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Cellular immunotherapy has emerged as one of the most potent approaches to treating cancer patients. Adoptive transfer of chimeric antigen receptor (CAR) T cells as well as the use of haploidentical natural killer (NK) cells can induce remission in patients with lymphoma and leukemia. Although the use of CAR T cells has been established, this approach is currently limited for wider use by the risk of severe adverse events, including cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome. Moreover, the risk of triggering graft vs host reactions in settings of allogeneic T cell infusion limits the use to autologous CAR T cells if advanced CRISPR engineering is not applied. In contrast, NK cell-based cancer immunotherapy has emerged as a safe approach even in allogeneic settings. However, efficient transduction of primary blood NK cells with vesicular stomatitis virus G glycoprotein (VSV-G) pseudotyped lentivirus commonly used for T cell modification remains challenging. This article presents a detailed method that significantly enhances the transduction efficiency of NK cells by utilizing a short-term culture in cytokine-supplemented medium. It also encompasses the preparation of high-titer and high-quality lentiviral particles for optimal NK cell transduction. Overall, this protocol details the step-by-step culture of NK cells in cytokine-supplemented medium, their transduction with VSV-G lentiviral vectors, and subsequent expansion for functional assays. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Isolation of NK cells from human peripheral blood mononuclear cells (PBMCs) Basic Protocol 2: NK cell expansion and transduction with lentivirus for generating CAR-NK cells Support Protocol 1: Plasmid amplification Support Protocol 2: Lentivirus preparation Support Protocol 3: Lentivirus titration., (© 2024 Wiley Periodicals LLC.)

A key step in developing engineered B cells for therapeutic purposes is evaluation in immunocompetent, large-animal models. Therefore, we developed methods to purify, expand, and differentiate non-human primate (NHP; rhesus macaque) B cells. After 7 days in culture, B cells expanded 10-fold, differentiated into a plasma cell phenotype (CD38, CD138), and secreted immunoglobulin G. Using single-cell sequencing and flow cytometry, we verified the presence of plasma cell genes in differentiated NHP B cells and unearthed less-recognized markers, such as CD59 and CD79A. In contrast with human cells, we found that the immune checkpoint molecule CD274 (PD-L1) and major histocompatibility complex (MHC) class I molecules were upregulated in NHP plasma cells in the transcriptional data. Lastly, we established the conditions for efficient transduction of NHP B cells with adeno-associated virus (AAV) vectors, achieving a delivery rate of approximately 60%. We envision that this work will accelerate proof-of-concept studies using engineered B cells in NHPs., Competing Interests: Declaration of interests R.G.J. and D.J.R. hold equity in and serve on the scientific advisory board of Be Biopharma, Inc. S.K. is currently an employee at Astellas Pharma, Y.H. is currently an employee of Sonoma Biotherapeutics, and C.L. is currently an employee of Sartorius AG., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Patient-derived organoids (PDOs) are now used to study many diseases, including prostate cancer. Here, we present a protocol for the transduction of human epithelial prostate cells and PDOs. We describe the steps for producing lentiviruses and transducing PDOs with high efficiency to obtain either overexpression or knockdown of specific genes. More generally, this protocol represents an efficient lentiviral transduction technique to study cell biology using various organoid models., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Recombinant adeno-associated virus (rAAV) vectors are currently the only proven vehicles for treating ophthalmological diseases through gene therapy. A wide range of gene therapy programs that target ocular diseases are currently being pursued. Nearly 20 years of research have gone into enhancing the efficacy of targeting retinal tissues and improving transgene delivery to specific cell types. The engineered AAV capsid, AAV2.7m8 is currently among the best capsids for transducing the retina following intravitreal (IVT) injection. However, adverse effects, including intraocular inflammation, have been reported following retinal administration of AAV2.7m8 vectors in clinical trials. Furthermore, we have consistently observed that AAV2.7m8 exhibits low packaging titers irrespective of the vector construct design. In this report, we found that AAV2.7m8 packages vector genomes with a higher degree of heterogeneity than AAV2. We also found that genome-loaded AAV2.7m8 stimulated the infiltration of microglia in mouse retinas following IVT administration, while the response to genome-loaded AAV2 and empty AAV2.7m8 capsids produced much milder responses. This finding suggests that IVT administration of AAV2.7m8 vectors may stimulate retinal immune responses in part because of its penchant to package and deliver non-unit length genomes., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Different screening methods are being developed to generate adeno-associated viral vectors (AAV) with the ability to bypass the blood-brain barrier (BBB) upon intravenous administration. Recently, the AAV9P31 stood out as the most efficient version among a library of peptide-displaying capsids selected in C57BL/6 mice using RNA-driven biopanning. In this work we have characterized in detail its biodistribution in different mouse strains (C57BL/6 and Balb/c), as well as in Sprague Dawley rats and non-human primates (Macaca fascicularis). Using GFP and NanoLuc reporter genes, we confirmed homogeneous infection and transgene expression across the CNS of mice injected intravenously with AAV9P31. A more restricted pattern was observed upon either intracerebroventricular or intraparenchymal injection. Following intravenous delivery, region- and cell-specific differential patterns of transduction were observed in the mouse brain, including a preferential transduction of astrocytes and neurons in the cerebral cortex and striatum, whereas neurons were the only transduced cell type in subcortical locations across the hippocampus, thalamus, hypothalamus, mesencephalon, brainstem and cerebellum. Furthermore, transduced microglial cells were never found in any CNS location. Peripheral organs transduced upon intravenous administration included lung, liver, peritoneum, heart and skeletal muscle. However, a comparable performance of AAV9P31 to bypass the BBB in rats and macaques was not observed, although a more limited neuronal transduction was found in the brainstem of rats upon intravenous delivery. Finally, intracerebroventricular delivery in macaques resulted in neuronal transduction in cortical, subcortical structures and cerebellum following a patchy pattern. In conclusion, the widespread CNS transduction obtained in mice upon intravenous delivery of AAV9P31 represents a powerful tool for modeling a wide variety of neurological disorders as well as an appealing choice for the evaluation of gene therapy-based therapeutics., (© 2024. The Author(s).)

