Tool For Gene Therapy Research Articles

Development of Lentiviral Packaging Cells and Scale Up of Production to Meet the Growing Demand in Cell and Gene Therapy.

Gamma-Retroviral (RVVs) and lentiviral vectors (LVVs) represent indispensable tools in somatic gene therapy, mediating the efficient, stable transfer of therapeutic genes into a variety of human target cells. LVVs, in contrast to RVVs, are capable of stably genetically modifying non-proliferating target cells, making them the superior instrument in cell and gene therapy. To date, the LVV manufacturing process employs human embryonic kidney cells (HEK293) and derivatives thereof transiently transfected with multiple plasmids encoding the required viral vector components. Alternatively, stable packaging cell lines were developed and engineered to express all vector components in trans. Currently, these cells are mostly cultured in cell stacks, where they grow adherently in 2D layers, limiting the scale-up of vector production. The production of viral vectors using stable suspension cell lines enables larger-scale production and higher yields under controlled conditions. Here, we review the improvements made to enhance vector safety and production yield. Current advancements in the establishment of stable packaging cell lines enabling inducible and constitutive LVV production are summarized and discussed. Manufacturing processes for lentiviral vectors using bioreactors with perfusion systems are required to meet the growing demand in cell and gene therapy and to reduce production and therapy costs.

Biotechnology for Global Sustainability: Innovations, Applications, and Challenges

Biotechnology, from a global perspective, involves applying biological systems, organisms, or derivatives to develop innovative products and processes that address global challenges and enhance the quality of life. As an interdisciplinary field bridging biology, technology, and engineering, biotechnology significantly impacts critical sectors, including healthcare, agriculture, environmental management, and industrial processes. In healthcare, biotechnology drives advancements in vaccines, gene therapy, personalized medicine, and diagnostic tools, improving disease prevention and treatment. Agricultural biotechnology focuses on developing genetically modified (GM) crops with higher yields, resistance to pests, and adaptability to climate change, ensuring food security. Additionally, it enhances livestock breeding and productivity. Environmental applications of biotechnology include bioremediation to combat pollution, development of biofuels to reduce reliance on fossil fuels, and creation of biodegradable materials to mitigate waste. In industrial processes, biotechnology optimizes manufacturing efficiency through the use of enzymes and microorganisms, contributing to sustainable production. Globally, biotechnology addresses pressing challenges like food insecurity, climate change, health inequality, and environmental degradation. Its role in sustainable development is critical, fostering eco-friendly solutions and improving the resilience of economies and societies. However, the field raises ethical and regulatory concerns, such as the risks of genetic engineering and the equitable distribution of benefits. International collaboration among governments, industries, and scientists is essential to ensure responsible innovation and maximize biotechnology’s potential to improve global well-being. As a transformative science, biotechnology continues to shape the future, balancing innovation with sustainability and equity.

Review on macromolecule-based magnetic theranostic agents for biomedical applications: targeted therapy and diagnostic imaging.

Clinical diagnostics and biological research are advanced by magnetic theranostic, which uses macromolecule-based magnetic theranostic agents for targeted therapy and diagnostic imaging. Within this review, the interaction of magnetic nanoparticles (MNPs) with biological macromolecules will be covered. The exciting potential of macromolecule-based magnetic theranostic agents to be used as a tool in drug delivery, photothermally therapy (PTT), gene therapy, hyperthermia therapy and photodynamic therapy (PDT) will be discussed. Innovative imaging technique: magnetic resonance imaging (MRI), magnetic particle imaging (MPI), fluorescence scanning, and photoacoustic scanning are revolutionizing biological diagnosis by potentially overcoming historical limitations. This review will cover the challenge of fabricating of macromolecule-based magnetic theranostic agents as a promising platform for theranostic that can combine therapies with diagnostics at subcellular level. Additionally, it looks at several chemical pathways leading to the process for generating MNPs, including the co-precipitation, the sol-gel, the hydrothermal synthesis, the polyol route, and the microemulsion technique. Eventually, the demands and prospects for magnetic theranostic are discussed, focusing on the requirement of further investigation to improve MNP structure towards biocompatible material and translation of these promising theranostic agents into clinical applications.

