Tool For Gene Therapy Research Articles

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.

Advances in CRISPR/Cas systems-based cell and gene therapy.

Cell and gene therapy are innovative biomedical strategies aimed at addressing diseases at their genetic origins. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) systems have become a groundbreaking tool in cell and gene therapy, offering unprecedented precision and versatility in genome editing. This chapter explores the role of CRISPR in gene editing, tracing its historical development and discussing biomolecular formats such as plasmid, RNA, and protein-based approaches. Next, we discuss CRISPR delivery methods, including viral and non-viral vectors, followed by examining the various engineered CRISPR variants for their potential in gene therapy. Finally, we outline emerging clinical applications, highlighting the advancements in CRISPR for breakthrough medical treatments.

Ultra-efficient delivery of CRISPR/Cas9 using ionic liquid conjugated polymers for genome editing-based tumor therapy.

Emerging CRISPR-Cas9 systems can rebuild DNA sequences in the genome in a spatiotemporal manner, offering a magic tool for biological research, drug discovery, and gene therapy. However, low delivery efficiency remains a major roadblock hampering the wide application of CRISPR-Cas9 gene editing talent. Herein, ionic liquid-conjugated polymers (IL-CPs) are explored as efficient platforms for CRISPR-Cas9 plasmid delivery and in vivo genome editing-based tumor therapy. Via molecular screening of IL-CPs, IL-CPs integrated with fluorination monomers (PBF) can encapsulate plasmids into hybrid nanoparticles and achieve over 90% delivery efficiency in various cells regardless of serum interference. In vitro and in vivo experiments demonstrate that PBF can mediate Cas9/PLK1 plasmids for intracellular delivery and therapeutic genome editing in tumor, achieving efficient tumor suppression. This work provides a new tool for safe and efficient CRISPR-Cas9 delivery and therapeutic genome editing, thus opening a new avenue for the development of ionic liquid polymeric vectors for genome editing and therapy.

Development of hematopoietic stem cell-targeted gene therapy

Hematopoietic stem cell (HSC)-targeted gene therapy is curative for various genetic blood diseases, and its efficacy has been demonstrated in recent clinical trials. HSCs have self-renewal and hematopoietic multipotency; therefore, repairing pathological mutations or defects in HSCs allows for a lifelong cure with a single treatment. Autologous HSC gene therapy has been developed by lentiviral gene addition or gene editing, and is an option for most patients because it does not require a compatible donor. Current HSC gene therapy is based on ex vivo methods, in which patient HSCs are harvested, genetically modified ex vivo, and autologously transplanted into patients. However, the complexity of this process and the high cost of treatment are hindering the spread of gene therapy. Therefore, in vivo HSC gene therapy is being developed to deliver gene therapy tools directly into bone marrow HSCs by administration without ex vivo culture.

A Replication-Competent Retroviral Vector Expressing the HERV-W Envelope Glycoprotein is a Potential Tool for Cancer Gene Therapy.

The fusogenic membrane glycoprotein (FMG) derived from the human endogenous retrovirus-W (HERV-W) exhibits fusogenic properties, making it a promising candidate for cancer gene therapy. When cells are transfected with HERV-W FMG, they can fuse with neighboring cells expressing the receptor, resulting in the formation of syncytia. These syncytia eventually undergo cell death within a few days. In addition, it has been observed that an HERV-W env mutant, which is truncated after amino acid 483, displays increased fusogenicity compared to the wild-type HERV-W env. In this study, we observed syncytium formation upon transfection of HeLa and TE671 human cancer cells with plasmids containing the HERV-W 483 gene. To explore the potential of a semi-replication-competent retroviral (s-RCR) vector encoding HERV-W 483 for FMG-mediated cancer gene therapy, we developed two replication-defective retroviral vectors: a gag-pol vector encoding HERV-W 483 (MoMLV-HERV-W 483) and an env vector encoding VSV-G (pCLXSN-VSV-G-EGFP). When MoMLV-HERV-W 483 and pCLXSN-VSV-G-EGFP were co-transfected into HEK293T cells to produce the s-RCR vector, gradual syncytium formation was observed. However, the titers of the s-RCR virus remained consistently low. To enhance gene transfer efficiency, we constructed an RCR vector encoding HERV-W 483 (MoMLV-10A1-HERV-W 483), which demonstrated replication ability in HEK293T cells. Infection of A549 and HT1080 human cancer cell lines with this RCR vector induced syncytium formation and subsequent cell death. Consequently, both the s-RCR vector and RCR encoding HERV-W 483 hold promise as valuable tools for cancer gene therapy.