Background Aims: Chimeric antigen receptor (CAR) T-cell products are commonly generated using lentiviral vector (LV) transduction. Optimal final formulation buffer (FFB) supporting LV stability during cryostorage is crucial for cost-effective manufacturing., Methods: To identify the ideal LV FFB composition for ex vivo CAR-T production, primary human T cells were transduced with vesicular stomatitis virus G-protein (VSV-G) -pseudotyped LVs (encoding a reporter gene or an anti-CD19-CAR). The formulations included phosphate-buffered saline (PBS), HEPES, or X-VIVO TM 15, and stabilizing excipients. The functional and viral particle titers and vector copy number were measured after LV cryopreservation and up to 24 h post-thaw incubation. CAR-Ts were produced with LVs in selected FFBs, and the resulting cells were characterized., Results: Post-cryopreservation, HEPES-based FFBs provided higher LV functional titers than PBS and X-VIVO TM 15, and 10% trehalose-20 mM MgCl 2 improved LV transduction efficiency in PBS and 50 mM HEPES. Thawed vectors remained stable at +4°C, while a ≤ 25% median decrease in the functional titer occurred during 24 h at room temperature. Tested excipients did not enhance LV post-thaw stability. CAR-Ts produced using LVs cryopreserved in 10% trehalose- or sucrose-20 mM MgCl 2 in 50 mM HEPES showed comparable transduction rates, cell yield, viability, phenotype, and in vitro functionality., Conclusion: A buffer consisting of 10% trehalose-20 mM MgCl 2 in 50 mM HEPES provided a feasible FFB to cryopreserve a VSV-G -pseudotyped LV for CAR-T-cell production. The LVs remained relatively stable for at least 24 h post-thaw, even at ambient temperatures. This study provides insights into process development, showing LV formulation data generated using the relevant target cell type for CAR-T-cell manufacturing., Competing Interests: Declaration of competing interest AK, VT, MK, VA-S, PK, HL, EL, HPL, and TKe were employed by the Kuopio Center for Gene and Cell Therapy (KCT) during this project. The remaining authors have no commercial, proprietary, or financial interest in the products or companies described in this article., (Copyright © 2024 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

To overcome limitations in the generalizability and efficiency of current AAV vectors, in this current study, we constructed an AAV variant library by the insertion of random heptapeptide sequences in the receptor-binding domain of the AAV9 capsid gene. We then applied a recently developed organ-on-a-chip in vitro model of the human blood-brain barrier (BBB) to iteratively enrich for variants that efficiently cross the BBB and transduce astrocyte cells. Through multiple rounds of screening, we obtained two candidate AAV variants, AAV-M6 and AAV-M8, which showed significantly higher BBB penetration efficiency than AAV9 or AAV-PHP.eB. Quantitative PCR (qPCR) assay showed that AAV-M6 could accumulate to a 5 times higher titer, while AAV-M8 reached a 3 times higher titer, than AAV-PHP.eB in the neural chamber of the model. The transduction assay further verified that the AAV-M6 candidate vector was able to infect HA-1800 cells after crossing the BBB, suggesting it could potentially transduce brain parenchymal cells after crossing the hCMEC/D3 layer at higher efficiency than AAV-PHP.eB. Molecular simulations suggested that the human receptor proteins, LY6D and M6PR, could bind the AAV-M6 heptapeptide insertion with high affinity. This study provides two promising candidate AAV vectors and demonstrates the use of this in vitro BBB model for scalable, high-throughput screening of gene therapies. These tools can drive investigations of the mechanisms underlying BBB permeability and the cell-type specificity of virus vectors.

