Viral Delivery System Research Articles

Establishment of enterically transmitted hepatitis virus animal models using lipid nanoparticle-based full-length viral genome RNA delivery system

BackgroundEnterically transmitted hepatitis viruses, such as hepatitis A virus (HAV) and hepatitis E virus (HEV), remain notable threats to public health. However, stable and reliable animal models of HAV and...

Generation of a human induced pluripotent stem cell line NTUHi004-A from a patient with Leigh syndrome harboring a homozygous missense mutation c.836 T > G (p.Met279Arg) in NDUFAF5 gene

Leigh syndrome is a rare autosomal recessive disorder showcasing a diverse range of neurological symptoms. Classical Leigh syndrome is associated with mitochondrial complex I deficiency, primarily resulting from biallelic mutations in the NDUFAF5 gene, encoding the NADH:ubiquinone oxidoreductase complex assembly factor 5. Using the Sendai virus delivery system, we generated an induced pluripotent stem cell line from peripheral blood mononuclear cells of a 47-years-old female patient who carried a homozygous NDUFAF5 c.836 T > G (p.Met279Arg) mutation. This cellular model serves as a tool for investigating the underlying pathogenic mechanisms and for the development of potential treatments for Leigh syndrome.

Challenges in Development and Qualification of PCR/dPCR Assays for Gene Therapy Biodistribution and Viral Shedding Assessment

Gene therapies are part of a larger class of advanced therapies that aim to treat disease via delivery of recombinant genetic material. A gene therapy product has two components, the delivery system (viral vector or non-viral) and the transgene (DNA or RNA). These therapies act via replacement of a non-functional gene, silencing of a disease-causing gene, or introduction of a new or modified gene with the goal of generating a therapeutic response in patients. Gene therapy biodistribution and viral vector shedding must be evaluated during non-clinical testing. Polymerase chain reaction (PCR) has emerged as the technique of choice to quantify the gene therapy product and the transferred genetic material in study samples. With increasing numbers of gene therapies in pre-clinical development, there has been a concomitant increase in the use of PCR in bioanalytical laboratories. A major challenge in this space is the lack of formal guidance for the development, characterization, and validation of PCR assays. This article will focus on the opportunities and challenges in developing and characterizing non-GLP, digital PCR assays for AAV gene therapy products. AAV vectors are currently the most common viral delivery system, however many of the insights presented will be applicable to other delivery systems.

Analytical ultracentrifugation sedimentation velocity for the characterization of recombinant adeno-associated virus vectors sub-populations

Recombinant adeno-associated virus virus-derived vectors (rAAVs) are among the most used viral delivery system for in vivo gene therapies with a good safety profile. However, rAAV production methods often lead to a heterogeneous vector population, in particular with the presence of undesired empty particles. Analytical ultracentrifugation sedimentation velocity (AUC-SV) is considered as the gold analytical technique allowing the measurement of relative amounts of each vector subpopulation and components like particle aggregates, based on their sedimentation coefficients. This letter presents the principle and practice of AUC experiments for rAAVs characterization. We discuss our results in the framework of previously published works. In addition to classical detection at 260nm, using interference optics in the ultracentrifuge can provide an independent estimate of weight percentages of the different populations of capsids, and of the genome size incorporated in rAAV particles.

Generation of induced pluripotent stem cells (IBMSi027-A) from a patient with hearing loss carrying WFS1 c.2051C > T (p.Ala684Val) variant

Pathogenic variants of the WFS1 gene can cause recessive-inherited Wolfram syndrome or dominant-inherited Wolfram-like syndrome with optic atrophy and hearing impairment. Using the Sendai virus delivery system, we generated induced pluripotent stem cells from the peripheral blood mononuclear cells of a female patient with the WFS1 pathogenic variant c.2051C > T (p.Ala684Val). The resulting induced pluripotent stem cells exhibited a normal karyotype and pluripotency, as confirmed using immunofluorescence staining, and differentiated into three germ layers in vivo. This cellular model provides a useful platform for investigating the pathogenic mechanisms of both blindness and deafness related to WFS1 variants.

