Gene Therapy Applications Research Articles

Nonviral nanoparticle gene delivery into the CNS for neurological disorders and brain cancer applications.

Nonviral nanoparticles have emerged as an attractive alternative to viral vectors for gene therapy applications, utilizing a range of lipid-based, polymeric, and inorganic materials. These materials can either encapsulate or be functionalized to bind nucleic acids and protect them from degradation. To effectively elicit changes to gene expression, the nanoparticle carrier needs to undergo a series of steps intracellularly, from interacting with the cellular membrane to facilitate cellular uptake to endosomal escape and nucleic acid release. Adjusting physiochemical properties of the nanoparticles, such as size, charge, and targeting ligands, can improve cellular uptake and ultimately gene delivery. Applications in the central nervous system (CNS; i.e., neurological diseases, brain cancers) face further extracellular barriers for a gene-carrying nanoparticle to surpass, with the most significant being the blood-brain barrier (BBB). Approaches to overcome these extracellular challenges to deliver nanoparticles into the CNS include systemic, intracerebroventricular, intrathecal, and intranasal administration. This review describes and compares different biomaterials for nonviral nanoparticle-mediated gene therapy to the CNS and explores challenges and recent preclinical and clinical developments in overcoming barriers to nanoparticle-mediated delivery to the brain. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Neurological Disease Therapeutic Approaches and Drug Discovery > Emerging Technologies Nanotechnology Approaches to Biology > Nanoscale Systems in Biology.

Application of Gene Therapy in Hemophilia.

Gene therapy refers to introducing normal exogenous genes into target cells to correct or compensate for the diseases caused by defective and abnormal genes for the purpose of therapy. It holds out hope of a cure for single-gene genetic diseases such as thalassemia, hemophilia, etc. At present, gene therapy is performed in two ways: introducing exogenous genes, and gene editing. A great number of clinical trials of gene therapy in hemophilia have been carried out using viral vectors to introduce foreign genes into target cells. However, the production of neutralizing antibodies following injection and the inability to prepare viral vectors in large quantities limit their application. Although gene-editing methods like CRISPR avoid the above problems, the potential risks of off-target effects are still unknown. More trials and evidence are needed to elucidate the safety and accuracy of gene therapy. This paper will review the bench and clinical work of gene therapy in hemophilia in recent years, and summarize the challenges and prospects of gene therapy, so as to provide directions for future scientific research in this field.

Adapter-Mediated Transduction with Lentiviral Vectors: A Novel Tool for Cell-Type-Specific Gene Transfer.

Selective gene delivery to a cell type of interest utilizing targeted lentiviral vectors (LVs) is an efficient and safe strategy for cell and gene therapy applications, including chimeric antigen receptor (CAR)-T cell therapy. LVs pseudotyped with measles virus envelope proteins (MV-LVs) have been retargeted by ablating binding to natural receptors while fusing to a single-chain antibody specific for the antigen of choice. However, the broad application of MV-LVs is hampered by the laborious LV engineering required for every new target. Here, we report the first versatile targeting system for MV-LVs that solely requires mixing with biotinylated adapter molecules to enable selective gene transfer. The analysis of the selectivity in mixed cell populations revealed transduction efficiencies below the detection limit in the absence of an adapter and up to 5000-fold on-to-off-target ratios. Flexibility was confirmed by transducing cell lines and primary cells applying seven different adapter specificities in total. Furthermore, adapter mixtures were applied to generate CAR-T cells with varying CD4/CD8-ratios in a single transduction step. In summary, a selective and flexible targeting system was established that may serve to improve the safety and efficacy of cellular therapies. Compatibility with a wide range of readily available biotinylated molecules provides an ideal technology for a variety of applications.

