Gene Therapy Delivery Research Articles

Plasmid Gene Therapy for Monogenic Disorders: Challenges and Perspectives

Monogenic disorders are a group of human diseases caused by mutations in single genes. While some disease-altering treatments offer relief and slow the progression of certain conditions, the majority of monogenic disorders still lack effective therapies. In recent years, gene therapy has appeared as a promising approach for addressing genetic disorders. However, despite advancements in gene manipulation tools and delivery systems, several challenges remain unresolved, including inefficient delivery, lack of sustained expression, immunogenicity, toxicity, capacity limitations, genomic integration risks, and limited tissue specificity. This review provides an overview of the plasmid-based gene therapy techniques and delivery methods currently employed for monogenic diseases, highlighting the challenges they face and exploring potential strategies to overcome these barriers.

Present insights into the progress in gene therapy delivery systems for central nervous system diseases.

Central nervous system (CNS) diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), spinal cord injury (SCI), and ischemic strokes and certain rare diseases, such as amyotrophic lateral sclerosis (ALS) and ataxia, present significant obstacles to treatment using conventional molecular pharmaceuticals. Gene therapy, with its ability to target previously "undruggable" proteins with high specificity and safety, is increasingly utilized in both preclinical and clinical research for CNS ailments. As our comprehension of the pathophysiology of these conditions deepens, gene therapy stands out as a versatile and promising strategy with the potential to both prevent and treat these diseases. Despite the remarkable progress in refining and enhancing the structural design of gene therapy agents, substantial obstacles persist in their effective and safe delivery within living systems. To surmount these obstacles, a diverse array of gene delivery systems has been devised and continuously improved. Notably, Adeno-Associated Virus (AAVs)-based viral gene vectors and lipid-based nanocarriers have each advanced the in vivo delivery of gene therapies to various extents. This review aims to concisely summarize the pathophysiological foundations of CNS diseases and to shed light on the latest advancements in gene delivery vector technologies. It discusses the primary categories of these vectors, their respective advantages and limitations, and their specialized uses in the context of gene therapy delivery.

Purification of AAV8 through a scalable two-step monolithic chromatography approach.

Adeno-associated viruses (AAV) are becoming increasingly popular as a powerful tool for gene delivery therapy applications. Although processes to produce AAV are established, future demand for this type of viral vector requires further development of manufacturing processes to make them more robust, scalable, and flexible to accommodate the rise of engineered capsids. This study focuses on designing and evaluating a two-step chromatography process for capturing and polishing AAV8 using monolith chromatography media. A cation-exchange-based capture step was established, using CIMmultus® SO3, resulting in a virus recovery of approximately 70 % of total capsids. This step also achieved high protein removal (>95 %) and considerable DNA clearance (>80 %). For the polishing step, three different CIMmultus® monoliths were evaluated: anion-exchange-based QA (strong anion exchange), multimodal-based PrimaS (weak anion exchange and hydrogen bonding) and multimodal-based PrimaT (weak anion exchange, hydrogen bonding and metal affinity) ligands. High viral vector genome recoveries, DNA and protein clearance and a three-fold full capsid enrichment were observed at a 1 mL column scale. Similar results were obtained during a scale-up to 8 mL and 4 mL monolith volumes for capture and polishing, respectively. These results provide a strong and robust alternative to conventional AAV purification methods, such as resin-based affinity chromatography and density gradient ultracentrifugation.