Oridonin employs interleukin 1 receptor type 2 to treat noise-induced hearing loss by blocking inner ear inflammation

NOD-like receptor protein 3 (NLRP3) inflammasomes trigger the inflammatory cascades and participate in various inflammatory diseases, including noise-induced hearing loss (NIHL) caused by oxidative stress. Recently, the anti-inflammatory traditional medicine oridonin (Ori) has been reported to provide hearing protection in mice after noise exposure by blocking the NLRP3-never in mitosis gene A-related kinase 7 (NEK7)-inflammasome complex assembly. Using RNA sequencing analysis, we further elucidated that interleukin 1 receptor type 2 (IL1R2) may be another crucial factor regulated by Ori to protect NIHL. We observed that IL1R2 expression was localized in spiral ganglion neurons, inner and outer hair cells, in Ori-treated mouse cochleae. Additionally, we confirmed that ectopic overexpression of IL1R2 in the inner ears of healthy mice using an adeno-associated virus delivery system significantly reduced noise-induced ribbon synapse lesions and hearing loss by blocking the “cytokine storm” in the inner ear. This study provides a novel theoretical foundation for guiding the clinical treatment of NIHL.

Engineered biocontainable RNA virus vectors for non-transgenic genome editing across crop species and genotypes

CRISPR/Cas genome-editing tools provide unprecedented opportunities for basic plant biology research and crop breeding. However, the lack of robust delivery methods has limited the widespread adoption of these revolutionary technologies in plant science. Here, we report an efficient, non-transgenic CRISPR/Cas delivery platform based on the engineered tomato spotted wilt virus (TSWV), an RNA virus with a host range of over 1000 plant species. We eliminated viral elements essential for insect transmission to liberate genome space for accommodating large genetic cargoes without sacrificing the ability to infect plant hosts. The resulting non-insect-transmissible viral vectors enabled effective and stable in planta delivery of Cas12a and Cas9 nucleases as well as adenine and cytosine base editors. In systemically infected plant tissues, the deconstructed TSWV-derived vectors induced efficient somatic gene mutations and base conversions in multiple crop species with little genotype dependency. Plants with heritable, bi-allelic mutations could be readily regenerated by culturing the virus-infected tissues in vitro without antibiotic selection. Moreover, we showed that antiviral treatment with ribavirin during tissue culture cleared the viral vectors in 100% of regenerated plants and further augmented the recovery of heritable mutations. Because many plants are recalcitrant to stable transformation, the viral delivery system developed in this work provides a promising tool to overcome gene delivery bottlenecks for genome editing in various crop species and elite varieties.

Chromatographic Purification of Viral Vectors for Gene Therapy Applications.

Chromatography has been a mainstay in the downstream processing and purification of biopharmaceutical medicines. Until now, this has largely involved the purification of protein products such as recombinant enzymes and monoclonal antibodies using large-scale column chromatography methods. The development of advanced therapeutic medicinal products (ATMP) is heralding in a new era of therapeutics for a range of indications. These new therapeutics use diverse substances ranging from live stem cell preparations to fragments of nucleic acid enclosed in a viral delivery system. With these new technologies come new challenges in their purification. In this chapter, the challenges faced in producing and purifying viral vectors capable of delivering life-altering gene therapy to the patient will be discussed. Current methods of chromatography capable of adaptation to meet these new challenges and advancements that may be needed to increase the purification capabilities for these new products will also be discussed.

Genetic transformation and gene delivery strategies in <i>Carica papaya</i> L.

Papaya (<italic>Carica papaya</italic> L.) is a popular tropical fruit of high commercial value due to its nutritional, medicinal and industrial properties. However, conventional breeding methods for papaya have several limitations, including a scarcity of natural genetic resources in the <italic>Carica</italic> genus and sexual incompatibility among closely-related species. To overcome these challenges, particle bombardment, agrobacterium-mediated transformation, and pollen tube-mediated transformation have been developed for generating genetically modified papaya with the desired traits over the past three decades. These transformation methods have been successfully employed to improve papaya's resistance to biotic and abiotic stress, fruit quality, and even to produce edible vaccines. Notably, papaya ringspot virus-resistant transgenic papaya is the first successful commercial application of genetic engineering technology in a fruit crop. Recently, CRISPR/Cas9-mediated genome editing technology and viral vector-based gene delivery system have emerged as promising tools for papaya genetic improvement and functional genomic studies in papaya. Genome editing will open a new era of precision breeding for papaya. This review aims to highlight past and recent gene transformation methods and their applications in papaya breeding, as well as future perspectives and challenges in improving papaya via genetic engineering.

Formulation for the Targeted Delivery of a Vaccine Strain of Oncolytic Measles Virus (OMV) in Hyaluronic Acid Coated Thiolated Chitosan as a Green Nanoformulation for the Treatment of Prostate Cancer: A Viro-Immunotherapeutic Approach.