Bibliometric analysis of global research trends in adeno-associated virus vector for gene therapy (1991-2022).

Gene therapy involves introducing and editing foreign genes in the body to treat and prevent genetic diseases. Adeno-associated virus (AAV) vector has become a widely used tool in gene therapy due to its high safety and transfection efficiency. This study employs bibliometric analysis to explore the foundation and current state of AAV vector application in gene therapy research. A total of 6,069 publications from 1991 to 2022 were analyzed, retrieved from the Science Citation Index Expanded (SCI-E) within the Web of Science Core Collection (WoSCC) of Clarivate Analytics. Institutions, authors, journals, references, and keywords were analyzed and visualized by using VOSviewer and CiteSpace. The R language and Microsoft Excel 365 were used for statistical analyses. The global literature on AAV vector and gene therapy exhibited consistent growth, with the United States leading in productivity, contributing 3,868 papers and obtaining the highest H-index. Noteworthy authors like Wilson JM, Samulski RJ, Hauswirth WW, and Mingozzi F were among the top 10 most productive and co-cited authors. The journal "Human Gene Therapy" published the most papers (n = 485) on AAV vector and gene therapy. Current research focuses on "gene editing," "gene structure," "CRISPR," and "AAV gene therapy for specific hereditary diseases." The application of AAV vector in gene therapy has shown continuous growth, fostering international cooperation among countries and institutions. The intersection of gene editing, gene structure, CRISPR, and AAV gene therapy for specific hereditary diseases and AAV vector represents a prominent and prioritized focus in contemporary gene therapy research. This study provides valuable insights into the trends and characteristics of AAV gene therapy research, facilitating further advancements in the field.

Expression and Functional Analysis of the Compact Thermophilic Anoxybacillus flavithermus Cas9 Nuclease

Research on Cas9 nucleases from different organisms holds great promise for advancing genome engineering and gene therapy tools, as it could provide novel structural insights into CRISPR editing mechanisms, expanding its application area in biology and medicine. The subclass of thermophilic Cas9 nucleases is actively expanding due to the advances in genome sequencing allowing for the meticulous examination of various microorganisms’ genomes in search of the novel CRISPR systems. The most prominent thermophilic Cas9 effectors known to date are GeoCas9, ThermoCas9, IgnaviCas9, AceCas9, and others. These nucleases are characterized by a varying temperature range of the activity and stringent PAM preferences; thus, further diversification of the naturally occurring thermophilic Cas9 subclass presents an intriguing task. This study focuses on generating a construct to express a compact Cas9 nuclease (AnoCas9) from the thermophilic microorganism Anoxybacillus flavithermus displaying the nuclease activity in the 37–60 °C range and the PAM preference of 5′-NNNNCDAA-3′ in vitro. Here, we highlight the close relation of AnoCas9 to the GeoCas9 family of compact thermophilic Cas9 effectors. AnoCas9, beyond broadening the repertoire of Cas9 nucleases, suggests application in areas requiring the presence of thermostable CRISPR/Cas systems in vitro, such as sequencing libraries’ enrichment, allele-specific isothermal PCR, and others.

Evaluation of the SH-SY5Y cell line as an in vitro model for potency testing of a neuropeptide-expressing AAV vector

Viral vectors have become important tools for basic research and clinical gene therapy over the past years. However, in vitro testing of vector-derived transgene function can be challenging when specific post-translational modifications are needed for biological activity. Similarly, neuropeptide precursors need to be processed to yield mature neuropeptides. SH-SY5Y is a human neuroblastoma cell line commonly used due to its ability to differentiate into specific neuronal subtypes. In this study, we evaluate the suitability of SH-SY5Y cells in a potency assay for neuropeptide-expressing adeno-associated virus (AAV) vectors. We looked at the impact of neuronal differentiation and compared single-stranded (ss) AAV and self-complementary (sc) AAV transduction at increasing MOIs, RNA transcription kinetics, as well as protein expression and mature neuropeptide production. SH-SY5Y cells proved highly transducible with AAV1 already at low MOIs in the undifferentiated state and even better after neuronal differentiation. Readouts were GFP or neuropeptide mRNA expression. Production of mature neuropeptides was poor in undifferentiated cells. By contrast, differentiated cells produced and sequestered mature neuropeptides into the medium in a MOI-dependent manner.