In vivo gene delivery to tissues using adeno-associated vector (AAVs) has revolutionized the field of gene therapy. Yet, while sensorineural hearing loss is one of the most common sensory disorders worldwide, gene therapy applied to the human inner ear is still in its infancy. Recent advances in the development recombinant AAVs have significantly improved their cell tropism and transduction efficiency across diverse inner ear cell types to a level that renders this tool valuable for conditionally manipulating gene expression in the context of developmental biology studies of the mouse inner ear. Here, we describe a protocol for in utero micro-injection of AAVs into the embryonic inner ear, using the AAV-PHP.eB and AAV-DJ serotypes that respectively target the sensory hair cells and the supporting cells of the auditory sensory epithelium. We also aimed to standardize procedures for imaging acquisition and image analysis to foster research reproducibility and allow accurate comparisons between studies. We find that AAV-PHP.eB and AAV-DJ provide efficient and reliable tools for conditional gene expression targeting cochlear sensory and supporting cells in the mouse inner ear, from late embryonic stages on., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Barbosa Spinola et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Allogeneic cell therapies, such as those involving macrophages or Natural Killer (NK) cells, are of increasing interest for cancer immunotherapy. However, the current techniques for genetically modifying these cell types using lenti- or gamma-retroviral vectors present challenges, such as required cell pre-activation and inefficiency in transduction, which hinder the assessment of preclinical efficacy and clinical translation. In our study, we describe a novel lentiviral pseudotype based on the Koala Retrovirus (KoRV) envelope protein, which we identified based on homology to existing pseudotypes used in cell therapy. Unlike other pseudotyped viral vectors, this KoRV-based envelope demonstrates remarkable efficiency in transducing freshly isolated primary human NK cells directly from blood, as well as freshly obtained monocytes, which were differentiated to M1 macrophages as well as B cells from multiple donors, achieving up to 80% reporter gene expression within three days post-transduction. Importantly, KoRV-based transduction does not compromise the expression of crucial immune cell receptors, nor does it impair immune cell functionality, including NK cell viability, proliferation, cytotoxicity as well as phagocytosis of differentiated macrophages. Preserving immune cell functionality is pivotal for the success of cell-based therapeutics in treating various malignancies. By achieving high transduction rates of freshly isolated immune cells before expansion, our approach enables a streamlined and cost-effective automated production of off-the-shelf cell therapeutics, requiring fewer viral particles and less manufacturing steps. This breakthrough holds the potential to significantly reduce the time and resources required for producing e.g. NK cell therapeutics, expediting their availability to patients in need., (© 2024. The Author(s).)

Targeted gene delivery to the brain is a critical tool for neuroscience research and has significant potential to treat human disease. However, the site-specific delivery of common gene vectors such as adeno-associated viruses (AAVs) is typically performed via invasive injections, which limit its applicable scope of research and clinical applications. Alternatively, focused ultrasound blood-brain-barrier opening (FUS-BBBO), performed noninvasively, enables the site-specific entry of AAVs into the brain from systemic circulation. However, when used in conjunction with natural AAV serotypes, this approach has limited transduction efficiency and results in substantial undesirable transduction of peripheral organs. Here, we use high throughput in vivo selection to engineer new AAV vectors specifically designed for local neuronal transduction at the site of FUS-BBBO. The resulting vectors substantially enhance ultrasound-targeted gene delivery and neuronal tropism while reducing peripheral transduction, providing a more than ten-fold improvement in targeting specificity in two tested mouse strains. In addition to enhancing the only known approach to noninvasively target gene delivery to specific brain regions, these results establish the ability of AAV vectors to be evolved for specific physical delivery mechanisms., (© 2024. The Author(s).)

Lentiviral vectors (LV) are efficient vehicles for in vivo gene delivery to the liver. LV integration into the chromatin of target cells ensures their transmission upon proliferation, thus allowing potentially life-long gene therapy following a single administration, even to young individuals. The glycoprotein of the vesicular stomatitis virus (VSV.G) is widely used to pseudotype LV, as it confers broad tropism and high stability. The baculovirus-derived GP64 envelope protein has been proposed as an alternative for in vivo liver-directed gene therapy. Here, we perform a detailed comparison of VSV.G- and GP64-pseudotyped LV in vitro and in vivo. We report that VSV.G-LV transduced hepatocytes better than GP64-LV, however the latter showed improved transduction of liver sinusoidal endothelial cells (LSEC). Combining GP64-pseudotyping with the high surface content of the phagocytosis inhibitor CD47 further enhanced LSEC transduction. Coagulation factor VIII (FVIII), the gene mutated in hemophilia A, is naturally expressed by LSEC, thus we exploited GP64-LV to deliver a FVIII transgene under the control of the endogenous FVIII promoter and achieved therapeutic amounts of FVIII and correction of hemophilia A mice., (© 2024. The Author(s).)

Background Aims: Gene therapy using lentiviral vectors (LVs) that harbor a functional β-globin gene provides a curative treatment for hemoglobinopathies including beta-thalassemia and sickle cell disease. Accurate quantification of the vector copy number (VCN) and/or the proportion of transduced cells is critical to evaluate the efficacy of transduction and stability of the transgene during treatment. Moreover, commonly used techniques for LV quantification, including real-time quantitative polymerase chain reaction (PCR) or fluorescence-activated cell sorting, require either a standard curve or expression of a reporter protein for the detection of transduced cells. In the present study, we describe a digital droplet PCR (ddPCR) technique to measure the lentiviral VCN in transduced hematopoietic stem and progenitor cells (HSPCs)., Methods: After HSPCs were transduced with an LV encoding the therapeutic β-globin (β A-T87Q ) gene, the integrated lentiviral sequence in the host genome was amplified with primers that targeted a sequence within the vector and the human RPP30 gene. The dynamic range of ddPCR was between 5 × 10 -3 ng and 5 × 10 -6 ng of target copy per reaction., Results: We found that the ddPCR-based approach was able to estimate VCN with high sensitivity and a low standard deviation. Furthermore, ddPCR-mediated quantitation of lentiviral copy numbers in differentiated erythroblasts correlated with the level of β A-T87Q protein detected by reverse-phase high-performance liquid chromatography., Conclusions: Taken together, the ddPCR technique has the potential to precisely detect LV copy numbers in the host genome, which can be used for VCN estimation, calculation of infectious titer and multiplicity of infection for HSPC transduction in a clinical setting., Competing Interests: Declaration of Competing Interest The authors have no commercial, proprietary or financial interest in the products or companies described in this article., (Copyright © 2024. Published by Elsevier Inc.)