Oncolytic viruses are reported as dynamite against cancer treatment nowadays. In the present work, a live attenuated oral measles vaccine (OMV) strain was used to formulate a polymeric surface-functionalized ligand-based nanoformulation (NF). OMV (half dose: not less than 500 TCID units; 0.25 mL) was encapsulated in thiolated chitosan and outermost coating with hyaluronic acid by ionic gelation method characterizing parameters was performed. CD44 high expression was confirmed in prostatic adenocarcinoma (PRAD) by GEPIA which extracted data of normal and cancer tissue from GTEx and TCGA. Bioinformatics tools confirmed the viral hemagglutinin capsid protein interaction with human Caspase-I, NLRP3, and TNF-α and viral fusion protein interaction with COX-II and Caspase-I after successful delivery of MV encapsulated in NFs due to high affinity of hyaluronic acid with CD44 on the surface of prostate cancer cells. Particle size = 275.6 mm, PDI = 0.372, and ±11.5 zeta potential were shown by zeta analysis, while the thiolated group in NFs was confirmed by FTIR and Raman analysis. SEM and XRD showed a spherical smooth surface and crystalline nature, respectively, while TEM confirmed virus encapsulation within nanoparticles, which makes it very useful in targeted virus delivery systems. The virus was released from NFs in a sustained but continuous release pattern till 48 h. The encapsulated virus titer was calculated as 2.34×107 TCID50/mL units, which showed syncytia formation on post-day infection 7. Multiplicities of infection 0.1, 0.5, 1, 3, 5, 10, 15, and 20 of HA-coated OMV-loaded NFs as compared to MV vaccine on PC3 was inoculated with IC50 of 5.1 and 3.52, respectively, and growth inhibition was seen after 72 h via MTT assay which showed apoptotic cancer cell death. Active targeted, efficacious, and sustained delivery of formulated oncolytic MV is a potent moiety in cancer treatment at lower doses with safe potential for normal prostate cells.

Combined anti-tumor efficacy of somatostatin fusion protein and vaccinia virus on tumor cells with high expression of somatostatin receptors

Somatostatin, a growth hormone-release inhibiting peptide, exerts antiproliferative and antiangiogenic effects on tumor cells. However, the short half-life of somatostatin limits its application in human therapy, and long-acting somatostatin fusion protein is also limited by its severe terminal degradation. Therefore, oncolytic virus delivery system was introduced to express somatostatin fusion protein and the anti-tumor effects of both somatostatin and oncolytic virus were combined to destroy tumor tissues. Here, a vaccinia VG9/(SST-14)2-HSA recombinant was constructed by replacing somatostatin fusion gene into TK locus of attenuated VG9 strain via homologous recombination. Results showed that vaccinia VG9/(SST-14)2-HSA possessed a combined anti-tumor effect on sstr-positive tumor cells in vitro. In the tumor burden models, BALB/c mice with complete immunity are most suitable for evaluating tumor regression and immune activation. Complete tumor regression was observed in 3 out of 10 mice treated with vaccinia VG9/TK− or VG9/(SST-14)2-HSA, and the survival of all mice in both groups was significantly prolonged. Besides, vaccinia VG9/(SST-14)2-HSA is more effective in prolonging survival than VG9/TK−. Vaccinia VG9/(SST-14)2-HSA exerts a combined anti-tumor efficacy including the oncolytic ability provided by the virus and the anti-tumor effect contributed by (SST-14)2-HSA, which is expected to become a potent therapeutic agent for cancer treatment.

Generation of induced pluripotent stem cells from a patient with hearing loss carrying OPA1 c.1468T>C (p.Cys490Arg) variant

Pathogenic variants of OPA1 have been associated with autosomal dominant optic atrophy (DOA), leading to optic, auditory, and other sensorineural neuropathies and myopathies. Using the Sendai virus delivery system, we generated induced pluripotent stem cells from the peripheral blood mononuclear cells of a female patient with the OPA1 pathogenic variant c.1468T>C (p.Cys490Arg). The resulting induced pluripotent stem cells exhibited a normal karyotype and pluripotency, as confirmed using immunofluorescence staining, and differentiated into three germ layers in vivo. This cellular model is a useful platform for investigating the pathogenic mechanisms of both blindness and deafness related to OPA1 variants.

Generation of a human induced pluripotent stem cell line NTUHi002-A from a patient with aceruloplasminemia harboring a homozygous splicing mutation c.607+1 delG in CP gene

Aceruloplasminemia is a rare autosomal recessive disorder caused by mutations in the CP gene, encoding the copper-binding protein ceruloplasmin. A mutation in the CP gene results in brain and systemic iron overload, which is classified as a rare subtype of neurodegeneration with brain iron accumulation (NBIA). Here, we used the Sendai virus delivery system to generate induced pluripotent stem cells from peripheral blood mononuclear cells of a patient carrying the CP c.607+1 delG homozygous splicing mutation. The generated cell line retained the original genotype, expressed pluripotency markers, and differentiated into cells of the three germ layers.