Conventional and Tropism-Modified High-Capacity Adenoviral Vectors Exhibit Similar Transduction Profiles in Human iPSC-Derived Retinal Organoids

Viral vector delivery of gene therapy represents a promising approach for the treatment of numerous retinal diseases. Adeno-associated viral vectors (AAV) constitute the primary gene delivery platform; however, their limited cargo capacity restricts the delivery of several clinically relevant retinal genes. In this study, we explore the feasibility of employing high-capacity adenoviral vectors (HC-AdVs) as alternative delivery vehicles, which, with a capacity of up to 36 kb, can potentially accommodate all known retinal gene coding sequences. We utilized HC-AdVs based on the classical adenoviral type 5 (AdV5) and on a fiber-modified AdV5.F50 version, both engineered to deliver a 29.6 kb vector genome encoding a fluorescent reporter construct. The tropism of these HC-AdVs was evaluated in an induced pluripotent stem cell (iPSC)-derived human retinal organoid model. Both vector types demonstrated robust transduction efficiency, with sustained transgene expression observed for up to 110 days post-transduction. Moreover, we found efficient transduction of photoreceptors and Müller glial cells, without evidence of reactive gliosis or loss of photoreceptor cell nuclei. However, an increase in the thickness of the photoreceptor outer nuclear layer was observed at 110 days post-transduction, suggesting potential unfavorable effects on Müller glial or photoreceptor cells associated with HC-AdV transduction and/or long-term reporter overexpression. These findings suggest that while HC-AdVs show promise for large retinal gene delivery, further investigations are required to assess their long-term safety and efficacy.

Mitochondrial-targeted therapies in traumatic brain injury: From bench to bedside.

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide, with limited effective therapeutic options currently available. Recent research has highlighted the pivotal role of mitochondrial dysfunction in the pathophysiology of TBI, making mitochondria an attractive target for therapeutic intervention. This review comprehensively examines advancements in mitochondrial-targeted therapies for TBI, bridging the gap from basic research to clinical applications. We discuss the underlying mechanisms of mitochondrial damage in TBI, including oxidative stress, impaired bioenergetics, mitochondrial dynamics, and apoptotic pathways. Furthermore, we highlight the complex interplay between mitochondrial dysfunction, inflammation, and blood-brain barrier (BBB) integrity, elucidating how these interactions exacerbate injury and impede recovery. We also evaluate various preclinical studies exploring pharmacological agents, gene therapy, and novel drug delivery systems designed to protect and restore mitochondrial function. Clinical trials and their outcomes are assessed to evaluate the translational potential of mitochondrial-targeted therapies in TBI. By integrating findings from benchto bedside, this review emphasizes promising therapeutic avenues and addresses remaining challenges. It also provides guidance for future research to pave the way for innovative treatments that improve patient outcomes in TBI.

UKHCDO gene therapy taskforce: Guidance for implementation of haemophilia gene therapy into routine clinical practice for adults.

2022 was a landmark year with two adeno-associated viral vectors (AAVs) receiving conditional marketing authorization from EMA for the treatment of persons with severe haemophilia A and severe to moderately severe haemophilia B and a third in 2024. Gene therapy is a transformative, irreversible treatment with long-lasting effects, necessitating development of new clinical pathways to ensure optimal outcomes. To develop a consensus framework and service specification for delivery of AAV gene therapy for haemophilia in adults within the UK using the hub-and-spoke model proposed by the European Association of Haemophilia and Allied Disorders and the European Haemophilia Consortium. The UK Haemophilia Centre Doctors Organisation (UKHCDO) set up a working party to develop expert consensus guidance, working with NHS England to ensure alignment with NHS England commissioning and the national service specification. These guidelines detail the patient pathway, counselling and governance requirements for the hub-and-spoke model. The national service specification requires the hub site to manage governance for AAV-based gene therapy. Proposed regional and national multidisciplinary teams will harmonize clinical practices incorporating expertise from various specialities and professional groups. Key requirements identified include standardized documentation and multidisciplinary collaboration. Nationally agreed patient information and counselling checklists will streamline the informed consent process and facilitate data collection for long-term safety and efficacy monitoring. These guidelines provide a structured framework for the delivery of liver-directed gene therapy. Whilst specific to the United Kingdom they provide a framework for the implementation of gene therapy in other countries for haemophilia and other monogenic disorders.

