Advances In Gene Therapy Research Articles

Future of Therapy for Inborn Errors of Immunity.

Over the past 20 years, the rapid evolution in the diagnosis and treatment of primary immunodeficiencies (PI) and the recognition of immune dysregulation as a feature in some have prompted the use of “inborn errors of immunity” (IEI) as a more encompassing term used to describe these disorders [1, 2] . This article aims to review the future of therapy of PI/IEI (referred to IEI throughout this paper). Historically, immune deficiencies have been characterized as monogenic disorders resulting in immune deficiencies affecting T cells, B cells, combination of T and B cells, or innate immune disorders. More recently, immunologists are also recognizing a variety of phenotypes associated with one genotype or similar phenotypes across genotypes and a role for incomplete penetrance or variable expressivity of some genes causing inborn errors of immunity [3]. The IUIS classification of immune deficiencies (IEIs) has evolved over time to include 10 categories, with disorders of immune dysregulation accounting for a new subset, some treatable with small molecule inhibitors or biologics. [1] Until recently, management options were limited to prompt treatment of infections, gammaglobulin replacement, and possibly bone marrow transplant depending on the defect. Available therapies have expanded to include small molecule inhibitors, biologics, gene therapy, and the use of adoptive transfer of virus-specific T cells to fight viral infections in immunocompromised patients. Several significant contributions to the field of clinical immunology have fueled the rapid advancement of therapies over the past two decades. Among these are educational efforts to recruit young immunologists to the field resulting in the growth of a world-wide community of clinicians and investigators interested in rare diseases, efforts to increase awareness of IEI globally contributing to international collaborations, along with advancements in diagnostic genetic testing, newborn screening, molecular biology techniques, gene correction, use of immune modulators, and ex vivo expansion of engineered T cells for therapeutic use. The development and widespread use of newborn screening have helped to identify severe combined immune deficiency (SCID) earlier resulting in better outcomes [4]. Continual improvements and accessibility of genetic sequencing have helped to identify new IEI diseases at an accelerated pace [5]. Advances in gene therapy and bone marrow transplant have made treatments possible in otherwise fatal diseases. Furthermore, the increased awareness of IEI across the world has driven networks of immunologists working together to improve the diagnosis and treatment of these rare diseases. These improvements in the diagnosis and treatment of IEI noted over the past 20 years bring hope for a better future for the IEI community. This paper will review future directions in a few of the newer therapies emerging for IEI. For easy reference, most of the diseases discussed in this paper are briefly described in a summary table, in the order mentioned within the paper (Appendix).

Heat Shock Protein 70 Inhibition Effect on Inflammatory Receptors via Nuclear Factor Kappa B on Melanoma Cell Lines, A-375 and C8161

The aim of this study was evaluation of effect of heat shock protein 70 on toll-like receptor 4 and toll-like receptor 2 in melanoma cell line, A-375. The melanoma cell lines were stimulated in time course with heat shock protein 70 protein and incubation was performed. After 48 h, the cells were collected and total messenger ribonucleic acid was extracted and the expression of toll-like receptor 2 and toll-like receptor 4 was determined by quantitative reverse transcription polymerase chain reaction. The results showed that the appearance of toll-like receptor 2, toll-like receptor 4 in melanoma cell line, A-375 decreased in exposure to heat shock protein 70. The results showed the expression of nuclear factor kappa B in melanoma cell line. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide evaluation results of melanoma cell death were compared at different times and statistical analysis showed a significant difference (p<0.001). The role of heat shock protein 70 has been identified as a tumor suppressor in melanoma cancer. Heat shock protein 70 is a new treatment for melanoma in the near future. Heat shock protein 70 can be considered as a potential target in gene therapy for patients with melanoma cancer and the advantages of gene therapy by heat shock protein 70 in this type of cancer over chemotherapy can reduce cytotoxic effects and its low invasiveness.

A Review on Gene Therapy

Gene therapy is an emerging concept that gives hope to people with highly fatal conditions. It’s almost three decades since the emergence of the idea of gene therapy. Since then, hundreds of medical trials have been conducted worldwide from which we not only have gained enough knowledge but have experienced the need for it in society. Even though the concept of gene therapy has experienced setbacks, success stories are increasing exponentially, proof of which are recommendations and approvals of gene therapy from various medical associations worldwide. Our knowledge has grown over the years, and during this period, we have come across various safety datahelp help us develop better gene therapy approaches. The chief concept of this procedure is to revamp the vehicles for delivery which typically are nanostructures, plasmids, and viruses. The USA and Europe have been pioneers in gene therapy for a very long period; various reports have come from Asian countries, including India, in recent times. Our knowledge of the concept of gene therapy is increasing day by day. New information and data are being analyzed regularly to help provide gene therapies for different diseases. New research has led to various new drug applications for approval at the FDA. In this review, various points, from the history of gene therapy and its requirement in today’s society to the most recent advances in gene therapy, have been discussed. It also covers the works and advances of gene therapy in India.

