to date. 50 nmGVs can be produced in bacteria, purified through centrifugation, and remain stable for months. Interstitially injected 50 nmGVs can extravasate into lymphatic tissues and gain access to critical immune cell populations, and electron microscopy images of lymph node tissues reveal their subcellular location in antigen-presenting cells adjacent to lymphocytes. The authors anticipate that 50 nmGVs can substantially broaden the range of cells accessible to current ultrasound technologies and may generate applications beyond biomedicine as ultrasmall stable gas-filled nanomaterials.

Ultrasound-mediated gene delivery specifically targets liver sinusoidal endothelial cells for sustained FVIII expression in hemophilia A mice

The ability to target the native production site of factor VIII (FVIII)— liver sinusoidal endothelial cells (LSECs)— can improve the outcome of hemophilia A (HA) gene therapy. By testing a matrix of ultrasound-mediated gene delivery (UMGD) parameters for delivering a GFP plasmid into the livers of HA mice, we were able to define specific conditions for targeted gene delivery to different cell types in the liver. Subsequently, two conditions were selected for experiments to treat HA mice via UMGD of an endothelial-specific human FVIII plasmid: low energy (LE; 50 W/cm2, 150μs PD) to predominantly target endothelial cells or high energy (HE; 110 W/cm2, 150 μs PD) to predominantly target hepatocytes. Both groups of UMGD-treated mice achieved persistent FVIII activity levels of ∼10% over 84 days post-treatment, however, half of the HE-treated mice developed low-titer inhibitors, while none of the LE mice did. Plasma transaminase levels and histological liver examinations revealed minimal transient liver damage that was lower in the LE group compared to the HE group. These results indicate that UMGD can safely target LSECs with a lower energy condition to achieve persistent FVIII gene expression. It demonstrates that this novel technology is highly promising for therapeutic correction of HA.

Synthesis of echogenic liposomes for sonoporation

Ultrasound contrast agents (UCAs), which are groups of engineered microbubbles, have been recently studied for drug delivery applications, since the cavitation of bubbles can increase the temporary permeability of nearby cells. However, the internal volume of UCAs is generally filled with gas, hence loading drug molecules into UCAs is limited. In this study, an echogenic liposome with a liquid and gas core is proposed as an alternative carrier of genetic material for ultrasound-mediated drug delivery. The structure of the synthesized echogenic liposome was analysed via transmission electron microscopy and confocal microscopy with fluorescent labels. The protection of siRNA by the echogenic liposomes was also verified by exposure to RNase. The results indicate that at least 10% of the total siRNA used in the experiment was successfully protected by the proposed echogenic liposome. Additionally, the release of siRNA from the liposomes could be successfully achieved with 1 W/cm2 ultrasound sonication at 1 MHz; parameters low enough to be used in generic ultrasound therapeutic systems. Although further studies to clarify the responses to incident ultrasound fields and to quantitatively analyse the internal liquid volume for drug loading are required, the proposed echogenic liposome has great potential for ultrasound-mediated gene delivery.

Non-invasive optogenetics with ultrasound-mediated gene delivery and red-light excitation

Optogenetics has revolutionized the capability of controlling genetically modified neurons in vitro and in vivo and has become an indispensable neuroscience tool. Using light as a probe for selective neuronal activation or inhibition and as a means to read out neural activity has dramatically enhanced our understanding of complex neural circuits. However, a common limitation of optogenetic studies to date is their invasiveness and spatiotemporal range. Direct viral injections into the brain tissue along with implantation of optical fibers and recording electrodes can disrupt the neuronal circuitry and cause significant damage. Conventional approaches are spatially limited around the site of the direct injection and insufficient in examining large networks throughout the brain. Lastly, optogenetics is currently not easily scalable to large animals or humans. Here, we demonstrate that optogenetic excitation can be achieved entirely non-invasively through the intact skull in mice. Using a needle-free combination of focused ultrasound-mediated viral delivery and extracorporeal illumination with red light, we achieved selective neuronal activation at depths up to 4 mm in the murine brain, confirmed through cFos expression and electrophysiology measurements within the treated areas. Ultrasound treatment significantly reduced freezing time during recall in fear conditioning experiments, but remote light exposure had a moderate effect on the freezing behavior of mice treated with viral vectors. The proposed method has the potential to open new avenues of studying, but also stimulating, neuronal networks, in an effort to elucidate normal or dysfunctional brain activity and treat neurological diseases. Finally, the same non-invasive methodology could be combined with gene therapy and applied to other organs, such as the eye and the heart.