CRISPR-Based Therapeutic Gene Editing for Duchenne Muscular Dystrophy: Advances, Challenges and Perspectives

Duchenne muscular dystrophy (DMD) is a severe neuromuscular disease arising from loss-of-function mutations in the dystrophin gene and characterized by progressive muscle degeneration, respiratory insufficiency, cardiac failure, and premature death by the age of thirty. Albeit DMD is one of the most common types of fatal genetic diseases, there is no curative treatment for this devastating disorder. In recent years, gene editing via the clustered regularly interspaced short palindromic repeats (CRISPR) system has paved a new path toward correcting pathological mutations at the genetic source, thus enabling the permanent restoration of dystrophin expression and function throughout the musculature. To date, the therapeutic benefits of CRISPR genome-editing systems have been successfully demonstrated in human cells, rodents, canines, and piglets with diverse DMD mutations. Nevertheless, there remain some nonignorable challenges to be solved before the clinical application of CRISPR-based gene therapy. Herein, we provide an overview of therapeutic CRISPR genome-editing systems, summarize recent advancements in their applications in DMD contexts, and discuss several potential obstacles lying ahead of clinical translation.

CD8+ T cell-associated genes MS4A1 and TNFRSF17 are prognostic markers and inhibit the progression of colon cancer.

BackgroundColon cancer (CC) is among the top three diseases with the highest morbidity and mortality rates worldwide. Its increasing incidence imposes a major global health burden. Immune checkpoint inhibitors, such as anti-PD-1 and anti-PD-L1, can be used for the treatment of CC; however, most patients with CC are resistant to immunotherapy. Therefore, identification of biomarkers that can predict immunotherapy sensitivity is necessary for selecting patients with CC who are eligible for immunotherapy.MethodsDifferentially expressed genes associated with the high infiltration of CD8+ T cells were identified in CC and para-cancerous samples via bioinformatic analysis. Kaplan–Meier survival analysis revealed that MS4A1 and TNFRSF17 were associated with the overall survival of patients with CC. Cellular experiments were performed for verification, and the protein expression of target genes was determined via immunohistochemical staining of CC and the adjacent healthy tissues. The proliferation, migration and invasion abilities of CC cells with high expression of target genes were determined via in vitro experiments.ResultsDifferential gene expression, weighted gene co-expression and survival analyses revealed that patients with CC with high expression of MS4A1 and TNFRSF17 had longer overall survival. The expression of these two genes was lower in CC tissues than in healthy colon tissues and was remarkably associated with the infiltration of various immune cells, including CD8+ T cells, in the tumour microenvironment (TME) of CC. Patients with CC with high expression of MS4A1 and TNFRSF17 were more sensitive to immunotherapy. Quantitative reverse transcription-polymerase chain reaction, western blotting and immunohistochemical staining validated the differential expression of MS4A1 and TNFRSF17. In addition, Cell Counting Kit-8, wound healing and transwell assays revealed that the proliferation, migration and invasion abilities of CC cells were weakened after overexpression of MS4A1 and TNFRSF17.ConclusionsThe core genes MS4A1 and TNFRSF17 can be used as markers to predict the sensitivity of patients with CC to immunotherapy and have potential applications in gene therapy to inhibit CC progression.

Plasmid DNA for Therapeutic Applications in Cancer.

Recently, the interest in using nucleic acids for therapeutic applications has been increasing. DNA molecules can be manipulated to express a gene of interest for gene therapy applications or vaccine development. Plasmid DNA can be developed to treat different diseases, such as infections and cancer. In most cancers, the immune system is limited or suppressed, allowing cancer cells to grow. DNA vaccination has demonstrated its capacity to stimulate the immune system to fight against cancer cells. Furthermore, plasmids for cancer gene therapy can direct the expression of proteins with different functions, such as enzymes, toxins, and cytotoxic or proapoptotic proteins, to directly kill cancer cells. The progress and promising results reported in animal models in recent years have led to interesting clinical results. These DNA strategies are expected to be approved for cancer treatment in the near future. This review discusses the main strategies, challenges, and future perspectives of using plasmid DNA for cancer treatment.

Pyruvate Kinase Deficiency: Current Challenges and Future Prospects.

