Treatment of Duschenne Muscular Dystrophy with exon skipping antisense oligonucleotides using novel polyethylenimine carriers

Duchenne muscular dystrophy (DMD) is a lethal disorder that is caused by mutations in the gene encoding the dystrophin protein. It has been established that exon skipping oligonucleotides (ESOs) are able to ameliorate the symptoms of DMD by modulating dystrophin pre-mRNA transcript splicing resulting in removal of the mutated exon from the mRNA transcript. It is estimated that up to 75% of DMD cases may be treatable using this ESO-based approach. However, the main obstacle that has limited the usefulness of ESOs in treatment of DMD, and most other diseases, is a lack of adequate carriers that enable ESO delivery to the target cell nuclei. Thus, for the ESO-based approach to ultimately provide clinical benefit in treating DMD, it is essential that new compounds be developed that improve the efficiency of ESO delivery to myonuclei.In this project, amine rich poly(ethylene imine) (PEI) based compounds were developed to improve the delivery of ESO’s to myonuclei of dystrophin-null muscle and recover the dystrophin protein. PEI is considered one of the most potent non-viral agents for nucleotide delivery; however, the feasibility of PEI-based compounds for the transfection of mature skeletal muscle has not been established. Alone, PEI is considered to be a cytotoxic compound, but the biocompatibility of PEI is significantly improved by incorporating nonionic linear polyethylene glycol (PEG) polymers into PEGPEI copolymers. This project demonstrates that PEG-PEI compounds are potent and versatile vectors capable of delivering ESO’s to skeletal muscle in vitro and in vivo. The PEG-PEI based compounds developed in this work serve as a platform for developing even more potent “next generation” PEG-PEI compounds that have a realistic potential for treatment of DMD.Ph.D., Biomedical Engineering -- Drexel University, 200

Formulation of polylactide-co-glycolic acid nanospheres for encapsulation and sustained release of poly(ethylene imine)-poly(ethylene glycol) copolymers complexed to oligonucleotides

Antisense oligonucleotides (AOs) have been shown to induce dystrophin expression in muscles cells of patients with Duchenne Muscular Dystrophy (DMD) and in the mdx mouse, the murine model of DMD. However, ineffective delivery of AOs limits their therapeutic potential. Copolymers of cationic poly(ethylene imine) (PEI) and non-ionic poly(ethylene glycol) (PEG) form stable nanoparticles when complexed with AOs, but the positive surface charge on the resultant PEG-PEI-AO nanoparticles limits their biodistribution. We adapted a modified double emulsion procedure for encapsulating PEG-PEI-AO polyplexes into degradable polylactide-co-glycolic acid (PLGA) nanospheres. Formulation parameters were varied including PLGA molecular weight, ester end-capping, and sonication energy/volume. Our results showed successful encapsulation of PEG-PEI-AO within PLGA nanospheres with average diameters ranging from 215 to 240 nm. Encapsulation efficiency ranged from 60 to 100%, and zeta potential measurements confirmed shielding of the PEG-PEI-AO cationic charge. Kinetic measurements of 17 kDa PLGA showed a rapid burst release of about 20% of the PEG-PEI-AO, followed by sustained release of up to 65% over three weeks. To evaluate functionality, PEG-PEI-AO polyplexes were loaded into PLGA nanospheres using an AO that is known to induce dystrophin expression in dystrophic mdx mice. Intramuscular injections of this compound into mdx mice resulted in over 300 dystrophin-positive muscle fibers distributed throughout the muscle cross-sections, approximately 3.4 times greater than for injections of AO alone. We conclude that PLGA nanospheres are effective compounds for the sustained release of PEG-PEI-AO polyplexes in skeletal muscle and concomitant expression of dystrophin, and may have translational potential in treating DMD

Efficient Nuclear Delivery and Nuclear Body Localization of Antisense Oligo-Nucleotides using Degradable Polymersomes

Delivery of antisense oligonucleotides, AON, presents many of the same challenges as delivery of any nucleic acid: charge, stability, cell uptake, endolysosomal escape, and entry into the nucleus. Here we demonstrate efficient delivery of AON after loading into biodegradable polymer vesicles or \u27polymersomes\u27. We focus on AON delivery to muscle cells in vitro and in vivo because of the emergence of AON in therapeutic strategies directed at muscular dystrophies. To first clarify uptake kinetics without the complications of typical multi-layered myotube cultures, we use micro-patterned C2C12 cells and show efficient uptake of AON-polymersomes. The biodegradable polymersomes break down and foster AON escape with the binding of fluorescent-AON into the nuclear bodies. Intramuscular injections of the polymersome-AON into the hind limbs of mdx-dystrophic mice show more efficient nuclear uptake than AON alone and also lead to dystrophin expression in the mdx mice. In sum, these neutral, degradable carriers of AON show promise in vivo

Perfusion-guided sonopermeation of neuroblastoma: a novel strategy for monitoring and predicting liposomal doxorubicin uptake