Applications of Ultrasound-Mediated Gene Delivery in Regenerative Medicine.

Research on the capability of non-viral gene delivery systems to induce tissue regeneration is a continued effort as the current use of viral vectors can present with significant limitations. Despite initially showing lower gene transfection and gene expression efficiencies, non-viral delivery methods continue to be optimized to match that of their viral counterparts. Ultrasound-mediated gene transfer, referred to as sonoporation, occurs by the induction of transient membrane permeabilization and has been found to significantly increase the uptake and expression of DNA in cells across many organ systems. In addition, it offers a more favorable safety profile compared to other non-viral delivery methods. Studies have shown that microbubble-enhanced sonoporation can elicit significant tissue regeneration in both ectopic and disease models, including bone and vascular tissue regeneration. Despite this, no clinical trials on the use of sonoporation for tissue regeneration have been conducted, although current clinical trials using sonoporation for other indications suggest that the method is safe for use in the clinical setting. In this review, we describe the pre-clinical studies conducted thus far on the use of sonoporation for tissue regeneration. Further, the various techniques used to increase the effectiveness and duration of sonoporation-induced gene transfer, as well as the obstacles that may be currently hindering clinical translation, are explored.

Transcutaneous ultrasound-mediated gene delivery into canine livers achieves therapeutic levels of factor VIII expression

AbstractA safe, effective, and inclusive gene therapy will significantly benefit a large population of patients with hemophilia. We used a minimally invasive transcutaneous ultrasound-mediated gene delivery (UMGD) strategy combined with microbubbles (MBs) to enhance gene transfer into 4 canine livers. A mixture of high-expressing, liver-specific human factor VIII (hFVIII) plasmid and MBs was injected into the hepatic vein via balloon catheter under fluoroscopy guidance with simultaneous transcutaneous UMGD treatment targeting a specific liver lobe. Therapeutic levels of hFVIII expression were achieved in all 4 dogs, and hFVIII levels were maintained at a detectable level in 3 dogs throughout the 60-day experimental period. Plasmid copy numbers correlated with hFVIII antigen levels, and plasmid-derived messenger RNA (mRNA) was detected in treated livers. Liver transaminase levels and histology analysis indicated minimal liver damage and a rapid recovery after treatment. These results indicate that liver-targeted transcutaneous UMGD is promising as a clinically feasible therapy for hemophilia A and other diseases.

A novel ultrasound-mediated nanodroplet-based gene delivery system for osteoporosis treatment

This project aimed to develop, optimize, and test an ultrasound-responsive targeted nanodroplet system for the delivery of osteoporosis-related silencing gene Cathepsin K small interfering RNA (CTSK siRNA) for osteoporosis treatment. The nanodroplet (ND) is composed of a gas core made from perfluorocarbon, stabilized with albumin, encapsulated with CTSK siRNA, and embedded with alendronate (AL) for bone targeting (CTSK siRNA-ND-AL). Following the development, the responsiveness of CTSK siRNA-ND-AL to a therapeutic ultrasound probe was examined. The results of biocompatibility tests with human bone marrow-derived mesenchymal stem cells proved no significant cell death (P > 0.05). When the CTSK siRNA-ND-AL was supplemented with human osteoclast precursors, they suppressed osteoclastogenesis. Thus, this project establishes the potential of nanotechnology and ultrasound to deliver genes into the osteoclasts. This research also presents a novel ultrasound responsive and targeted nanodroplet platform that can be used as a gene and drug delivery system for various diseases including cancer.