Pyruvate kinase deficiency (PKD) is a rare autosomal recessive disease marked by chronic hemolytic anemia of various severity and frequent complications including gallstones, splenomegaly, iron overload, and others. Disease phenotype is highly heterogeneous and changes over time with children, adolescents and adult patients displaying different transfusion requirement and rates of complications. The diagnosis relies on the initial clinical suspicion in a patient with chronic hemolysis and exclusion of other more common congenital forms of hemolytic anemias; it is supported by the demonstration of reduced PK enzyme activity, and further confirmed by the detection of (homozygous or compound heterozygous) mutations of PKLR gene. Therapy is mainly supportive, with vitamin supplementation and transfusions (based on symptoms and patient growth rather than on fixed Hb thresholds). Splenectomy is widely performed, although it is less effective than in membrane defects and carries thrombotic and infectious risk. In the last decade, the allosteric PK enzyme activator mitapivat showed dramatic clinical benefit in clinical trials and gene therapy is also being studied to substitute the defective enzyme. In this review, we provide an insight in the current challenges of PKD diagnosis and management and discuss the future application of novel drugs and gene therapy, including a focus on quality of life.

Artificial primary-miRNAs as a platform for simultaneous delivery of siRNA and antisense oligonucleotide for multimodal gene regulation.

Self-assembled nucleic acid nanostructures have been widely explored for gene therapy applications due to their unique advantages. Their roles are not limited to offer intracellular delivery platforms but additionally provide a biological function to induce targeted gene regulation. Here, we report a self-assembled artificial primary-miRNA (pri-miRNA) for achieving simultaneous multimodal gene regulation. Artificial pri-miRNAs are designed to play a role as substrate RNAs to recruit and interact with Drosha/DGCR8 (Microprocessor). Incorporation of functional RNA motifs and site-specific chemical modification of the primary miRNA are utilized for the biogenesis of two individual gene-regulating oligonucleotides. Once they are cleaved by the endogenous Drosha/DGCR8 complex, basal strands and pre-miRNA can be generated inside of cells. In this study, we integrated basal strands with either SMN2 ASO or anti-miR21 to induce multimodal gene regulation. Microprocessing and subsequent gene regulation were first evaluated by measuring the activity of reporter pre-miRNA. Chemical modification on the primary miRNA was optimized through a series of in vitro Drosha cleavage tests and targeted gene silencing in cells. Primary miRNA with the basal ASO or anti-miR strands showed a successful in vitro activity and resulted in simultaneous multimodal gene regulation in cells. Artificial primary miRNA may offer synergistic therapeutic effects for treating various diseases, including spinal muscular atrophy and cancer.

Application of Gene Therapy for Treatment of Atrial Fibrillation

Atrial fibrillation is the most common arrhythmia globally and is associated with an increased risk for various other complications. Ablation, one of the most widely used treatments, is irreversible and can damage tissue surrounding the ablation line. Furthermore, many people with structural heart abnormalities or other conditions cannot be effectively treated. The complexity of atrial fibrillation and the uniqueness of each case make a universally effective treatment nearly impossible. The present paper describes the current treatments for atrial fibrillation and assesses the potential of gene therapy as a new clinical treatment. Further research into gene therapy could provide more targeted and effective treatments for atrial fibrillation and many other genetic conditions.

Consideration of quality by design principles in the use of sterilizing grade filters for clarification and final sterile filtration in adeno-associated virus applications