New MiniPromoter Ple389 (ADORA2A) drives selective expression in medium spiny neurons in mice and non-human primates

Compact cell type-specific promoters are important tools for basic and preclinical research and clinical delivery of gene therapy. In this work, we designed novel MiniPromoters to target D1 and D2 type dopaminoceptive medium spiny neurons in the striatum by manually identifying candidate regulatory regions or employing the OnTarget webserver. We then empirically tested the designs in rAAV-PHP.B for specificity and robustness in three systems: intravenous injection in mice, intracerebroventricular injection in mice, and intracerebroventricular injection in non-human primates. Twelve MiniPromoters were designed from eight genes: seven manually and five using OnTarget. When delivered intravenously in mice, three MiniPromoters demonstrated highly selective expression in the striatum, with Ple389 (ADORA2A) showing high levels of dopamine D2-receptor cell co-localization. The same three MiniPromoters also displayed enriched expression in the striatum when delivered intracerebroventricularly in mice with high levels of DARPP32 co-localization. Finally, Ple389 (ADORA2A) was intracerebroventricularly injected in non-human primates and showed enriched expression in the striatum as in the mouse. Ple389 (ADORA2A) demonstrated expression in the medium spiny neurons in all three systems tested and exhibited the highest level of D2-MSNs and DARPP32 co-labeling in mice, demonstrating its potential as a tool for gene therapy approaches for Parkinson and Huntington disease treatment.

Brief Comparison of the Efficacy of Cationic and Anionic Liposomes as Nonviral Delivery Systems.

In recent decades, the development and application of nonviral vectors, such as liposomes and lipidic nanoparticles, for gene therapy and drug delivery have seen substantial progress. The interest in the physicochemical properties and structures of the complexes liposome/DNA and liposome/RNA is due to their potential to substitute viruses as carriers of drugs or genetic material into cells with minimal cytotoxicity, which could lead to their use in gene therapy. Initially, cationic liposomes were utilized as nonviral DNA delivery vectors; subsequently, different molecules, such as polymers, were incorporated to enhance transfection efficiency. Additionally, liposome/protein complexes have been developed as nonviral vectors for the treatment of diseases. The most relevant internalization pathways of these vectors and the few transfection results obtained using targeted and nontargeted liposomes are discussed below. The high cytotoxicity of cationic liposomes represents a significant challenge for the development of gene therapy and drug delivery. Anionic liposomes offer a promising alternative to address the limitations of conventional cationic liposomes, including immune response, short circulation time, and low toxicity. This review will discuss the advantages of cationic liposomes and the novel anionic liposome-based systems that have emerged as a result. The advent of novel designs and manufacturing techniques has facilitated the development of innovative systems, designated as lipid nanoparticles (LNPs), which serve as highly efficacious regulators of the immune system.

Advancements in p53-Based Anti-Tumor Gene Therapy Research.

The p53 gene is one of the genes most closely associated with human tumors and has become a popular target for tumor drug design. Currently, p53-based gene therapy techniques have been developed, but these therapies face challenges such as immaturity, high safety hazards, limited efficacy, and low patient acceptance. However, researchers are no less enthusiastic about the treatment because of its theoretical potential to treat cancer. In this paper, the advances in p53-based gene therapy and related nucleic acid delivery technologies were reviewed and prospected in order to support further development in this field.

Gene Therapy in Cardiovascular Disease: Recent Advances and Future Directions in Science: A Science Advisory From the American Heart Association.

Cardiovascular disease remains the foremost cause of morbidity and mortality globally, affecting millions of individuals. Recent discoveries illuminate the substantial role of genetics in cardiovascular disease pathogenesis, encompassing both monogenic and polygenic mechanisms and identifying tangible targets for gene therapies. Innovative strategies have emerged to rectify pathogenic variants that cause monogenic disorders such as hypertrophic, dilated, and arrhythmogenic cardiomyopathies and hypercholesterolemia. These include delivery of exogenous genes to supplement insufficient protein levels caused by pathogenic variants or genome editing to correct, delete, or modify mutant sequences to restore protein function. However, effective delivery of gene therapy to specified cells presents formidable challenges. Viral vectors, notably adeno-associated viruses and nonviral vectors such as lipid and engineered nanoparticles, offer distinct advantages and limitations. Additional risks and obstacles remain, including treatment durability, tissue-specific targeting, vector-associated adverse events, and off-target effects. Addressing these challenges is an ongoing imperative; several clinical gene therapy trials are underway, and many more first-in-human studies are anticipated. This science advisory reviews core concepts of gene therapy, key obstacles, patient risks, and ongoing research endeavors to enable clinicians to understand the complex landscape of this emerging therapy and its remarkable therapeutic potential to benefit cardiovascular disease.