Ultrasound-mediated gene delivery of factor VIII plasmids for hemophilia A gene therapy in mice

Gene therapy offers great promises for a cure of hemophilia A resulting from factor VIII (FVIII) gene deficiency. We have developed and optimized a non-viral ultrasound-mediated gene delivery (UMGD) strategy. UMGD of reporter plasmids targeting mice livers achieved high levels of transgene expression predominantly in hepatocytes. Following UMGD of a plasmid encoding human FVIII driven by a hepatocyte-specific promoter/enhancer (pHP-hF8/N6) into the livers of hemophilia A mice, a partial phenotypic correction was achieved in treated mice. In order to achieve persistent and therapeutic FVIII gene expression, we adopted a plasmid (pHP-hF8-X10) encoding an FVIII variant with significantly increased FVIII secretion. By employing an optimized pulse-train ultrasound condition and immunomodulation, the treated hemophilia A mice achieved 25%–150% of FVIII gene expression on days 1–7 with very mild transient liver damage, as indicated by a small increase of transaminase levels that returned to normal within 3 days. Therapeutic levels of FVIII can be maintained persistently without the generation of inhibitors in mice. These results indicate that UMGD can significantly enhance the efficiency of plasmid DNA transfer into the liver. They also demonstrate the potential of this novel technology to safely and effectively treat hemophilia A.

Ultrasound-mediated gene delivery into suspended plant cells using polyethyleneimine-coated mesoporous silica nanoparticles

Sonoporation, ultrasound-mediated membrane perforation can potentially puncture plasma membrane and rigid cell wall on presumably reversible basis which benefit gene transfection and plant biotechnology. Herein, positively charged poly-ethyleneimine (PEI)-coated mesoporous silica nanoparticles (MSNs) with an average diameter of 100 ± 8.7 nm was synthesized for GUS-encoding plasmid delivery into the suspended tobacco cells using the ultrasound treatment. The overall potential of PEI-MSN for DNA adsorption was measured at 43.43 μg DNA mg−1 PEI-MSNs. It was shown that high level of sonoporation may adversely upset the cell viability. Optimal conditions of ultrasonic treatment are obtained as 8 min at 3 various intensities of 160, 320 and 640 W. Histochemical staining assay was used to follow the protein expression. It was shown that PEI-coated MSNs efficiently transfer the GUS-encoding plasmid DNA into the tobacco cells. The results of this study showed that ultrasonic treatment provides an economical and straightforward approach for gene transferring into the plant cells without any need to complicated devices and concerns about safety issues.

Echogenic, Ultrasound-Sensitive Chitosan Nanodroplets for Spatiotemporally Controlled DKK-2 Gene Delivery to Prostate Cancer Cells.

PurposeTo synthesize echogenic chitosan/perfluorohexane nanodroplets (CNDs) for DKK-2 gene delivering in a spatiotemporally controlled manner in vitro.MethodsThe characteristics, contrast-enhanced ultrasound imaging, DNA binding and DNase protection capacity, DKK-2 gene transfection and effects on LNCaP cells of these CNDs were investigated.ResultsThe obtained CNDs showed positive surface charges and could attract the genetic cargo with negative surface charges to form nanocomplexes. Agarose gel electrophoresis confirmed binding of the CNDs and pDNA. DKK-2 pDNA-loaded CNDs, in combination with ultrasound, ruptured and released DKK-2 pDNA, entering LNCaP cells through nano-scale pores in the cell membrane, which further reduced the proliferation of LNCaP cells.ConclusionThese stable and safe CNDs may be a promising choice to achieve efficient ultrasound-mediated gene delivery to specific tissues in a spatiotemporally controlled manner.

Sonodelivery in Skeletal Muscle: Current Approaches and Future Potential.

There are currently multiple approaches to facilitate gene therapy via intramuscular gene delivery, such as electroporation, viral delivery, or direct DNA injection with or without polymeric carriers. Each of these methods has benefits, but each method also has shortcomings preventing it from being established as the ideal technique. A promising method, ultrasound-mediated gene delivery (or sonodelivery) is inexpensive, widely available, reusable, minimally invasive, and safe. Hurdles to utilizing sonodelivery include choosing from a large variety of conditions, which are often dependent on the equipment and/or research group, and moderate transfection efficiencies when compared to some other gene delivery methods. In this review, we provide a comprehensive look at the breadth of sonodelivery techniques for intramuscular gene delivery and suggest future directions for this continuously evolving field.