Gene therapy for kidney disease: targeting cystinuria.

The aim of this study was to summarize recent findings in kidney gene therapy while proposing cystinuria as a model kidney disease target for genome engineering therapeutics. Despite the advances of gene therapy for treating diseases of other organs, the kidney lags behind. Kidney-targeted gene delivery remains an obstacle to gene therapy of kidney disease. Nanoparticle and adeno-associated viral vector technologies offer emerging hope for kidney gene therapy. Cystinuria represents a model potential target for kidney gene therapy due to its known genetic and molecular basis, targetability, and capacity for phenotypic rescue. Although gene therapy for kidney disease remains a major challenge, new and evolving technologies may actualize treatment for cystinuria and other kidney diseases.

Advances in gene therapy for neurogenetic diseases: a brief review.

Neurogenetic diseases are neurological conditions with a genetic cause (s). There are thousands of neurogenetic diseases, and most of them are incurable. The development of bioinformatics and elucidation of the mechanism of pathogenesis have allowed the development of gene therapy approaches, which show great potential in treating neurogenetic diseases. Viral vectors delivery, antisense oligonucleotides, gene editing, RNA interference, and burgeoning viroid delivery technique are promising gene therapy strategies, and commendable therapeutic effects in the treatment of neurogenetic diseases have been achieved (Fig.1). This review highlights a sampling of advances in gene therapies for neurogenetic disorders. Fig. 1 Examples of gene therapy strategies used in the treatment of neurogenetic diseases. The schematic diagram shows different gene therapy approaches used for treating a sampling of neurogenetic disorders, such as ASO therapy, gene editing, gene augmentation, and RNA interference.

Editorial: Nanotechnology for Precision Cancer Therapy: Advances in Gene Therapy, Immunotherapy, and 3D Bioprinting

EDITORIAL article Front. Nanotechnol., 14 October 2021Sec.Biomedical Nanotechnology https://doi.org/10.3389/fnano.2021.776898

Development of alternative gene transfer techniques for exvivo and invivo gene therapy in a canine model.

IntroductionGene therapy have recently attracted much attention as a curative therapeutic option for inherited single gene disorders such as hemophilia. Hemophilia is a hereditary bleeding disorder caused by the deficiency of clotting activity of factor VIII (FVIII) or factor IX (FIX), and gene therapy for hemophilia using viral vector have been vigorously investigated worldwide. Toward further advancement of gene therapy for hemophilia, we have previously developed and validated the efficacy of novel two types of gene transfer technologies using a mouse model of hemophilia A. Here we investigated the efficacy and safety of the technologies in canine model. Especially, validations of technical procedures of the gene transfers for dogs were focused.MethodsGreen fluorescence protein (GFP) gene were transduced into normal beagle dogs by ex vivo and in vivo gene transfer techniques. For ex vivo gene transfer, blood outgrowth endothelial cells (BOECs) derived from peripheral blood of normal dogs were transduced with GFP gene using lentivirus vector, propagated, fabricated as cell sheets, then implanted onto the omentum of the same dogs. For in vivo gene transfer, normal dogs were subjected to GFP gene transduction with non-viral piggyBac vector by liver-targeted hydrodynamic injections.ResultsNo major adverse events were observed during the gene transfers in both gene transfer systems. As for ex vivo gene transfer, histological findings from the omental biopsy performed 4 weeks after implantation revealed the tube formation by implanted GFP-positive BOECs in the sub-adipose tissue layer without any inflammatory findings, and the detected GFP signals were maintained over 6 months. Regarding in vivo gene transfer, analyses of liver biopsy samples revealed more than 90% of liver cells were positive for GFP signals in the injected liver lobes 1 week after gene transfers, then the signals gradually declined overtime.ConclusionsTwo types of gene transfer techniques were successfully applied to a canine model, and the transduced gene expressions persisted for a long term. Toward clinical application for hemophilia patients, practical assessments of therapeutic efficacy of these techniques will need to be performed using a dog model of hemophilia and FVIII (or FIX) gene.

Understanding the Genetic and Molecular Basis of Familial Hypertrophic Cardiomyopathy and the Current Trends in Gene Therapy for Its Management.