Mechanically mediated cargo delivery to cells using microfluidic devices.

Drug delivery technologies, which are a crucial area of research in the field of cell biology, aim to actively or passively deliver drugs to target cells to enhance therapeutic efficacy and minimize off-target effects. In recent years, with advances in drug development, particularly, the increasing demand for macromolecular drugs (e.g., proteins and nucleic acids), novel drug delivery technologies and intracellular cargo delivery systems have emerged as promising tools for cell and gene therapy. These systems include various viral- and chemical-mediated methods as well as physical delivery strategies. Physical methods, such as electroporation and microinjection, have shown promise in early studies but have not been widely adopted due to concerns regarding efficiency and cellular viability. Recently, microfluidic technologies have provided new opportunities for cargo delivery by allowing for precise control of fluid dynamic parameters to achieve efficient and safe penetration of cell membranes, as well as for foreign material transport. Microfluidics-based mechanical delivery methods utilize biophysical phenomena, such as cell constriction and fluid shear, and are associated with high throughput and high transfection efficiency. In this review, we summarize the latest advancements in microfluidic mechanical delivery technologies, and we discuss constriction- and fluid shear-induced delivery strategies. Furthermore, we explore the potential application of artificial intelligence in optimizing cargo delivery technologies, aiming to provide theoretical support and practical guidance for the future development of novel cellular drug delivery technologies.

Alternative NADH dehydrogenase: A complex I backup, a drug target, and a tool for mitochondrial gene therapy

Alternative NADH dehydrogenase, also known as type II NADH dehydrogenase (NDH-2), catalyzes the same redox reaction as mitochondrial respiratory chain complex I. Specifically, it oxidizes reduced nicotinamide adenine dinucleotide (NADH) while simultaneously reducing ubiquinone to ubiquinol. However, unlike complex I, this enzyme is non-proton pumping, comprises of a single subunit, and is resistant to rotenone. Initially identified in bacteria, fungi and plants, NDH-2 was subsequently discovered in protists and certain animal taxa including sea squirts. The gene coding for NDH-2 is also present in the genomes of some annelids, tardigrades, and crustaceans. For over two decades, NDH-2 has been investigated as a potential substitute for defective complex I. In model organisms, NDH-2 has been shown to ameliorate a broad spectrum of conditions associated with complex I malfunction, including symptoms of Parkinson's disease. Recently, lifespan extension has been observed in animals expressing NDH-2 in a heterologous manner. A variety of mechanisms have been put forward by which NDH-2 may extend lifespan. Such mechanisms include the activation of pro-longevity pathways through modulation of the NAD+/NADH ratio, decreasing production of reactive oxygen species (ROS) in mitochondria, or then through moderate increases in ROS production followed by activation of defense pathways (mitohormesis). This review gives an overview of the latest research on NDH-2, including the structural peculiarities of NDH-2, its inhibitors, its role in the pathogenicity of mycobacteria and apicomplexan parasites, and its function in bacteria, fungi, and animals.

Systemic Toxicity of Recombinant Adeno-Associated Virus Gene Therapy Vectors.

Recombinant adeno-associated virus (rAAV) vectors have emerged as a promising tool for gene therapy. However, the systemic administration of rAAV vectors is not without risks, particularly for dose levels >1 × 1014 viral genome per kilogram of body weight (vg/kg). rAAV-associated toxicities can variably manifest either acutely or in a delayed manner. Acute toxicities often present shortly after administration and can include severe immune responses, hepatotoxicity, and thrombotic microangiopathy (TMA). Delayed toxicities, on the other hand, may emerge weeks to months post-treatment, potentially involving chronic liver damage or prolonged immune activation. Thrombotic microangiopathy is often associated with complement activation and endothelial damage. The activation of the complement system can additionally trigger a cascade of inflammatory responses, exacerbating systemic toxicity. While many of these toxicities are reversible with appropriate medical intervention, there have been instances where the adverse effects were severe enough to lead to fatalities. Both human and animal studies have reported these adverse effects, highlighting the critical importance of thorough preclinical testing. However, a differential toxicity profile associated with systemic AAV administration exists between humans and nonhuman primates (NHPs), in which certain toxicities reported in humans are yet to be observed in NHPs, and vice versa. This review aims to explore the recent literature on systemic rAAV toxicities, focusing on dose levels, the role of the complement activation pathway, endothelial injury, TMA, hepatotoxicity, and the bidirectional translational safety profiles from both human and animal studies.