Echogenic PEGylated PEI-Loaded Microbubble As Efficient Gene Delivery System.

BackgroundCancer stem cells (CSCs) are responsible for cancer therapeutic resistance and metastasis. To date, in addition to surgery, chemotherapy, and radiotherapy, gene delivery has emerged as a potential therapeutic modality for ovarian cancer. Efficient and safe targeted gene delivery is complicated due to the tumor heterogeneity barrier. Ultrasound (US)-stimulated microbubbles (MBs) have demonstrated a method of enabling non-invasive targeted gene delivery.PurposeThe purpose of our study was to show the utility of poly(ethylene glycol)-SS-polyethylenimine-loaded microbubbles (PSP@MB) as an ultrasound theranostic and redox-responsive agent in a gene delivery system.Patients and methodsPSP nanoparticles were conjugated to the MB surface through biotin–avidin linkage, increasing the gene-loading efficiency of MB. The significant increase in the release of genes from the PSP@MB complexes was achieved upon ultrasound exposure. The positive surface charge in PSP@MB can condense the plasmid through electrostatic interactions; agarose-gel electrophoresis further confirmed the ability of PSP@MB to condense plasmids. The morphology, particle sizes and zeta potential of PSP@MB were characterized by transmission electron microscopy and dynamic light scattering.ResultsLaser confocal microscopy showed that the combination of ultrasound with PSP@MB could promote the cellular uptake of plasmids. Plasmids which encode enhanced green fluorescence protein (EGFP) reporter genes or luciferase reporter genes were delivered to CSCs in vitro and to subcutaneous xenografts in vivo via the combination of ultrasound with PSP@MB. Gene transfection efficiency was evaluated by fluorescence microscopy and In Vivo Imaging Systems. This study demonstrated that the combination of ultrasound with PSP@MB can remarkably promote gene delivery to solid tumors as well as diminishing the toxicity towards normal tissues in vivo. The combination of PSP@MB and the use of ultrasound can efficiently enhance accumulation, extravasation and penetration into solid tumors.ConclusionTaken together, our study showed that this novel PSP@MB and ultrasound-mediated gene delivery system could efficiently target CSCs.

Transcutaneous Ultrasound-Mediated Nonviral Gene Delivery to the Liver in a Porcine Model

Ultrasound (US)-mediated gene delivery (UMGD) of nonviral vectors was demonstrated in this study to be an effective method to transfer genes into the livers of large animals via a minimally invasive approach. We developed a transhepatic venous nonviral gene delivery protocol in combination with transcutaneous, therapeutic US (tUS) to facilitate significant gene transfer in pig livers. A balloon catheter was inserted into the pig hepatic veins of the target liver lobes via jugular vein access under fluoroscopic guidance. tUS exposure was continuously applied to the lobe with simultaneous infusion of pGL4 plasmid (encoding a luciferase reporter gene) and microbubbles. tUS was delivered via an unfocused, two-element disc transducer (H105) or a novel focused, single-element transducer (H114). We found applying transcutaneous US using H114 and H105 with longer pulses and reduced acoustic pressures resulted in an over 100-fold increase in luciferase activity relative to untreated lobes. We also showed effective UMGD by achieving focal regions of >105 relative light units (RLUs)/mg protein with minimal tissue damage, demonstrating the feasibility for clinical translation of this technique to treat patients with genetic diseases.

Nonviral ultrasound-mediated gene delivery in small and large animal models.