Hypertrophic cardiomyopathy (HCM) is a genetically acquired disease of cardiac myocytes. Studies show that 70% of this disease is a result of different mutations in various sarcomere genes. This review aims to discuss several genetic mutations, epigenetic factors, and signal transduction pathways leading to the development of HCM. In addition, this article elaborates on recent advances in gene therapies and their implications for managing this condition. We start by discussing the founding mutations in HCM and their effect on power stroke generation. The less explored field of epigenetics including methylation, acetylation, and the role of different micro RNAs in the development of cardiac muscle hypertrophy has been highlighted in this article. The signal transduction pathways that lead to gene transcription, which in turn lead to increased protein synthesis of cardiac muscle fibers are elaborated. Finally, the microscopic events leading to the pathophysiologic macro events of cardiac failure, and the current experimental trials of gene therapy models, and the clustered regularly interspaced short palindromic repeats (CRISPR) type 2 system proteins, are discussed. We have concluded our discussion by emphasizing the need for more studies on epigenomics and experimental designs for gene therapy in HCM patients. This review focuses on the process of HCM from initial mutation to the development of phenotypic expression and various points of intervention in cardiac myocardial hypertrophy development.

Recent Advances in Gene Therapy for Cardiac Tissue Regeneration.

Cardiovascular diseases (CVDs) are responsible for enormous socio-economic impact and the highest mortality globally. The standard of care for CVDs, which includes medications and surgical interventions, in most cases, can delay but not prevent the progression of disease. Gene therapy has been considered as a potential therapy to improve the outcomes of CVDs as it targets the molecular mechanisms implicated in heart failure. Cardiac reprogramming, therapeutic angiogenesis using growth factors, antioxidant, and anti-apoptotic therapies are the modalities of cardiac gene therapy that have led to promising results in preclinical studies. Despite the benefits observed in animal studies, the attempts to translate them to humans have been inconsistent so far. Low concentration of the gene product at the target site, incomplete understanding of the molecular pathways of the disease, selected gene delivery method, difference between animal models and humans among others are probable causes of the inconsistent results in clinics. In this review, we discuss the most recent applications of the aforementioned gene therapy strategies to improve cardiac tissue regeneration in preclinical and clinical studies as well as the challenges associated with them. In addition, we consider ongoing gene therapy clinical trials focused on cardiac regeneration in CVDs.

Gene therapy in retinal diseases: A review.

Over 2 million people worldwide are suffering from gene-related retinal diseases, inherited or acquired, and over 270 genes have been identified which are found to be responsible for these conditions. This review article touches upon the mechanisms of gene therapy, various enzymes of the visual cycle responsible for different genetic diseases, Luxturna—the first US Food and Drug Administration (FDA)-approved therapeutic gene product, and several ongoing trials of gene therapy for age-related macular degeneration. Gene therapy has tremendous potential for retinal conditions due to its ease of accessibility, immune-privileged status, and tight blood-retinal barriers, limiting systemic side effects of the drug. In recent years, advances in gene therapy in retinal conditions have increasing significantly, with progress in cell-specific targeting and transduction efficiency of gene products through the use of adeno-associated viral vectors (AAVs), suggesting that even greater success in future clinical trials is possible.

Functionalized PDA/DEX-PEI@HA nanoparticles combined with sleeping-beauty transposons for multistage targeted delivery of CRISPR/Cas9 gene

CRISPR/Cas9 system has been used as the most powerful gene editing tool for precision medicine and advanced gene therapy. However, its wide applications are limited by the poor biosafety of lentivirus delivery vectors though with high-efficiency transduction. To construct a safer vector and promote genome integration, the CRISPR/Cas9 gene is cloned into a plasmid-based non-viral safe vector Sleeping-Beauty (SB) transposon in this study to obtain pT2SpCas9. Meanwhile, PDA/DEX-PEI@HA (PDPH) nanoparticles are constructed to facilitate the precise CRISPR/Cas9 targeting delivery, by using polydopamine (PDA) as the carrier, hyaluronic acid (HA) as the cell-targeting ligand and dexamethasone (DEX) as the nuclear localization signal (NLS). The results showed that PDPH could deliver pDNA efficiently into the cell and further into the nucleus. The transfection efficiency of PDPH is much higher than that of NPs without HA and DEX. Remarkably, the cytotoxicity of PDPH is negligible in comparison to PEI25k and PEI10k. Western blots showed that after the transfection of PDPH/pT2SpCas9-Nanog/SB11, Nanog protein in HeLa cells is knocked out, and the proliferation and migration abilities of tumor cells are significantly decreased. This study demonstrates that PDA/DEX-PEI25k@HA/pT2SpCas9 (PDPH25 K/pT2SpCas9) has the great potential as a non-viral gene vector for CRISPR/Cas9 delivery and clinical medication.