The development of gene therapy applications using recombinant adeno-associated viruses (AAV) as a gene delivery vector has gained significant momentum in recent years. Although there are numerous applications for potential therapies to treat unmet medical needs, the ability to implement robust AAV manufacturing processes continues to be a bottleneck to fully realizing the potential of AAV based therapeutics. This includes the requirement to understand the characteristics (quality attributes) that are critical to product quality and patient safety, and to define these in appropriate detail for regulatory submissions such as chemistry, manufacturing and controls (CMC) information. Sterilizing grade filtration plays an important role in AAV manufacturing, both for drug substance (DS) as well as final drug product (DP). However, to fulfill regulatory expectations for provision of CMC information, it is recommended that the principles of quality by design (QbD) are considered during the early product development stages of the AAV manufacturing process. Both prior knowledge and experimental data should be used to identify potential critical quality attributes (CQAs). Enhanced knowledge should result from increased experience, and ultimately the critical process parameters (CPPs) and critical material attributes (CMAs) that can impact the CQAs can then be defined. The objective is to define a manufacturing control strategy that includes appropriate control of CPPs and CMAs to assure the CQAs of the drug product. Here, the application of QbD principles for sterilizing grade filtration in both DS clarification and DP final fill steps of a typical AAV manufacturing process is described. Both experimental and data review approaches are included to help understand what a typical design space could look like, from which appropriate controls can be implemented. Ultimately, the expectations from regulatory agencies for assurance of product quality and patient safety are aligned with the requirement for a robust manufacturing control strategy to ensure consistent product quality is achieved.

M6A modification of mRNA in skin diseases.

N6-methyladenosine (m6A) is the predominant post-transcriptional modification for eukaryotic mRNA. It's regulated by methyltransferases, demethylases, and m6A binding proteins, and plays an important role in regulating splicing, translation, and degradation of mRNA. Skin diseases, especially immune skin diseases and skin tumors, have a complicated pathogenesis and are refractory to treatment, seriously affecting the patient quality of life. Recent studies have revealed that m6A and its regulatory proteins can affect the development of numerous skin diseases. The m6A modification was found to be involved in skin accessory development, including hair follicle and sweat gland formation. The level of m6A modification was significantly altered in a variety of skin diseases including melanoma, cutaneous squamous cell carcinoma, Merkel cell carcinoma, and psoriasis, and affected a variety of biological processes including cell proliferation and differentiation migration. The m6A and its regulatory proteins may become potential molecular markers or therapeutic targets for skin diseases, and have promising clinical applications in early diagnosis, efficacy determination, prognosis prediction, and gene therapy of skin diseases.

Progress in Intradermal and Transdermal Gene Therapy with Microneedles

Gene therapy is one of the most widely studied treatments and has the potential to treat a variety of intractable diseases. The skin's limited permeability, as the body's initial protective barrier, drastically inhibits the delivery effect of gene medicine. Given the potential adverse effects and physicochemical features of the medications, improving generic drug penetration into the skin barrier and achieving an effective level of target tissues remains a challenge. Microneedles have made tremendous improvements in aided gene transfer and medication delivery as a unique method. Microneedles offer the advantage of being minimally invasive and painless, as well as the ability to distribute gene medicines straight through the stratum corneum. Microneedles have been used to penetrate skin tissue with various nucleic acids and medicines in recent years, allowing for a wide range of applications in the treatment of skin ailments. This review focuses on skin-related disorders and immunity, and it primarily discusses the progress of microneedle transdermal gene therapy in recent years. It also complements the current major vectors and related microneedle gene therapy applications.

Optimizing mRNA purification conditions by using a high-throughput screening approach

To support the development of mRNA-based therapies, Thermo Fisher Scientific has developed a platform chromatography solution for the purification of mRNA. The POROS™ Oligo (dT)25 Affinity Resin helps to address the selectivity and capacity requirements for the large-scale manufacturing of mRNA used in vaccine and gene therapy applications. Typically, mRNA binds to the Oligo (dT)25 affinity resin using high ionic strength conditions and neutral pH and is eluted from the column using low ionic strength solutions such as water. Although water works well for most mRNA constructs, a need to identify alternative elution buffers to optimize mRNA recovery exists. This article describes experiments conducted to optimize mRNA purification using high-throughput screening.