Placental gene therapy in nonhuman primates: a pilot study of maternal, placental, and fetal response to non-viral, polymeric nanoparticle delivery of IGF1.

Currently, there are no placenta-targeted treatments to alter the in utero environment for administration to pregnant women who receive a diagnosis of fetal growth restriction (FGR). Water-soluble polymers have a distinguished record of clinical relevance outside of pregnancy. We have demonstrated the effective delivery of polymer-based nanoparticles containing a non-viral human insulin-like growth factor 1 (IGF1) transgene to correct placental insufficiency in small animal models of FGR. Our goals were to extend these studies to a proof-of-concept study in the pregnant macaque, establish feasibility of nanoparticle-mediated gene therapy delivery to trophoblasts, and investigate the acute maternal, placental, and fetal responses to treatment. Pregnant macaques underwent ultrasound-guided intraplacental injections of nanoparticles (GFP- or IGF1-expressing plasmid under the control of the trophoblast-specific PLAC1 promoter complexed with a HPMA-DMEAMA co-polymer) at approximately gestational day 100 (term = 165 days). Fetectomy was performed 24 h (GFP; n = 1), 48 h (IGF1; n = 3) or 10 days (IGF1; n = 3) after nanoparticle delivery. Routine pathological assessment was performed on biopsied maternal tissues and placental and fetal tissues. Maternal blood was analyzed for complete blood count (CBC), immunomodulatory proteins and growth factors, progesterone (P4), and estradiol (E2). Placental ERK/AKT/mTOR signaling was assessed using Western blot and qPCR. Fluorescent microscopy and in situ hybridization confirmed placental uptake and transient transgene expression in villous syncytiotrophoblast. No off-target expression was observed in either maternal or fetal tissues. Histopathological assessment of the placenta recorded observations not necessarily related to the IGF1 nanoparticle treatment. In maternal blood, CBCs, P4, and E2 remained within the normal range for pregnant macaques across the treatment period. Changes to placental ERK and AKT signaling at 48 h and 10 days after IGF1 nanoparticle treatment indicated an upregulation in placental homeostatic mechanisms to prevent overactivity in the normal pregnancy environment. The lack of adverse maternal reaction to nanoparticle-mediated IGF1 treatment, combined with changes in placental signaling to maintain homeostasis, indicates no deleterious impact of treatment during the acute phase of study.

Applications and Developments of Gene Therapy Drug Delivery Systems for Neurological Disorders

AbstractNeurological diseases (NDs) such as Alzheimer's disease, Parkinson's disease, ischemic strokes, spinal cord injuries, and other similar conditions that continue to pose a substantial health and economic burden on a global scale. It is crucial to tackle the difficulties provided by current medications due to the adverse effects and its immunological reactions to develop improved treatments for neurodegenerative illnesses. Gene therapy is currently being extensively used in preclinical and clinical studies for various diseases because of its ability to enhance the delivery and effectiveness of treatments. Various gene delivery techniques, including messenger RNA, small interfering RNA, antisense oligonucleotides, microRNA, CRISPR/Cas9 system, and plasmid DNA, have been created to address these difficulties. The goal of this study is to provide a clear overview of the pathophysiological underpinnings of NDs illnesses while also illuminating recent developments in gene delivery vector technologies. It goes over the main classifications of these vectors, their individual benefits and drawbacks, and their specific applications in the delivery of gene therapy.