Proof of Gene Doping in a Mouse Model with a Human Erythropoietin Gene Transferred Using an Adenoviral Vector.

Despite the World Anti-Doping Agency (WADA) ban on gene doping in the context of advancements in gene therapy, the risk of EPO gene-based doping among athletes is still present. To address this and similar risks, gene-doping tests are being developed in doping control laboratories worldwide. In this regard, the present study was performed with two objectives: to develop a robust gene-doping mouse model with the human EPO gene (hEPO) transferred using recombinant adenovirus (rAdV) as a vector and to develop a detection method to identify gene doping by using this model. The rAdV including the hEPO gene was injected intravenously to transfer the gene to the liver. After injection, the mice showed significantly increased whole-blood red blood cell counts and increased expression of hematopoietic marker genes in the spleen, indicating successful development of the gene-doping model. Next, direct and potentially indirect proof of gene doping were evaluated in whole-blood DNA and RNA by using a quantitative PCR assay and RNA sequencing. Proof of doping could be detected in DNA and RNA samples from one drop of whole blood for approximately a month; furthermore, the overall RNA expression profiles showed significant changes, allowing advanced detection of hEPO gene doping.

White Matter Pathology as a Barrier to Gangliosidosis Gene Therapy.

The gangliosidoses are a family of neurodegenerative lysosomal storage diseases that have recently seen promising advances in gene therapy. White matter deficits are well established components of gangliosidosis pathology that are now receiving more attention because they are partially refractory to correction by gene therapy. After a brief synopsis of normal myelinogenesis, this review outlines current viewpoints on the origin of white matter deficits in the gangliosidoses and potential obstacles to treating them effectively by gene therapy. Dysmyelinogenesis (failure of myelin sheaths to form properly) is proposed as the predominant contributor to white matter pathology, but precise mechanistic details are not well understood. The involvement of neuronal storage deficits may extend beyond secondary demyelination (destruction of myelin due to axonal loss) and contribute to dysmyelinogenesis. Preclinical studies in animal models of the gangliosidoses have substantially improved lifespan and quality of life, leading to the initiation of several clinical trials. However, improvement of white matter pathology has lagged behind other metrics and few evidence-based explanations have been proposed to date. Research groups in the field are encouraged to include myelin-specific investigations in future gene therapy work to address this gap in knowledge.

Recent advances in gene therapy for atrial fibrillation.

Atrial fibrillation (AF) is the most common heart rhythm disorder in adults and a major cause of stroke. Unfortunately, current treatments for AF are suboptimal as they are not targeting the molecular mechanisms underlying AF. In this regard, gene therapy is emerging as a promising approach for mechanism-based treatment of AF. In this review, we summarize recent advances and challenges in gene therapy for this important cardiovascular disease.

Emerging gene therapies for enhancing the hemostatic potential of platelets.

Platelet transfusions are an integral component of balanced hemostatic resuscitation protocols used to manage severe hemorrhage following trauma. Enhancing the hemostatic potential of platelets could lead to further increases in the efficacy of transfusions, particularly for non-compressible torso hemorrhage or severe hemorrhage with coagulopathy, by decreasing blood loss and improving overall patient outcomes. Advances in gene therapies, including RNA therapies, are leading to new strategies to enhance platelets for better control of hemorrhage. This review will highlight three approaches for creating modified platelets using gene therapies: (i) direct transfection of transfusable platelets ex vivo, (ii) in vitro production of engineered platelets from platelet-precursor cells, and (iii) modifying the bone marrow for in vivo production of modified platelets. In summary, modifying platelets to enhance their hemostatic potential is an exciting new frontier in transfusion medicine, but more preclinical development as well as studies testing the safety and efficacy of these agents are needed.

Production of Adeno-Associated Virus Vectors in Cell Stacks for Preclinical Studies in Large Animal Models.

Adeno-associated virus (AAV) vectors are among the most clinically advanced gene therapy vectors, with three AAV gene therapies approved for humans. Clinical advancement of novel applications for AAV involves transitioning from small animal models, such as mice, to larger animal models, including dogs, sheep, and nonhuman primates. One of the limitations of administering AAV to larger animals is the requirement for large quantities of high-titer virus. While suspension cell culture is a scalable method for AAV vector production, few research labs have the equipment (e.g., bioreactors) or know how to produce AAV in this manner. Moreover, AAV titers are often significantly lower when produced in suspension HEK 293 cells as compared to adherent HEK293 cells. Described here is a method for producing large quantities of high-titer AAV using cell stacks. A detailed protocol for titering AAV as well as methods for validating vector purity are also described. Finally, representative results of AAV-mediated transgene expression in a sheep model are presented. This optimized protocol for large-scale production of AAV vectors in adherent cells will enable molecular biology laboratories to advance the testing of their novel AAV therapies in larger animal models.