Neuroblastoma (NB) is the most common extracranial solid tumor in infants and children, and imposes significant morbidity and mortality in this population. The aggressive chemoradiotherapy required to treat high-risk NB results in survival of less than 50%, yet is associated with significant long-term adverse effects in survivors. Boosting efficacy and reducing morbidity are therefore key goals of treatment for affected children. We hypothesize that these may be achieved by developing strategies that both focus and limit toxic therapies to the region of the tumor. One such strategy is the use of targeted image-guided drug delivery (IGDD), which is growing in popularity in personalized therapy to simultaneously improve on-target drug deposition and assess drug pharmacodynamics in individual patients. IGDD strategies can utilize a variety of imaging modalities and methods of actively targeting pharmaceutical drugs, however in vivo imaging in combination with focused ultrasound is one of the most promising approaches already being deployed for clinical applications. Over the last two decades, IGDD using focused ultrasound with microbubble ultrasound contrast agents (UCAs) has been increasingly explored as a method of targeting a wide variety of diseases, including cancer. This technique, known as sonopermeation, mechanically augments vascular permeability, enabling increased penetration of drugs into target tissue. However, to date, methods of monitoring the vascular bioeffects of sonopermeation in vivo are lacking. UCAs are excellent vascular probes in contrast-enhanced ultrasound (CEUS) imaging, and are thus uniquely suited for monitoring the effects of sonopermeation in tumors. Methods: To monitor the therapeutic efficacy of sonopermeation in vivo, we developed a novel system using 2D and 3D quantitative contrast-enhanced ultrasound imaging (qCEUS). 3D tumor volume and contrast enhancement was used to evaluate changes in blood volume during sonopermeation. 2D qCEUS-derived time-intensity curves (TICs) were used to assess reperfusion rates following sonopermeation therapy. Intratumoral doxorubicin (and liposome) uptake in NB was evalauted ex vivo along with associated vascular changes. Results: In this study, we demonstrate that combining focused ultrasound therapy with UCAs can significantly enhance chemotherapeutic payload to NB in an orthotopic xenograft model, by improving delivery and tumoral uptake of long-circulating liposomal doxorubicin (L-DOX) nanoparticles. qCEUS imaging suggests that changes in flow rates are highly sensitive to sonopermeation and could be used to monitor the efficacy of treatment in vivo. Additionally, initial tumor perfusion may be a good predictor of drug uptake during sonopermeation. Following sonopermeation treatment, vascular biomarkers show increased permeability due to reduced pericyte coverage and rapid onset of doxorubicin-induced apoptosis of NB cells but without damage to blood vessels. Conclusion: Our results suggest that significant L-DOX uptake can occur by increasing tumor vascular permeability with microbubble sonopermeation without otherwise damaging the vasculature, as confirmed by in vivo qCEUS imaging and ex vivo analysis. The use of qCEUS imaging to monitor sonopermeation efficiency and predict drug uptake could potentially provide real-time feedback to clinicians for determining treatment efficacy in tumors, leading to better and more efficient personalized therapies. Finally, we demonstrate how the IGDD strategy outlined in this study could be implemented in human patients using a single case study

The Evolution and Recent Trends in Acoustic Targeting of Encapsulated Drugs to Solid Tumors: Strategies beyond Sonoporation

Despite recent advancements in ultrasound-mediated drug delivery and the remarkable success observed in pre-clinical studies, no delivery platform utilizing ultrasound contrast agents has yet received FDA approval. The sonoporation effect was a game-changing discovery with a promising future in clinical settings. Various clinical trials are underway to assess sonoporation’s efficacy in treating solid tumors; however, there are disagreements on its applicability to the broader population due to long-term safety issues. In this review, we first discuss how acoustic targeting of drugs gained importance in cancer pharmaceutics. Then, we discuss ultrasound-targeting strategies that have been less explored yet hold a promising future. We aim to shed light on recent innovations in ultrasound-based drug delivery including newer designs of ultrasound-sensitive particles specifically tailored for pharmaceutical usage

Advances in Ultrasound Mediated Gene Therapy Using Microbubble Contrast Agents

<p>Microbubble ultrasound contrast agents have the potential to dramatically improve gene therapy treatments by enhancing the delivery of therapeutic DNA to malignant tissue. The physical response of microbubbles in an ultrasound field can mechanically perturb blood vessel walls and cell membranes, enhancing drug permeability into malignant tissue. In this review, we discuss literature that provided evidence of specific mechanisms that enhance <i>in vivo</i> gene delivery utilizing microbubble contrast agents, namely their ability to 1) improving cell membrane permeability, 2) modulate vascular permeability, and 3) enhance endocytotic uptake in cells. Additionally, we review novel microbubble vectors that are being developed in order to exploit these mechanisms and deliver higher gene payloads with greater target specificity. Finally, we discuss some future considerations that should be addressed in the development of next-generation microbubbles in order to improve <i>in vivo</i> microbubble gene delivery. Overall, microbubbles are rapidly gaining popularity as efficient gene carriers, and combined with their functionality as imaging contrast agents, they represent powerful theranostic tools for image guided gene therapy applications.</p

Cavitation-Enhanced Drug Delivery and Immunotherapy

Welcome to this special issue on Cavitation-Enhanced Drug Delivery and Immunotherapy—a rapidly evolving area that has been buoyed in recent years by the development of methods harnessing the activity of ultrasound-stimulated bubbles known as cavitation [...