Neuroimaging Applications for the Delivery and Monitoring of Gene Therapy for Central Nervous System Diseases.

Neurological disease due to single-gene defects represents a targetable entity for adeno-associated virus (AAV)-mediated gene therapy. The delivery of AAV-mediated gene therapy to the brain is challenging, owing to the presence of the blood-brain barrier. Techniques in gene transfer, such as convection-enhanced intraparenchymal delivery and image-guided delivery to the cerebrospinal fluid spaces of the brain, have led the field into highly accurate delivery techniques, which provide correction of genetic defects in specific brain regions or more broadly. These techniques commonly use magnetic resonance imaging (MRI), computed tomography, and fluoroscopic guidance. Even more, the neuroimaging changes evaluated by MRI, MR spectroscopy, diffusion tensor imaging, and functional MRI can serve as important biomarkers of therapy effect and overall disease progression. Here, we discuss the role of neuroimaging in delivering AAV vectors and monitoring the effect of gene therapy.

Advances in glial cell line-derived neurotrophic factor

Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor with significant research value due to its profound effects on dopaminergic, cholinergic, and motor neurons. Initially identified for its neuroprotective role in dopaminergic neurons, GDNF has been found to support various neuronal populations and plays a crucial role in neural development, maintenance, and repair. This review provides a comprehensive overview of the molecular characteristics, tissue distribution, physiological function of GDNF and its protective effect on a series of diseases, emphasizing its potential therapeutic applications. Meanwhile, this study discusses the challenges in delivering GDNF across the blood-brain barrier and explores current strategies to enhance its clinical efficacy, including the use of gene therapy and innovative delivery methods. In summary, the review underscores the promise of GDNF as a therapeutic agent for neurodegenerative diseases and nerve injuries, highlighting the need for further research to translate these findings into clinical practice.

Identification of a novel neutralization epitope in rhesus AAVs

Adeno-associated viruses (AAVs) are popular gene therapy delivery vectors, but their application can be limited by anti-vector immunity. Both preexisting neutralizing antibodies (NAbs) and post-administration NAbs can limit transgene expression and reduce the clinical utility of AAVs. The development of novel AAVs will advance our understanding of AAV immunity and may also have practical applications. In this study, we identified five novel AAV capsids from rhesus macaques. RhAAV4282 exhibited 91.4% capsid sequence similarity with AAV7 and showed similar tissue tropism with slightly diminished overall signal. Despite this sequence homology, RhAAV4282 and AAV7 showed limited cross-neutralization. We determined a cryo-EM structure of the RhAAV4282 capsid at 2.57Å resolution and identified a small segment within the hypervariable region IV, involving seven amino acids that formed a shortened external loop in RhAAV4282 compared with AAV7. We generated RhAAV4282 and AAV7 mutants that involved swaps of this region and showed that this region partially determined neutralization phenotype. We termed thisregion the hypervariable region IV neutralizing epitope (HRNE). Our data suggests that modification of the HRNE can lead to AAVs with altered neutralization profiles.

Advancements of Nano-biotechnology in Public Health Sector: Benefits and Challenges

Nano-biotechnology is the discipline of creating and using nanoscale devices to study biological sciences. It has become an important frontier in many important areas, especially in the treatment of diseases and drug use. Nano-biotechnology has great potential to improve biological research and thus support global health care. Many new nanodevices and nanoparticles will be used to benefit human health in the future. Healthcare as a human right often becomes the focus of technological innovations. Technological advances have made it easier to deliver goods efficiently, on time, reliable and cost-effectively. Advances in nanoscience facilitated the expansion of new generations of nanostructures. They all have unique features that make their applications perfect. Nanotechnology has continued to impact health since its inception and has had a significant impact on the evolution of health, leading to better outcomes. Over the last 20 years, the world has witnessed the advancement of nanotechnology into living spaces, with the influence of many research studies in various medical fields. The arena of nanomedicine emerges as the amalgamation of nanotechnology along with nanocarriers/nanosystems in the medical field, like in the prevention, prognosis and prophylaxis of various infectious diseases. It has been noticed that in the diagnosis and therapeutics, advanced nanosystems have been found to be more appropriate in comparison to conventional ones. The present review mainly highlights the merits and demerits of nanosystems in the areas of cancer management, gene therapy, genetic diseases, and drug delivery. Though nanotechnology has great potential, it is still underutilized. Further efforts are needed to overcome these limitations and exploit the potential to reform the healthcare sector in the future.