E2A, VA RNA I, and L4-22k adenoviral helper genes are sufficient for AAV production in HEK293 cells

The replication-defective adeno-associated virus (AAV) is extensively utilized as a research tool or vector for gene therapy. The production process of AAV remains intricate, expensive, and mechanistically underexplored. With the aim of enhancing AAV manufacturing efficiencies in mammalian cells, we revisited the questions and optimization surrounding the requirement of the various adenoviral helper genes in enabling AAV production. First, we refined the minimal set of adenoviral genes in HEK293 AAV production to E2A, L4-22 K /33 K, and VA RNA I. These findings challenge the previously accepted necessity of adenoviral E4orf6 in AAV production. In addition, we identified L4-22 K genes as crucial helpers for AAV production. Next, a revised minimal adenoviral helper plasmid comprising E2A, L4-22 K, and VA RNA I genes was designed and demonstrated to yield high titer and potent AAV preps in HEK293 transient transfection. Lastly, stable packaging cells harboring inducible E2A and L4-22 K genes were shown to maintain AAV production yields comparable to transient transfection over a culture period of ∼10weeks. Combined, these findings further our understanding of adenoviral helper function in mammalian AAV production and provide novel plasmid and cell-line reagents with an improved safety profile for potential broad applicability in the research and gene therapy community.

Engineering of HEK293T Cell Factory for Lentiviral Production by High-Throughput Selected Genes.

Lentiviral vectors (LVs) are crucial tools in gene therapy and bioproduction, but high-yield LV production systems are urgently needed. Using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 high-throughput screening, we identified nine critical genes (LDAH, GBP3, BPIFC, NHLRC1, NHLRC3, ZNF425, TTC37, LRRC4B, and SPINK6) from 17,501 genes that limit LV packaging and formation. Knocking out these genes in HEK293T cells significantly increased virus production, with LDAH knockout exhibiting a 6.63-fold increase. Studies on multigene knockouts demonstrated that the cumulative effects of different gene knockouts can significantly enhance lentivirus production in HEK293T cells. Triple knockout of GBP3, BPIFC, and LDAH increased LV titer by ∼8.33-fold, and knockout (or knockdown) of GBP3, NHLRC1, and NHLRC3 increased LV titer by ∼6.53-fold. This study established HEK293T cell lines with multiple genes knockout for efficient LV production, providing reliable technical support for LV production and application and offering new perspectives for studying LV packaging mechanisms and related virus research.

Adeno-Associated Virus 5 Protein Particles Produced by E. coli Cell-Free Protein Synthesis.

Recombinant adeno-associated viruses (rAAVs) have emerged as important tools for gene therapy and, more recently, vaccine development. Nonetheless, manufacturing can be costly and time-consuming, emphasizing the importance of alternative production platforms. We investigate the potential of E. coli-based cell-free protein synthesis (CFPS) to produce recombinant AAV5 virus-like particles (VLPs). AAV5 virus protein 3 (VP3) constructs, both with and without Strep-tag II, were expressed with CFPS. Lower reaction temperatures resulted in increased solubility, with the untagged variant containing nearly 90% more soluble VLP VP3 protein at 18 °C than at 37 °C. Affinity chromatography of N-terminally Strep(II)-tagged VP3 enabled successful isolation with minimal processing. DLS and TEM confirmed the presence of ∼20 nm particles. Furthermore, the N-terminally tagged AAV5 VP3 VLPs were biologically active, successfully internalizing into HeLa cells. This study describes an innovative approach to AAV VLP production using E. coli-based CFPS, demonstrating its potential for rapid and biologically active AAV VLP synthesis.