Background: Transfection is an important analytical method for studying gene expression in the cellular environment. There are some barriers to efficient DNA transfection in host cells, including circumventing the plasma membrane, escaping endosomal compartmentalization, autophagy, immune sensing pathways, and translocating the nuclear envelope. Therefore, it would be very useful to introduce an optimum transfection approach to achieve a high transfection efficiency in the Vero cell line. The aim of this study was to compare various transfection techniques and introduce a highly efficient method for gene delivery in Vero cells., Methods: In the current study, three transfection methods were used, including chemical transfection, electroporation, and lentiviral vector transduction, to obtain the optimum transfection conditions in the Vero cell line. Vero cells were cultured and transfected with chemical transfection reagents, electroporation, or HIV-1-based lentivectors under different experimental conditions. Transfection efficiency was assessed using flow cytometry and fluorescence microscopy to detect GFP-positive cells., Results: Among the tested methods, TurboFect™ chemical transfection exhibited the highest efficiency. Optimal transfection conditions were achieved using 1 µg DNA and 4 µL TurboFect™ in 6 × 10 4 Vero cells., Conclusion: TurboFect™, a cationic polymer transfection reagent, demonstrated superior transfection efficiency in Vero cells compared with electroporation and lentivirus particles, and is the optimal choice for chemical transfection in the Vero cell line., (© 2024. The Author(s).)

Background: Adeno-associated virus (AAV)-based gene therapy is a promising strategy to treat muscle diseases. However, this strategy is currently confronted with challenges, including a lack of transduction efficiency across the entire muscular system and toxicity resulting from off-target tissue effects. Recently, novel myotropic AAVs named MyoAAVs and AAVMYOs have been discovered using a directed evolution approach, all separately demonstrating enhanced muscle transduction efficiency and liver de-targeting effects. However, these newly discovered AAV variants have not yet been compared., Methods: In this study, we performed a comparative analysis of these various AAV9-derived vectors under the same experimental conditions following different injection time points in two distinct mouse strains., Results: We highlight differences in transduction efficiency between AAV9, AAVMYO, MyoAAV2A and MyoAAV4A that depend on age at injection, doses and mouse genetic background. In addition, specific AAV serotypes appeared more potent to transduce skeletal muscles including diaphragm and/or to de-target heart or liver., Conclusions: Our study provides guidance for researchers aiming to establish proof-of-concept approaches for preventive or curative perspectives in mouse models, to ultimately lead to future clinical trials for muscle disorders., (© 2024. The Author(s).)

Manufacturing of recombinant adeno-associated virus (AAV) vectors produces three types of capsids: full, intermediate, and empty. While there are different opinions about the impact of intermediate and empty capsids on safety and efficacy of AAV products, they are generally considered impurities because they are not the intended fully intact vector product. The presence of these impurities could impact product efficacy due to potential competition with fully packaged AAVs for cellular transduction, as well as have potential implications to patient safety due to increased capsid load during dosing. To determine the impact of intermediate capsids on potency, an AAV preparation was separated into fractions enriched for full, intermediate, or empty capsids. Using a matrix of in vitro (infectivity, gene expression, biological activity) and in vivo potency assays to determine potency as a function of capsid content, our results indicate that while intermediate capsids contribute to the vector genome titer of the product and are equally as infectious as full capsids, they do not contribute to the potency of the AAV product. This study confirms the criticality of reducing and controlling the level of intermediate capsids to ensure a more efficacious AAV product., (© 2024. The Author(s).)

In this study, we introduce electrospun polydioxanone (PDO) nonwoven fabrics as a platform for the delivery of adeno-associated virus (AAV) vectors for transduction and genome editing by adhering them to organ surfaces, including the heart. AAV vectors were loaded onto the PDO fabrics by soaking the fabrics in a solution containing AAV vectors. In vitro, the amount of AAV vectors loaded onto the fabrics could be adjusted by changing their concentration in the solution, and the number of cells expressing the green fluorescent protein (GFP) encoded by the AAV vectors increased in correlation with the increasing amount of loaded AAV vectors. In vivo, both transduction and genome editing resulted in the observation of GFP expression around AAV vector-loaded PDO fabrics attached to the surfaces of mouse hearts, indicating effective transduction and expression at the target site. These results demonstrate the great potential of electrospun PDO nonwoven fabrics carrying therapeutic AAV vectors for gene therapy.