Transcriptional switch of hepatocytes initiates macrophage recruitment and T-cell suppression in endotoxemia

The liver plays crucial roles in the regulation of immune defense during acute systemic infections. However, the roles of liver cellular clusters and intercellular communication in the progression of endotoxemia have not been well-characterized. Single-cell RNA sequencing analysis was performed, and the transcriptomes of 19,795 single liver cells from healthy and endotoxic mice were profiled. The spatial and temporal changes in hepatocytes and non-parenchymal cell types were validated by multiplex immunofluorescence staining, bulk transcriptomic sequencing, or flow cytometry. Furthermore, we used an adeno-associated virus delivery system to confirm the major mechanisms mediating myeloid cell infiltration and T-cell suppression in septic murine liver. We identified a proinflammatory hepatocyte (PIH) subpopulation that developed primarily from periportal hepatocytes and to a lesser extent from pericentral hepatocytes and played key immunoregulatory roles in endotoxemia. Multicellular cluster modeling of ligand-receptor interactions revealed that PIHs play a crucial role in the recruitment of macrophages via the CCL2-CCR2 interaction. Recruited macrophages (RMs) released cytokines (e.g., IL6, TNFα, and IL17) to induce the expression of inhibitory ligands, such as PD-L1, on hepatocytes. Subsequently, RM-stimulated hepatocytes led to the suppression of CD4+ and memory T-cell subsets partly via the PD-1/PD-L1 interaction in endotoxemia. Furthermore, sinusoidal endothelial cells expressed the highest levels of proapoptotic and inflammatory genes around the periportal zone. This pattern of gene expression facilitated increases in the number of fenestrations and infiltration of immune cells in the periportal zone. Our study elucidates unanticipated aspects of the cellular and molecular effects of endotoxemia on liver cells at the single-cell level and provides a conceptual framework for the development of novel therapeutic approaches for acute infection. The liver plays a crucial role in the regulation of immune defense during acute systemic infections. We identified a proinflammatory hepatocyte subpopulation and demonstrated that the interactions of this subpopulation with recruited macrophages are pivotal in the immune response during endotoxemia. These novel findings provide a conceptual framework for the discovery of rational therapeutic targets in acute infection.

HGG-16. DISSECTING THE EFFECT OF ATM DELETION ON RADIOSENSITIVITY IN DIFFUSE MIDLINE GLIOMAS WITH H3K27M MUTATION

Abstract Diffuse midline gliomas (DMGs) are responsible for a large proportion of childhood brain tumor deaths. Currently, radiation therapy is thought to be one of the most effective treatment options, but more than 90% of children still die within 2 years of diagnosis. DMGs are defined by somatic histone 3 K27M (H3K27M) mutations that have been shown to promote the G0/G1 to S cell cycle transition. The majority of DMGs also contain loss-of-function mutations in TP53. Prior research demonstrated that orthotopic xenograft and primary mouse models of non-H3K27M-mutated gliomas with inactivation of p53 are preferentially radiosensitized by inactivation of Ataxia Telangiectasia Mutated (ATM), a kinase that mediates DNA repair in response to DNA damage caused by radiation. The high frequency of mutations that deregulate p53 in DMGs raises the possibility that H3K27M-mutant DMGs may also be radiosensitized by ATM inhibition, representing a unique therapeutic opportunity. Here, we hypothesize that H3K27M-mutant DMGs that have loss of function of p53 will be radiosensitized by loss of ATM. To test this hypothesis, we used the RCAS-TVA viral gene delivery system to generate genetically-faithful primary mouse models of H3K27M-mutant DMG with p53 deletion, and we used Cre recombinase to delete Atm in the tumor cells of these mice and generated littermate controls that retained Atm. Mice were imaged weekly via luciferase-based bioluminescence to track tumor development and irradiated with three daily fractions of 10 Gy after tumor detection. We subsequently quantified the survival of mice without neurological decline following irradiation. In separate cohorts, we collected primary tumors after irradiation to verify H3K27M expression and to assess cell cycle arrest and mechanisms of cell death. These studies will elucidate mechanisms by which ATM inactivation can radiosensitize H3K27M-mutant DMGs with nonfunctioning p53, which will guide the design of clinical trials testing ATM inhibitors in DMG patients.

Sevoflurane impairs m6A-mediated mRNA translation and leads to fine motor and cognitive deficits.