Efficient AAV9 Purification Using a Single-Step AAV9 Magnetic Affinity Beads Isolation.

Adeno-associated viruses (AAVs) have emerged as promising tools for gene therapy due to their safety and efficacy in delivering therapeutic genes or gene editing sequences to various tissues and organs. AAV serotype 9 (AAV9), among AAV serotypes, stands out for its ability to efficiently target multiple tissues, thus holding significant potential for clinical applications. However, existing methods for purifying AAVs are cumbersome, expensive, and often yield inconsistent results. In this study, we explore a novel purification strategy utilizing Dynabeads™ CaptureSelect™ magnetic beads. The AAV9 magnetic beads capture AAV9 with high specificity and recovery between 70 and 90%, whereas the AAVX magnetic beads did not bind to the AAV9. Through continuous interaction with AAVs in solution, these beads offer enhanced clearance of genomic DNA and plasmids even in the absence of endonuclease. The beads could be regenerated at least eight times, and the used beads could be stored for up to six months and reused without a significant reduction in recovery. The potency of the AAV9-purified vectors in vivo was comparable to that of iodixanol purified vectors.

A modified glycosylase base editor without predictable DNA off-target effects.

Glycosylase base editor (GBE) can induce C-to-G transversion in mammalian cells, showing great promise for the treatment of human genetic disorders. However, the limited efficiency of transversion and the possibility of off-target effects caused by Cas9 restrict its potential clinical applications. In our recent study, we have successfully developed TaC9-CBE and TaC9-ABE by separating nCas9 and deaminase, which eliminates the Cas9-dependent DNA off-target effects without compromising editing efficiency. We developed a novel GBE called TaC9-GBEYE1, which utilizes the deaminase and UNG-nCas9 guided by TALE and sgRNA, respectively. TaC9-GBEYE1 showed comparable levels of on-target editing efficiency to traditional GBE at 19 target sites, without any off-target effects caused by Cas9 or TALE. The TaC9-GBEYE1 is a safe tool for gene therapy.

PlasmidHunter: accurate and fast prediction of plasmid sequences using gene content profile and machine learning.

Plasmids are extrachromosomal DNA found in microorganisms. They often carry beneficial genes that help bacteria adapt to harsh conditions. Plasmids are also important tools in genetic engineering, gene therapy, and drug production. However, it can be difficult to identify plasmid sequences from chromosomal sequences in genomic and metagenomic data. Here, we have developed a new tool called PlasmidHunter, which uses machine learning to predict plasmid sequences based on gene content profile. PlasmidHunter can achieve high accuracies (up to 97.6%) and high speeds in benchmark tests including both simulated contigs and real metagenomic plasmidome data, outperforming other existing tools.

Bio-Nano Toolbox for Precision Alzheimer's Disease Gene Therapy.

Alzheimer's disease (AD) is the most burdensome aging-associated neurodegenerative disorder, and its treatment encounters numerous failures during drug development. Although there are newly approved in-market β-amyloid targeting antibody solutions, pathological heterogeneity among patient populations still challenges the treatment outcome. Emerging advances in gene therapies offer opportunities for more precise personalized medicine; while, major obstacles including the pathological heterogeneity among patient populations, the puzzled mechanism for druggable target development, and the precision delivery of functional therapeutic elements across the blood-brain barrier remain and limit the use of gene therapy for central neuronal diseases. Aiming for "precision delivery" challenges, nanomedicine provides versatile platforms that may overcome the targeted delivery challenges for AD gene therapy. In this perspective, to picture a toolbox for AD gene therapy strategy development, the most recent advances from benchtop to clinics are highlighted, possibly available gene therapy targets, tools, and delivery platforms are outlined, their challenges as well as rational design elements are addressed, and perspectives in this promising research field are discussed.

Targeted modulation of gene expression through receptor-specific delivery of small interfering RNA peptide conjugates.