Assessment of the Effect of Overexpression of Death-Associated Protein Kinases 3 Using PEGFPN1 on Gastric Adenocarcinoma Cell Line (MKN45)

Background: Gastric cancer (GC) is one of the most common malignancies worldwide. An in-depth understanding of the molecular mechanisms that underlies tumor GC will lead to breakthroughs in the targeted treatment of GC. Based on multiple lines of evidence, death-associated protein kinase 3 (DAPK3) regulates both programmed cell death including apoptosis and autophagy. The widespread experimental evidence raises the possibility of using DAPK-based gene therapy strategies. Objectives: The aim of this study was to investigate the effect of overexpression of DAPK3 using the PEGFPN1 vector on the gastric adenocarcinoma cell line (MKN45). Methods: The MKN45 cell lines were cultured in a DMEM culture medium and, then, the recombinant vector PEGFPN1-DAPK3 was transfected into the cells by lipofectamine 2000. The effects of the overexpression of the DAPK3 gene on MKN45 cells were evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), flow cytometry, and real-time quantitative reverse transcription PCR (qRT-PCR) techniques. Results: Our findings indicated that overexpression of DAPK3 in MKN45 cells not only affects the expression of apoptosis-related genes but also changes the expression of autophagy-related genes. Additionally, overexpression of DAPK3 reduces the metabolic activity of cells. Conclusions: The overexpression of the DAPK3 gene can lead to cell death by both inducing apoptosis and autophagy pathways in the gastric adenocarcinoma cell line (MKN45). This anti-cancer activity may describe a hopeful strategy in the application of novel gene therapy for the treatment of gastric adenocarcinoma; however, further research is required to examine the clinical effectiveness of this strategy in GC treatment.

Effects of cell seeding technique and cell density on BMP-2 production in transduced human mesenchymal stem cells.

Small animal models have demonstrated the efficacy of ex vivo regional gene therapy using scaffolds loaded with BMP-2-expressing mesenchymal stem cells (MSCs). Prior to clinical translation, optimization of seeding techniques of the transduced cells will be important to minimize time and resource expenditure, while maximizing cell delivery and BMP-2 production. No prior studies have investigated cell-seeding techniques in the setting of transduced cells for gene therapy applications. Using BMP-2-expressing transduced adipose-derived MSCs and a porous ceramic scaffold, this study compared previously described static and dynamic seeding techniques with respect to cell seeding efficiency, uniformity of cell distribution, and in vitro BMP-2 production. Static and negative pressure seeding techniques demonstrated the highest seeding efficiency, while orbital shaking was associated with the greatest increases in BMP-2 production per cell. Low density cell suspensions were associated with the highest seeding efficiency and uniformity of cell distribution, and the greatest increases in BMP-2 production from 2 to 7 days after seeding. Our results highlight the potential for development of an optimized cell density and seeding technique that could greatly reduce the number of MSCs needed to produce therapeutic BMP-2 levels in clinical situations. Further studies are needed to investigate in vivo effects of cell seeding techniques on bone healing.

CRISPR/Cas9 Gene Editing of Hematopoietic Stem and Progenitor Cells for Gene Therapy Applications

CRISPR/Cas9 Gene Editing of Hematopoietic Stem and Progenitor Cells for Gene Therapy Applications

CRISPR/Cas9 Gene Editing of Hematopoietic Stem and Progenitor Cells for Gene Therapy Applications.

CRISPR/Cas9 is a highly versatile and efficient gene-editing tool adopted widely to correct various genetic mutations. The feasibility of gene manipulation of hematopoietic stem and progenitor cells (HSPCs) in vitro makes HSPCs an ideal target cell for gene therapy. However, HSPCs moderately lose their engraftment and multilineage repopulation potential in ex vivo culture. In the present study, ideal culture conditions are described that improves HSPC engraftment and generate an increased number of gene-modified cells in vivo. The current report displays optimized in vitro culture conditions, including the type of culture media, unique small molecule cocktail supplementation, cytokine concentration, cell culture plates, and culture density. In addition to that, an optimized HSPC gene-editing procedure, along with the validation of the gene-editing events, are provided. For in vivo validation, the gene-edited HSPCs infusion and post-engraftment analysis in mouse recipients are displayed. The results demonstrated that the culture system increased the frequency of functional HSCs in vitro, resulting in robust engraftment of gene-edited cells in vivo.