Astrocytes constitute a major part of the central nervous system and the delineation of their activity patterns is conducive to a better understanding of brain network dynamics. This study aimed to develop a magnetic resonance imaging (MRI)-based method in order to monitor the brain-wide or region-specific astrocytes in live animals. Adeno-associated virus (AAVs) vectors carrying the human glial fibrillary acidic protein (GFAP) promoter driving the EGFP-AQP1 (Aquaporin-1, an MRI reporter) fusion gene were employed. The following steps were included: constructing recombinant AAV vectors for astrocyte-specific expression, detecting MRI reporters in cell culture, brain regions, or whole brain following cell transduction, stereotactic injection, or tail vein injection. The astrocytes were detected by both fluorescent imaging and Diffusion-weighted MRI. The novel AAV mutation (Site-directed mutagenesis of surface-exposed tyrosine (Y) residues on the AAV5 capsid) significantly increased fluorescence intensity (p < 0.01) compared with the AAV5 wild type. Transduction of the rAAV2/5 carrying AQP1 induced the titer-dependent changes in MRI contrast in cell cultures (p < 0.05) and caudate-putamen (CPu) in the brain (p < 0.05). Furthermore, the MRI revealed a good brain-wide alignment between AQP1 levels and ADC signals, which increased over time in most of the transduced brain regions. In addition, the rAAV2/PHP.eB serotype efficiently introduced AOP1 expression in the whole brain via tail vein injection. This study provides an MRI-based approach to detect dynamic changes in astrocytes in live animals. The novel in vivo tool could help us to understand the complexity of neuronal and glial networks in different pathophysiological conditions., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Human induced pluripotent stem cells (hiPSCs) are a potential source of somatic cells for cell therapies due to their ability to self-renew and differentiate into various cells of the body. To date, the clinical application of hiPSCs has been limited due to safety issues. The present study aims to standardize the safety procedure of the derivation of GMP-compliant induced pluripotent stem cell (iPSC) lines from human fibroblasts. The hiPSC lines were generated using the nonintegrative Sendai virus method to incorporate Yamanaka reprogramming factors (OCT3/4, SOX2, KLF4 and c-MYC) into cells. A constant temperature was maintained during the cell culture, including all stages of the culture after transduction with Sendai virus. Pluripotency was proved in six independently generated hiPSC lines from adult female (47 years old) and male (57 years old) donors' derived fibroblasts via alkaline phosphatase live (ALP) staining, qPCR, and immunocytochemistry. The hiPSC lines showed a gradual decrease in the presence of the virus with each subsequent passage, and this reduction was specific to the hiPSC line. The frequency and probability of chromosomal aberrations in hiPSCs were dependent on both the iPSC clone identity and sex of the donor. In summary, the generation of hiPSC for clinical applications requires safety standards application (biosafety protocol, quality control of hiPSC lines, viral and genetic integrity screening) from the first stages of the clonal selection of hiPSC from the same donor.

Transformed lung organoids have extensive applications in lung cancer modeling and drug screening. Traditional two-dimensional (2D) cultures fail to propagate a large subpopulation of murine primary tumors in vitro. However, three-dimensional (3D) air-liquid interface (ALI) cultures, which are employed to grow normal lung organoids, can be used to efficiently culture cancerous lung tumor cells. Here, we detail a procedure for cultivating genetically modified lung organoids in 3D-ALI cultures. This protocol contains two parts. The first part describes how to transduce lung epithelial cells, which are either freshly sorted from lungs or from actively growing murine organoids, with virus in order to modify gene expression. The target lung cells are incubated with virus for 1-2 h for transduction. Then, the transduced cells are thoroughly washed and mixed with stromal support cells and Matrigel and are loaded into transwell inserts for culture and validated for genetic modifications through downstream assays. The second part describes how to isolate tumor cells growing orthotopically in genetically engineered mouse models to produce organoid cell lines that can be used for ex vivo drug discovery assays. For this protocol, tumors are isolated from lungs of mice, finely chopped and washed. Then, tumor chunks are mixed with Matrigel for 3D-ALI culture. Finally, organoids budding from tumor chunks are trypsinized and passaged to establish an organoid line. Together these two protocols provide a promising platform to study the genesis, progression, and treatment of lung cancer., (© 2024. The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Baculovirus-mediated gene expression in mammalian cells, BacMam, is a useful alternative to transient transfection for recombinant protein production in various types of mammalian cell lines. We decided to establish BacMam in our lab in order to streamline our workflows for gene expression in insect and mammalian cells, as it is straightforward to parallelize the baculovirus generation for both types of eukaryotic cells. This chapter provides a step-by-step description of the protocols we use for the generation of the recombinant BacMam viruses, the transduction of mammalian cell cultures, and optimization of the protein production conditions through small-scale expression and purification tests., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Lentiviral gene transfer represents a versatile and powerful method for genetic transduction of many cell lines and primary cells including "hard-to-transfect" cells. As a consequence of the integration of the recombinant lentiviral vector into the cellular genome, the transgene is stably maintained, and long-term producing cells are established. Here, we describe the current state of the art and give details for lab-scale production of lentiviral vectors as well as for infection and titration of the viral vectors., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Cellular senescence is characterized by cell cycle arrest and senescence-associated secretory phenotypes. Cellular senescence can be caused by various stress stimuli such as DNA damage, oxidative stress, and telomere attrition and is related to several chronic diseases, including atherosclerosis, Alzheimer's disease, and osteoarthritis. Chromobox homolog 4 (CBX4) has been shown to alleviate cellular senescence in human mesenchymal stem cells and is considered a possible target for senomorphic treatment. Here, we explored whether CBX4 expression is associated with replicative senescence in WI-38 fibroblasts, a classic human senescence model system. We also examined whether and how regulation of CBX4 modifies the senescence phenotype and functions as an antisenescence target in WI-38. During the serial culture of the WI-38 primary fibroblast cell line to a senescent state, we found increased expression of senescence markers, including senescence β -galactosidase (SA- β gal) activity, protein expression of p16, p21, and DPP4, and decreased proliferation marker EdU; moreover, CBX4 protein expression declined. With knockdown of CBX4, SA- β gal activity and p16 protein expression increased, and EdU decreased. With the activation of CBX4, SA- β gal activity, p16, and DPP4 protein decreased. In addition, CBX4 knockdown increased, while CBX4 activation decreased, gene expression of both CDKN2A (encoding the p16 protein) and DPP4. Genes related to DNA damage and cell cycle pathways were regulated by CBX4. These results demonstrate that CBX4 can regulate replicative senescence in a manner consistent with a senomorphic agent., Competing Interests: All authors declare that there are no conflicts of interest associated with this study., (Copyright © 2022 Yu-Hsiu Chen et al.)