Ultrasound-mediated gene delivery (sonoporation) is a minimally invasive, nonviral and clinically translatable method of gene therapy. This method offers a favorable safety profile over that of viral vectors and is less invasive as compared with other physical gene delivery approaches (e.g., electroporation). We have previously used sonoporation to overexpress transgenes in different skeletal tissues in order to induce tissue regeneration. Here, we provide a protocol that could easily be adapted to address various other targets of tissue regeneration or additional applications, such as cancer and neurodegenerative diseases. This protocol describes how to prepare, conduct and optimize ultrasound-mediated gene delivery in both a murine and a porcine animal model. The protocol includes the preparation of a microbubble-DNA mix and in vivo sonoporation under ultrasound imaging. Ultrasound-mediated gene delivery can be accomplished within 10 min. After DNA delivery, animals can be followed to monitor gene expression, protein secretion and other transgene-specific outcomes, including tissue regeneration. This procedure can be accomplished by a competent graduate student or technician with prior experience in ultrasound imaging or in performing in vivo procedures.

Photothermal-Enhanced Phase-Transition Nanodroplets for Ultrasound-Mediated Diagnosis and Gene Transfection.

Gene therapy is one of the promising solutions in cancer therapeutics. Ultrasound-mediated gene delivery showed great potential as a noninvasive strategy for gene therapy. However, the efficiency of gene transfection and incorporation of multiple functions remain key challenges in the development of gene delivery systems. In this study, we developed perfluoropentane (PFP) and gold nanorods (AuNRs) loading nanodroplets for photothermal-enhanced ultrasound-mediated imaging and gene transfection. The nanodroplet theranostic system was formulated with fluorinated cationic poly(aspartamide) based polymer that encapsulated PFP, AuNRs, and plasmid DNA and was stabilized with a negatively charged poly(glutamic acid)-g-MeO-poly(ethylene glycol) (PGA-g-mPEG) coating. The nanodroplets presented good stability, biocompatibility, and DNA binding stability. Upon treatment with both near-infrared and ultrasound energy, the photothermal and ultrasound-responsive system exerted a synergistic effect, in which strong adsorption of light induced hyperthermia that promoted the phase transition of PFP and the following ultrasound irradiation, generating strong acoustic cavitation and sonoporation, thus leading to enhanced ultrasound contrast imaging and gene transfection efficiency both in vitro and in vivo.

Microbubbles for Nucleic Acid Delivery in Liver Using Mild Sonoporation.

Ultrasound-mediated gene delivery is an interesting approach, which could help in increasing gene transfer in deep tissues. Moreover, it allows for performing experiments guided by the image to determine which elements are required. Microbubbles complexed with a eukaryotic expression cassette are excellent agents as they are responsive to ultrasounds and, upon oscillation, can destabilize membranes to enhance gene transfer. Here, we describe the preparation of positively charged microbubbles, plasmid free of antibiotic resistance marker, their combination and the conditions of ultrasound-mediated liver transfection post-systemic administration in mice. This association allowed us to obtain a superior liver gene expression at least over 8months after a single injection.

Nucleic Acid Delivery System by the Combination of Lipid bubbles and Ultrasound

RNA interference (RNAi)-based therapy has gained attention because of its potent genesilencing effect and high specificity. However, the efficient delivery of nucleic acids to the target site is a major challenge to the clinical implementation. Recently, ultrasound-mediated gene delivery systems have been developed and attracted interest due to its safety and site-specificity. By the combination with contrast agents, called microbubbles, not only the delivery effects but also the imaging effects are significantly enhanced. We developed lipid bubbles (LBs) entrapping an ultrasound contrast gas to enhance the efficacy of ultrasound-mediated delivery and imaging. In this review, we summarize ultrasound-mediated nucleic acid delivery systems and discuss the possibility of combining LBs and ultrasound for RNAi-based therapies. We prepared polyethylene glycol-modified liposomes and entrapped an echo-contrast gas within the liposomes. Small interfering RNA (siRNA) were transfected into cells and muscles using LBs and ultrasound. Moreover, we also developed nucleic acid-loaded LBs using cholesterol-conjugated siRNA or positively-charged lipid for an efficient systemic delivery of siRNA and microRNA. The usability of LBs for RNA delivery system was evaluated by the silencing effects of target genes and the therapeutic effects on ischemia hind limb. A combination of LBs and therapeutic ultrasound was able to enhance the gene silencing effects by siRNA. Nucleic acid-loaded LBs were able to efficiently deliver siRNA or microRNA by systemic administration. A combination of LBs and diagnostic ultrasound also enhanced the imaging efficiency. Using a hindlimb ischemia mouse model, microRNA-loaded LBs could lead to increased angiogenic factors and improved blood flow. Ultrasound technology is widely used in clinical settings not only for diagnosis but also for therapy. Ultrasonic devices are being actively developed. Computer-controlled ultrasound systems can provide precise exposure to the target site. The combination of precise ultrasound exposure and LBs might be useful for target site-specific nucleic acids delivery, and holds potential to be developed into a beneficial therapeutic and diagnostic system for various diseases.