Prototype mouse models for researching SEND-based mRNA delivery and gene therapy.

One of the major challenges of gene therapy-an approach to treat diseases caused by faulty genes-is a lack of technologies that deliver healthy gene copies to target tissues and cells. Some commonly used approaches include viral vectors or coating therapeutic nucleic acids with lipid-based nanoparticles to pass through cell membranes, but these technologies have had limited success. A revolutionary tool, the CRISPR-Cas gene-editing system, offers tremendous promise, but it too suffers from problems with delivery. Another tool, called 'SEND' (for 'selective endogenous encapsidation for cellular delivery'), seems to offer a better solution. The SEND system uses endogenous genetic components to package mRNA cargoes to deliver them to other cells via virus-like particles (VLPs). The SEND-VLP tool has enormous potential as a gene-therapy tool, if the endogenous components of SEND can be repurposed to produce VLPs containing therapeutic cargoes. However, several aspects of this newly identified phenomenon are not yet fully understood. Genetically engineered mouse (GEM) models, expressing different combinations of SEND components in a controllable and inducible fashion, could serve as valuable tools to understand more about this tool and to repurpose it for gene-therapy applications. In this Perspective, we discuss how GEM models and mouse molecular genetics tools could be used for SEND-VLP research.

PLGA-based nanoparticles for the treatment of cancer: current strategies and perspectives

Research on cancer treatment is always of great importance because of the extensive and difficult treatment options and side effects of chemotherapeutic agents. Due to this, novel techniques for cancer treatment are the need of the day. Nowadays, nanotechnology is of great interest for its applications as diagnostic tools, theragnostic, contrasting agents, and vehicles for delivering drugs. Nanoparticles (NPs) are made up of biocompatible and biodegradable polymers that improve the pharmacokinetic and pharmacodynamic properties of drugs, reduce side effects, improve stability, prolong the release of drug, and reduce the dosing frequency. Poly (lactic-co-glycolic acid) (PLGA) is FDA-approved synthetic polymer which can be used to formulate NPs that can be targeted to a specific site for the safe and effective delivery of drugs. PLGA-based NPs can be used for a variety of cancer therapies including tumor-targeted drug delivery, gene therapy, hyperthermia, and photodynamic therapy. This article discusses the method of preparation, characterization, encapsulation of chemotherapeutic drugs, effect of physicochemical properties of PLGA- based NPs, and how we can exploit these aspects through various methods of preparation for drug loading, biodistribution, target specificity, and their use in cancer treatment. Along with these targeting strategies, gene therapy, cancer immunotherapy, and various applications have also been discussed. This article also aims to discuss the incorporation of diagnostic tools and therapeutic moiety in one versatile formulation of PLGA-NPs and the difficulties faced in translating this promising tool to clinical use.

Gene-Based Therapeutics for Parkinson's Disease.

Parkinson’s disease (PD) is a complex multifactorial disorder that is not yet fully surmised, and it is only when such a disease is tackled on multiple levels simultaneously that we should expect to see fruitful results. Gene therapy is a modern medical practice that theoretically and, so far, practically, has demonstrated its capability in joining the battle against PD and other complex disorders on most if not all fronts. This review discusses how gene therapy can efficiently replace current forms of therapy such as drugs, personalized medicine or invasive surgery. Furthermore, we discuss the importance of enhancing delivery techniques to increase the level of transduction and control of gene expression or tissue specificity. Importantly, the results of current trials establish the safety, efficacy and applicability of gene therapy for PD. Gene therapy’s variety of potential in interfering with PD’s pathology by improving basal ganglial circuitry, enhancing dopamine synthesis, delivering neuroprotection or preventing neurodegeneration may one day achieve symptomatic benefit, disease modification and eradication.