Aims: Adenoviruses that have CNGRCVSGCAGRC peptide inserted into fiber (AdFNGR) or hexon (AdHNGR) protein, respectively, showed increased transduction of endothelial cells. In this study we investigated if cysteines within the CNGRCVSGCAGRC sequence inserted into Ad serotype 5 Ad5 fiber or hexon protein form disulfide bond(s) and whether they play a role in retargeting potential of AdFNGR and AdHNGR., Methods: Transduction efficiency of adenoviruses was done by counting infected cells under the microscope. Adenovirus attachment and internalization were measured by qPCR. Flow cytometry was used to evaluate the expression of CD13 and integrins. Gene knockdown was achieved by transfection of small interfering RNA. Mass spectrometry was used for determining disulfide bonds in adenovirus fiber and hexon protein. Molecular modeling was use to predict interaction of CNGRCVSGCAGRC peptide and CD13., Key Findings: AdFNGR and AdHNGR attach better to CD13 and/or αvβ3 integrin-positive cells than Adwt. Reducing disulfide bonds using DTT decreased transduction efficiency and attachment of both AdFNGR and AdHNGR. Cysteins from CNGRCVSGCAGRC peptide within AdHNGR do not form disulfide bonds. Knockdown of αvβ3 integrin reduced increased transduction efficiency of both AdFNGR and AdHNGR, while CD13 knockdown had no effect, indicating that retargeting properties of these viruses rely mainly on αvβ3 integrin expression., Significance: Insertion site of NGR-containing peptides as well as NGR flanking residues are critical for receptor binding affinity/specificity and transduction efficiency of NGR retargeted adenoviral vectors., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Osteoarthritis (OA) is a chronic degenerative disease featured by cartilage erosion and inflammation. Luteolin, a member of the flavonoid family, has been shown to exert anti-inflammatory and antioxidative activities. However, the potential biological effects and underlying mechanism of luteolin on chondrocytes and OA progression remain largely elusive. In this study, the potential effect and mechanism of luteolin on OA were investigated in vitro and in vivo . Our data revealed that luteolin inhibited H 2 O 2 -induced cell death, apoptosis, oxidative stress, programmed necrosis, and inflammatory mediator production in primary murine chondrocytes. In addition, luteolin could activate the AMPK and Nrf2 pathways, and AMPK serves as a positive upstream regulator of Nrf2. In vivo results demonstrated the therapeutic effects of luteolin on OA in the DMM mouse model. Collectively, our findings showed that luteolin might serve as a novel and effective treatment for OA and provided a new research direction for clinical OA therapies., Competing Interests: The authors declare that they have no conflict of interest., (Copyright © 2022 Zhiqiang Zhou et al.)

The recombinant adeno‑associated virus 8 (rAAV8) vector is a widely used tool in basic research and clinical trials. The cytomegalovirus immediate‑early enhancer/chicken β‑actin (CAG) promoter is a synthetic promoter used in adenoviral constructs with a wide spectrum and notable efficiency. The thyroxine binding globulin (TBG) promoter is a liver‑specific promoter, which directs transgene expression in hepatocytes. However, the transduction efficiency of the rAAV vector is dependent on both the administration routes and the promoter elements. In the present study, the transduction efficiency in the liver following intraperitoneal (IP) and intravenous (IV) injections of rAAV8 with the CAG, TBG669 and TBG410 promoters was compared. Enhanced green fluorescent protein (EGFP) expression was used as the biomarker to indicate efficiency. Among the three different promoters, CAG exhibited the highest efficiency from both IV and IP injections. Following IV administration, EGFP expression, induced by the CAG promoter, was 67‑fold higher compared with that in the TBG410 promoter group and 26‑fold higher compared with that in the TBG669 promoter group. EGFP protein expression was higher with IV injection compared with that for IP injection for both the CAG and TBG669 promoters (P<0.05). With the CAG promoter, EGFP protein expression was 1.5‑fold higher with the use of IV injection than with IP injection. With the TBG410 promoter, no differences were observed between the two administrations. In conclusion, these findings demonstrated that the CAG promoter was much more efficient at driving gene expression in the liver compared with that for the TBG promoters in rAAV8. In addition, IP administration produced comparable efficiency for gene delivery via the rAAV8 vector, particularly with the promoter TBG410.