Echogenic Chitosan Nanodroplets for Spatiotemporally Controlled Gene Delivery.

To attain attractive ultrasound-responsive gene delivery, a new kind of echogenic chitosan nanodroplets (CND) was developed to explore the potential to deliver genes in a spatiotemporally controlled manner. Self-assembled amphiphilic chitosan micelles of nanoscale size were fabricated to encapsulate hydrophobic perfluoropentane into the inner cores. The resulting CND presented a positive surface charge, enabling the formation of nano-complexes with genetic cargo through electrostatic interactions. Agarose-gel electrophoresis further confirmed the ability of CND to bind DNA. CND was also observed to protect DNA from degradation by nucleases. A temperature-dependent droplet-to-bubble conversion was also demonstrated. More importantly, our study revealed that CND in combination with ultrasound could significantly enhance gene delivery. In conclusion, our study demonstrated a novel carrier with great potential for efficient ultrasound-mediated gene delivery to specific tissues in a spatiotemporally controlled manner.

Abstract 020: Regression of Atherosclerosis Through Manipulation of Vascular Macrophages; a Novel Gene-therapy Approach

Aggressive lipid lowering halts atherosclerotic plaque progression but does not lead to bulk plaque regression in humans. We have previously reported that gene transfection of the HDL protein apoAI into macrophages (MΦ-apoAI) decrease the rate of plaque development in atherosclerosis-prone mice. In this study, we hypothesized that MΦ-apoAI can promote regression of atherosclerosis synergistically with lipid-lowering. Atherogenic mice (LDLR -/- and LDLR -/- /MΦ-apoAI) were fed a high-fat diet to promote stage II/III atherosclerotic lesions (as baseline comparator), and then switched to an extreme lipid-lowering intervention (chow with MTTP inhibitor). After 3 weeks on the lipid-lowering diet, both groups showed reduced systemic and lesion inflammation, and reduced macrophage content on histology when compared to baseline. Upon lipid-lowering, vascular ultrasound showed that LDLR -/- /MΦ-apoAI mice has 21% improvement in aortic pulse transit time, and a 37.1% improvement in instantaneous aortic compliance (a surrogate marker for arterial elastic modulus), compared to LDLR -/- mice. Histologic evidence showed that LDLR -/- /MΦ-apoAI mice on lipid-lowering diet also had reduced lesion size (-24%), reduced macrophage content (-27.5%), increased M2/M1 ratio (+51%) and reduced necrotic core area (-28.6%), compared to LDLR -/- mice. Using transplantation of bone marrow cells from MΦ-apoAI mice into recipient mice with established lesions, we show that newly recruited cells are not major contributors to the regression afforded by MΦ-apoAI. Thus, to study the kinetics of pre-existing cells in the lesion during regression, we developed a contrast ultrasound-mediated gene delivery system to tag and trace lesion cells in LDLR -/- mice, which revealed that lesion macrophages expressing apoAI specifically migrate to mediastinal LN in a CCR7-dependent manner during regression. Expression of apoAI in pre-existing lesion macrophages, promotes regression of atherosclerosis beyond lipid-lowering alone, and increases CCR7-dependent egress of macrophages to adjacent lymph nodes. Ultrasound-mediated gene delivery can be a useful device to study the kinetics of cell in the artery wall and also represent a novel approach with application to human therapy.