Small interfering RNA (siRNA) has emerged as a valuable tool to address RNA interference (RNAi) to modulate gene expression also in therapy. However, challenges such as inefficient cell targeting and rapid degradation in biological systems have limited its success. To address these issues, the development of a receptor-specific shuttle system represents a promising solution. [F7,P34]-NPY analogues were modified by solid-phase peptide synthesis, enabling non-covalent conjugation with siRNA. This modification yielded an efficient siRNA vehicle capable of binding and transporting its cargo into target cells without adversely affecting receptor activation or cell viability. Mass spectrometry and gel shift assays confirmed successful and stable siRNA binding under various conditions. Microscopy experiments further demonstrated the co-internalization of labeled peptides and siRNA in Hepa1c1 cells, highlighting the stability of the complex. In vitro quantitative RT-PCR experiments, targeting the TSC22D4 gene to normalize systemic glucose homeostasis and insulin resistance, revealed a functional peptide-based siRNA shuttle system with the ability to decrease mRNA expression to approximately 40%. These findings strengthen the potential of receptor-specific siRNA shuttle systems as efficient tools for gene therapy that offer a possibility for reducing side effects.

Gene targeting in adult organs using in vivo cleavable donor plasmids for CRISPR-Cas9 and CRISPR-Cas12a

The CRISPR-Cas system for in vivo genome editing is a powerful tool for gene therapy against several diseases. We have previously developed the pCriMGET_9-12a system, an in vivo cleavable donor plasmid for precise targeted knock-in of exogenous DNA by both Cas9 and Cas12a. Here, we show that the pCriMGET_9-12a system can be applied for in vivo in-frame knock-in of exogenous DNA in adult mouse liver by hydrodynamic delivery of the targeting plasmids. The in vivo cleavable pCriMGET_9-12a donor plasmids significantly increased the knock-in efficiency of both CRISPR-Cas9 and CRISPR-Cas12a in the adult mouse liver compared to uncleavable donor plasmids. This strategy also achieved in-frame reporter gene knock-in without indel mutations. Therefore, in vivo gene targeting using the pCriMGET_9-12a system may contribute to the establishment of safer, more precise, versatile and efficient gene therapy methods in adult organs.

Accelerated Development of AAV Purification Process Using a High-Throughput and Automated Crossflow System

Adeno-associated viruses (AAV) are currently predominant viral transfer tools for gene therapy, and efforts are being made to design faster and more efficient methods and technologies for their manufacturing. The early selection of high-performing filters is essential for developing an ultrafiltration and diafiltration (UF/DF) process, especially when feed material is scarce, and timelines are short. However, few methods and technologies exist to enable process optimization with multiple variations in a single run. In this study, we explored the potential of Ambr® Crossflow for high-throughput, automated screening of different membrane materials, pore sizes and different process conditions for the UF/DF step of AAV8. The best overall performance was obtained with a 100 kDa PES flat sheet cassette. The UF/DF process was further transferred to a larger scale to the Sartoflow® Smart Tangential Flow Filtration (TFF) system using a 100 kDa PES Sartocon® Slice 200 cassette and compared to a 100 kDa PES hollow fiber. Virus recovery, permeate flux and total protein removal values of the flat sheet cassette were similar to those achieved in small-scale devices, and higher than those of the hollow fiber, thus demonstrating similar performance at a larger process scale. The high-throughput, automated method described herein allowed to screen multiple materials and process parameters of a UF/DF process in a time- and resource-efficient way, making it a useful tool to accelerate early-stage downstream process development of AAV.