Agrobacterium-mediated transient gene expression is a rapid and useful approach for characterizing functions of gene products in planta. However, the practicability of the method in the model liverwort Marchantia polymorpha has not yet been thoroughly described. Here we report a simple and robust method for Agrobacterium-mediated transient transformation of Marchantia thalli and its applicability. When thalli of M. polymorpha were co-cultured with Agrobacterium tumefaciens carrying β-glucuronidase (GUS) genes, GUS staining was observed primarily in assimilatory filaments and rhizoids. GUS activity was detected 2 days after infection and saturated 3 days after infection. We were able to transiently co-express fluorescently tagged proteins with proper localizations. Furthermore, we demonstrate that our method can be used as a novel pathosystem to study liverwort-bacteria interactions. We also provide evidence that air chambers support bacterial colonization., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Neuroblastoma, the most common extracranial solid tumor of childhood, is thought to arise from neural crest-derived immature cells. The prognosis of patients with high-risk or recurrent/refractory neuroblastoma remains quite poor despite intensive multimodality therapy; therefore, novel therapeutic interventions are required. We examined the expression of a cell adhesion molecule CD146 (melanoma cell adhesion molecule [MCAM]) by neuroblastoma cell lines and in clinical samples and investigated the anti-tumor effects of CD146-targeting treatment for neuroblastoma cells both in vitro and in vivo. CD146 is expressed by 4 cell lines and by most of primary tumors at any stage. Short hairpin RNA-mediated knockdown of CD146, or treatment with an anti-CD146 polyclonal antibody, effectively inhibited growth of neuroblastoma cells both in vitro and in vivo, principally due to increased apoptosis via the focal adhesion kinase and/or nuclear factor-kappa B signaling pathway. Furthermore, the anti-CD146 polyclonal antibody markedly inhibited tumor growth in immunodeficient mice inoculated with primary neuroblastoma cells. In conclusion, CD146 represents a promising therapeutic target for neuroblastoma., (© 2021 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Background and Aims: Hepatitis C virus (HCV) leads to a high rate of chronic infection and T cell dysfunction. Although it is well known that chronic antigenic stimulation is a driving force for impaired T cell functions, the precise mechanisms underlying immune activation-induced T cell dysfunctions during HCV infection remain elusive., Approach and Results: Here, we demonstrated that circulating CD4 + T cells from patients who are chronically HCV-infected exhibit an immune activation status, as evidenced by the overexpression of cell activation markers human leukocyte antigen-antigen D-related, glucose transporter 1, granzyme B, and the short-lived effector marker CD127 - killer cell lectin-like receptor G1 + . In contrast, the expression of stem cell-like transcription factor T cell factor 1 and telomeric repeat-binding factor 2 (TRF2) are significantly reduced in CD4 + T cells from patients who are chronically HCV-infected compared with healthy participants (HP). Mechanistic studies revealed that CD4 + T cells from participants with HCV exhibit phosphoinositide 3-kinase/Akt/mammalian target of rapamycin signaling hyperactivation on T cell receptor stimulation, promoting proinflammatory effector cell differentiation, telomeric DNA damage, and cellular apoptosis. Inhibition of Akt signaling during T cell activation preserved the precursor memory cell population and prevented inflammatory effector cell expansion, DNA damage, and apoptotic death. Moreover, knockdown of TRF2 reduced HP T cell stemness and triggered telomeric DNA damage and cellular apoptosis, whereas overexpression of TRF2 in CD4 T cells prevented telomeric DNA damage., Conclusions: These results suggest that modulation of immune activation through inhibiting Akt signaling and protecting telomeres through enhancing TRF2 expression may open therapeutic strategies to fine tune the adaptive immune responses in the setting of persistent immune activation and inflammation during chronic HCV infection., (© 2021 by the American Association for the Study of Liver Diseases.)

Phenylketonuria (PKU) is the most common inborn error of metabolism of the liver, and results from mutations of both alleles of the phenylalanine hydroxylase gene (PAH). As such, it is a suitable target for gene therapy via gene delivery with a recombinant adeno-associated virus (AAV) vector. Here we use the synthetic AAV vector Anc80 via systemic administration to deliver a functional copy of a codon-optimized human PAH gene, with or without an intron spacer, to the Pah enu2 mouse model of PKU. Dose-dependent transduction of the liver and expression of PAH mRNA were present with both vectors, resulting in significant and durable reduction of circulating phenylalanine, reaching near control levels in males. Coat color of treated Pah enu2 mice reflected an increase in pigmentation from brown to the black color of control animals, further indicating functional restoration of phenylalanine metabolism and its byproduct melanin. There were no adverse effects associated with administration of AAV up to 5 × 10 12 VG/kg, the highest dose tested. Only minor and/or transient variations in some liver enzymes were observed in some of the AAV-dosed animals which were not associated with pathology findings in the liver. Finally, there was no impact on cell turnover or apoptosis as evaluated by Ki-67 and TUNEL staining, further supporting the safety of this approach. This study demonstrates the therapeutic potential of AAV Anc80 to safely and durably cure PKU in a mouse model, supporting development for clinical consideration., (© 2021 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