Automating Laboratory Processes by Connecting Biotech and Robotic Devices—An Overview of the Current Challenges, Existing Solutions and Ongoing Developments

The constantly growing interest and range of applications of advanced cell, gene and regenerative therapies raise the need for efficient production of biological material and novel treatment technologies. Many of the production and manipulation processes of such materials are still manual and, therefore, need to be transferred to a fully automated execution. Developers of such systems face several challenges, one of which is mechanical and communication interfaces in biotechnological devices. In the present state, many devices are still designed for manual use and rarely provide a connection to external software for receiving commands and sending data. However, a trend towards automation on the device market is clearly visible, and the communication protocol, Open Platform Communications Data Access (OPC DA), seems to become established as a standard in biotech devices. A rising number of vendors offer software for device control and automated processing, some of which even allow the integration of devices from multiple manufacturers. The high, application-specific need in functionalities, flexibility and adaptivity makes it difficult to find the best solution and, in many cases, leads to the creation of new custom-designed software. This report shall give an overview of existing technologies, devices and software for laboratory automation of biotechnological processes. Furthermore, it presents an outlook for possible future developments and standardizations.

Non-Viral Vector-Mediated Gene Therapy for ALS: Challenges and Future Perspectives.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease, for which no effective treatment is yet available to either slow or terminate it. Recent advances in gene therapy renew hope for developing an effective approach to control this disease. Non-viral vectors, such as lipid- and polymer-based nanoparticles, cationic polymers, and exosomes, can effectively transfer genes into primary neurons. The resulting gene expression can be long-term, stable, and without immunological complications, which is essential for the effective management of neurological disorders. This Review will first describe the current research and clinical stage of novel therapies for ALS. It will then touch on the journey of non-viral vector use in ALS, subsequently highlighting the application of non-viral vector-mediated gene therapy. The bottlenecks in the translation of non-viral vectors for ALS treatment are also discussed, including the biological barriers of systemic administration and the issues of "when, where, and how much?" for effective gene delivery. The prospect of employing emerging techniques, such as CRISPR-Cas9 gene editing, stem cell methodology, and low-intensity focused ultrasound for fueling the transport of non-viral vectors to the central nervous system for personalized gene therapy, is briefly discussed in the context of ALS. Despite the challenging road that lies ahead, with the current expansion in interest and technological advancement in non-viral vector-delivered gene therapy for ALS, we hold hope that the field is headed toward a positive future.

Gene Therapy in the Anterior Eye Segment.

This review provides comprehensive information about the advances in gene therapy in the anterior segment of the eye, including cornea, conjunctiva, lacrimal gland, and trabecular meshwork. We discuss gene delivery systems, including viral and non-viral vectors as well as gene editing techniques, mainly CRISPR-Cas9, and epigenetic treatments, including antisense and siRNA therapeutics. We also provide a detailed analysis of various anterior segment diseases where gene therapy has been tested with corresponding outcomes. Disease conditions include corneal and conjunctival fibrosis and scarring, corneal epithelial wound healing, corneal graft survival, corneal neovascularization, genetic corneal dystrophies, herpetic keratitis, glaucoma, dry eye disease, and other ocular surface diseases. Although most of the analyzed results on the use and validity of gene therapy at the ocular surface have been obtained in vitro or using animal models, we also discuss the available human studies. Gene therapy approaches are currently considered very promising as emerging future treatments of various diseases, and this field is rapidly expanding.

Usher Syndrome in the Inner Ear: Etiologies and Advances in Gene Therapy.

Hearing loss is the most common sensory disorder with ~466 million people worldwide affected, representing about 5% of the population. A substantial portion of hearing loss is genetic. Hearing loss can either be non-syndromic, if hearing loss is the only clinical manifestation, or syndromic, if the hearing loss is accompanied by a collage of other clinical manifestations. Usher syndrome is a syndromic form of genetic hearing loss that is accompanied by impaired vision associated with retinitis pigmentosa and, in many cases, vestibular dysfunction. It is the most common cause of deaf-blindness. Currently cochlear implantation or hearing aids are the only treatments for Usher-related hearing loss. However, gene therapy has shown promise in treating Usher-related retinitis pigmentosa. Here we review how the etiologies of Usher-related hearing loss make it a good candidate for gene therapy and discuss how various forms of gene therapy could be applied to Usher-related hearing loss.