Engineering lentivirus envelope VSV-G for liver targeted delivery of IDOL-shRNA to ameliorate hypercholesterolemia and atherosclerosis

Lentiviral vectors (LVs) have been widely used as a tool for gene therapies. However, tissue-selective transduction after systemic delivery remains a challenge. Inducible degrader of low-density lipoprotein receptor is an attractive target for treating hypercholesterolemia. Here, a liver-targeted lentiviral vector CS8-LV-shIDOL is developed by incorporating a hepatocyte-targeted peptide derived from circumsporozoite protein (CSP) into the lentivirus envelope for liver-targeted delivery of IDOL-shRNA to alleviate hypercholesterolemia. Tail-vein injection of CS8-LV-shIDOL results in extremely high accumulation in liver, and nearly undetectable in other organs in mice. Besides, it shows superior therapeutic efficacy in lowering serum LDL-C and reducing atherosclerotic lesions over unmodified LV-shIDOL in hyperlipidemic mice. Mechanically, the envelope-engineered CS8-LV-shIDOL can enter liver cells via low density lipoprotein receptor-related protein (LRP). Thus, this study provides a novel approach for liver-targeted delivery of IDOL-shRNA to treat hypercholesterolemia by using an envelope-engineered lentiviral vector, and this delivery system has great potential for liver-targeted transgene therapy.

Development of Cas13a-based therapy for cancer treatment.

Gene therapy has become a major focus of current biomedical research. CRISPR (Clustered Regularly Inter spaced Short Palindromic Repeats) systems have been extensively researched for disease treatment applications through genome editing specificity. Compared with Cas9 (CRISPR-associated proteins, Cas), a commonly used tool enzyme for genome editing, Cas13a exhibits RNA-dependent endonuclease activity, including collateral cleavage without obvious potential genetic risks. With its high specificity, Cas13a has significantly improved the sensitivity of viral diagnosis and shown potential to eliminate viruses. However, its efficacy in tumor therapy has not been determined. This review introduces the mechanism and research developments associated with the CRISPR-Cas13a system in tumor treatments and its potential to be used as a new tool for gene therapy. We hope more research would apply Cas13a-based therapy in cancer treatment in the future.

Tumor-targeted delivery of SNHG15 siRNA using a ZIF-8 nanoplatform: Towards a more effective prostate cancer therapy

Small interfering RNAs (siRNAs) can be used as a powerful tool in gene therapy to downregulate the expression of specific disease related genes. Some properties however, such as instability, and low penetration into cells can limit their efficacy, and thus reduce their therapeutic potential. Metal-organic frameworks (MOFs) such as zeolitic imidazolate framework-8 (ZIF-8), which consist of organic bridging ligands and metal cations (Zn), have a very high binding affinity with nucleic acids including siRNAs. In this study, we designed a PEGylated ZIF-8 platform that was equipped with epithelial cell adhesion molecule (EpCAM) aptamer for the targeted delivery of siRNA molecules, in order to knockdown SNHG15 in both a prostate cancer (PC) cell line, and a human PC xenograft mouse model. SNHG15 is a long noncoding RNA, with oncogenic roles in different cancers including PC. The results indicated that the depletion of SNHG15 by Apt-PEG-siRNA@ZIF-8 nanoplatfrom inhibited cell proliferation and colony formation, and increased apoptosis in PC cells. This nanoparticle facilitated the release of siRNAs into the tumor environment in vivo, and subsequently reduced the tumor growth, with no side effects observed in vital organs. We have therefore developed a novel siRNA nano-delivery system for targeted prostate cancer treatment; however further studies are required before it can be tested in clinical trials.

Helical reconstruction of VP39 reveals principles for baculovirus nucleocapsid assembly

Baculoviruses are insect-infecting pathogens with wide applications as biological pesticides, in vitro protein production vehicles and gene therapy tools. Its cylindrical nucleocapsid, which encapsulates and protects the circular double-stranded viral DNA encoding proteins for viral replication and entry, is formed by the highly conserved major capsid protein VP39. The mechanism for VP39 assembly remains unknown. We use electron cryomicroscopy to determine a 3.2 Å helical reconstruction of an infectious nucleocapsid of Autographa californica multiple nucleopolyhedrovirus, revealing how dimers of VP39 assemble into a 14-stranded helical tube. We show that VP39 comprises a distinct protein fold conserved across baculoviruses, which includes a Zinc finger domain and a stabilizing intra-dimer sling. Analysis of sample polymorphism shows that VP39 assembles in several closely-related helical geometries. This VP39 reconstruction reveals general principles for baculoviral nucleocapsid assembly.