Kidney-Targeting Mesoscale Nanoparticles for Delivery of Gene Therapy in CKD

Kidney-Targeting Mesoscale Nanoparticles for Delivery of Gene Therapy in CKD

Vesiclemania: ILGD INSTAND 2024 - A German extravaganza of tiny packages!

The 2024 IGLD INSTAND Symposium brought together leading experts in EV research. Key themes included the importance of methodological rigor and standardization, emphasized through the latest MISEV 2023 guidelines, and advances in EV isolation technologies. Several presentations explored the clinical applications of EVs, including their use in stem cell treatments, liquid biopsies, and as gene therapy delivery systems using EV-mediated CRISPR/Cas9. Discussions also focused on the interplay between EVs and the immune system, such as their role in modulating viral infections and their therapeutic potential in pathological situations and cancer. In conclusion, the event emphasized the need for inter-laboratory reproducibility and collaborative research to ensure EV technologies are primed for clinical translation, underscoring the importance of collaborative efforts to drive innovations in the field.

Nanodiamond: A Promising Carbon‐Based Nanomaterial for Therapeutic and Regenerative Dental Applications

AbstractNanodiamonds (NDs) are utilized for various purposes in dentistry, such as tissue regeneration, gene therapy, and medication delivery. Despite the examination of their potential for dental uses, there is currently no published review summarizing the conducted investigations. Additional investigations are required to comprehend the biological compatibility of NDs in dentistry. NDs have been proven effective for a variety of applications in dentistry because of unique qualities such as target cell selectivity, nanometer size, and fluorescence. Nanodiamonds find various applications in dentistry, encompassing roles in directed tissue regeneration, reinforcing polymers, and administering medications for treating infections and cancer. Researchers have also suggested the potential use of nanodiamonds as coatings for antibacterial or bioactive dental implants. Nevertheless, investigations on their biological compatibility have been scarce and insufficient so far, necessitating significantly further studies to envision the potential use of NDs in dentistry. It is believed that within the ten years to come, these emerging substances will discover several kinds of daily usage in dental practice.

Gene therapy for Duchenne Muscular Dystrophy: assessing the readiness of Italian centres of expertise.

Duchenne muscular dystrophy (DMD) is a heritable disorder that causes a rapid and progressive loss of ambulatory skills. There is no curative therapy for this pathology, that is currently managed with a combination of physiotherapy and pharmacological interventions limiting the progression of the disease (e.g. corticosteroids, cardiac medications). However, a new opportunity is represented by gene therapy, a promising treatment that, however, requires significant expertise during the whole delivery of care and a solid organisational infrastructure. An organisational strategy that could effectively support its delivery to DMD patients in Italy is the hub-and-spoke model. However, an accurate portrait of the present network of DMD centres of expertise in Italy and of their readiness in the delivery of gene therapy is paramount, to facilitate access to this experimental medicine in the future. In this context, the present study aimed to map the DMD centres of expertise in Italy and later evaluate their preparedness in terms of gene therapy delivery. For this purpose, a series of items was proposed to 30 centres in Italy, of which 20 responded. After assessing the readiness of the involved centres in terms of patient preparation, therapy infusion, close surveillance, and long-term follow-up, we proposed a suitable organizational model, namely a flexible hub-and-spoke model, for the delivery of gene therapy in the Italian DMD network and solutions to tackle the challenges emerged from the survey. Overall, the present study detected an adequate readiness of the Italian DMD centres of expertise, despite observing a significant room for improvement in digital infrastructures, culture, and training.