Clinical surgical practices have found that children who undergo multiple anesthesia may have an increased risk of deficiencies in cognition and fine motor control. Here, we report that YT521-B homology domain family 1 (YTHDF1), a critical reader protein for N6-methyladenosine-modified mRNA, was significantly downregulated in the prefrontal cortex of young mice after multiple sevoflurane anesthesia exposures. Importantly, sevoflurane led to a decrease in protein synthesis in mouse cortical neurons that was fully rescued by YTHDF1, suggesting that anesthesia may affect early brain development by affecting m6A-dependent mRNA translation. Transcriptome-wide experiments showed that numerous mRNA targets related to synaptic functions in the prefrontal mouse cortex were associated with m6A methylation and YTHDF1. In particular, we found that synaptophysin, a critical presynaptic protein, was specifically modified by m6A methylation and associated with YTHDF1, and m6A methylation of synaptophysin decreased with multiple sevoflurane exposures. Importantly, we showed that fine motor control skills and cognitive functions were impaired in mice with multiple anesthesia exposures, and these effects were fully reversed by reintroducing YTHDF1 through a blood-brain barrier (BBB)-crossing viral delivery system. Finally, we found that the fine motor skills in children who underwent prolonged anesthesia were compromised 6months after surgery. Our findings indicated that impairment in the translational regulation of mRNA via N6-methyladenosine methylation is a potential mechanism underlying the effects of anesthesia on neural development in the young brain. 1. N6-methyladenosine (m6A) modifications were involved in anesthesia-induced neurotoxicity. 2. Sevoflurane impairs m6A-mediated mRNA translation and leads to fine motor deficits in young mice. 3. YTHDF1, a m6A reader protein, rescued sevoflurane-induced protein synthesis inhibition and fine motor deficits in young mice.

Use of nanotechnology in the diagnosis and treatment of coronavirus

Coronavirus is a beta virus that has caused a worldwide pandemic since December 2019. Many treatments such as antiviral drugs, immunosuppressive drugs, neutralizing antibodies, and monoclonal antibodies have been tested on coronavirus disease 2019 (COVID-19) that most of them were effective. Given that nanotechnology-based approaches have been successful in detection and treatment of viral systems such as human immunodeficiency virus (HIV), influenza A virus subtype H1N1 and Middle East respiratory syndrome coronavirus (MERS-CoV), they also seem to be effective in detecting and treating COVID-19. Nanotechnology is used in various methods for early and rapid diagnosis of the disease. Nanoparticles can be used in products for the diagnosis, treatment and prevention of COVID-19. These substances are very effective in the controlled delivery of antiviral drugs and biomolecules and they are also used in the manufacture of personal safety equipment, widely, and the production of anti-virus coatings for surfaces, air filters and the production of vaccines. In general, nanomaterial can play an important role in controlling the disease, based on strategies to prevent the virus from entering the host cell, inhibiting virus replication, virus delivery systems, and nano-based vaccines. Nanotechnology is a multidisciplinary tool that can offer a variety of solutions based on disease prevention, diagnosis and treatment strategies.

From the Literature

From the Literature

An induced pluripotent stem cells line (ZZUNEUi022-A) derived from urine cells of healthy male human.

Urine cells (or renal tubular cells) can be isolated from human urine samples efficiently. This noninvasive and cost-effective method to collect biological sample provide us favorable access to donor cells from human. In the present study, we generate ZZUNEUi022-A, a urine cells-derived induced pluripotent stem cell (iPSC) line, from a 29-year-old healthy male via Sendai virus delivery system. ZZUNEUi022-A showed stable karyotype, and could differentiate into three germ layers (ectoderm, mesoderm, and endoderm) readily in an embryoid body formation model.

Generation of a human induced pluripotent stem cell (iPSC) line (IBMS-iPSC-070-02) from a patient with neurodegeneration with brain iron accumulation (NBIA) having compound heterozygous mutations in PANK2 gene.

Neurodegeneration with brain iron accumulation (NBIA) is a genetically and phenotypically heterogeneous group of inherited neurodegenerative disorder characterized by basal ganglia iron deposition. Mutations in Pantothenate Kinase 2 (PANK2) are major genetic causes for patients with NBIA. The location of PANK2 in the mitochondria suggests mutant PANK2 causing mitochondrial dysfunction in the pathogenesis of NBIA. Here, we used the Sendai virus delivery system to generate induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells of a female patient having compound heterozygous mutations in PANK2. This cellular model could provide a platform for pathophysiological studies of NBIA in the future.