The pharmacokinetic profile of AAV particles following intrathecal delivery has not yet been clearly defined. The present study evaluated the distribution profile of adeno-associated virus serotype 5 (AAV5) viral vectors following lumbar intrathecal injection in mice. After a single bolus intrathecal injection, viral DNA concentrations in mouse whole blood, spinal cord, and peripheral tissues were determined using quantitative polymerase chain reaction (qPCR). The kinetics of AAV5 vector in whole blood and the concentration over time in spinal and peripheral tissues were analyzed. Distribution of the AAV5 vector to all levels of the spinal cord, dorsal root ganglia, and into systemic circulation occurred rapidly within 30 min following injection. Vector concentration in whole blood reached a maximum 6 h postinjection with a half-life of approximately 12 h. Area under the curve data revealed the highest concentration of vector distributed to dorsal root ganglia tissue. Immunohistochemical analysis revealed AAV5 particle colocalization with the pia mater at the spinal cord and macrophages in the dorsal root ganglia (DRG) 30 min after injection. These results demonstrate the widespread distribution of AAV5 particles through cerebrospinal fluid and preferential targeting of DRG tissue with possible clearance mechanisms via DRG macrophages.

Background: Pre-existing immunities hamper the application of human adenovirus (HAdV) vectors in gene therapy or vaccine development. Fowl adenovirus (FAdV)-based vector might represent an alternative., Methods: An intermediate plasmid containing FAdV-4 fiber genes, pMD-FAV4Fs, was separated from FAdV-4 adenoviral plasmid pKFAV4GFP. An overlap extension polymerase chain reaction (PCR) was employed for fiber modification in pMD-FAV4Fs, and the modified fibers were restored to generate new adenoviral plasmids through restriction-assembly. FAdV-4 vectors were rescued and amplified in chicken LMH cells. Fluorescence microscopy and flow cytometry were used to evaluate the gene transfer efficiency. The amount of viruses binding to cells was determined by a real-time PCR. A plaque-forming assay and one-step growth curve were used to evaluate virus growth., Results: Four sites in the CD-, DE-, HI- and IJ-loop of fiber1 knob could tolerate the insertion of exogenous peptide. The insertion of RGD4C peptide in the fiber1 knob significantly promoted FAdV-4 transduction to human adherent cells such as 293, A549 and HEp-2, and the insertion to the IJ-loop demonstrated the best performance. The replacement of the fiber2 knob of FAdV-4 with that of HAdV-35 improved the gene transfer to human suspension cells such as Jurkat, K562 and U937. Fiber-modified FAdV-4 vectors could transduce approximately 80% human cells at an acceptable multiplicity of infection. Enhanced gene transfer mainly resulted from increased virus binding. Fiber modifications did not significantly influence the growth of recombinant FAdV-4 in packaging cells., Conclusions: As a proof of principle, it was feasible to enhance gene transduction of FAdV-4 vectors to human cells by modifying the fibers., (© 2021 The Authors. The Journal of Gene Medicine published by John Wiley & Sons Ltd.)

Preserving morphological features that are important for cell function and structure is a critical parameter for in vitro experiments with rat cardiomyocytes. Lentiviral vectors are commonly used as gene transfer tool because of its high flexibility, efficiency to deliver expression cassettes and versatility of transducing quiescent cells. The tropism of the recombinant viral particle can be determined depending on the virus envelope, which shows a specific binding to cell surface receptors on the target cell. The combination of promoter arrangement and viral envelope must be optimized to achieve a greater transduction efficiency and a higher transgene expression. In this study we explored the optimization of promoters and heterologous envelopes to transduce primary culture of neonatal rat ventricular myocytes. Our results suggest a robust expression driven by the cytomegalovirus promoter, and high efficiency transduction mediated by VSV-G envelope with no apparent compromising ultrastructural features of genetically modified cells., (Copyright © 2021 Elsevier Inc. All rights reserved.)

One approach to improve the utility of adeno-associated virus (AAV)-based gene therapy is to engineer the AAV capsid to (i) overcome poor transport through tissue barriers and (ii) redirect the broadly tropic AAV to disease-relevant cell types. Peptide- or protein-domain insertions into AAV surface loops can achieve both engineering goals by introducing a new interaction surface on the AAV capsid. However, we understand little about the impact of insertions on capsid structure and the extent to which engineered inserts depend on a specific capsid context to function. Here, we examine insert-capsid interactions for the engineered variant AAV9-PHP.B. The 7-amino-acid peptide insert in AAV9-PHP.B facilitates transport across the murine blood-brain barrier via binding to the receptor Ly6a. When transferred to AAV1, the engineered peptide does not bind Ly6a. Comparative structural analysis of AAV1-PHP.B and AAV9-PHP.B revealed that the inserted 7-amino-acid loop is highly flexible and has remarkably little impact on the surrounding capsid conformation. Our work demonstrates that Ly6a binding requires interactions with both the PHP.B peptide and specific residues from the AAV9 HVR VIII region. An AAV1-based vector that incorporates a larger region of AAV9-PHP.B-including the 7-amino-acid loop and adjacent HVR VIII amino acids-can bind to Ly6a and localize to brain tissue. However, unlike AAV9-PHP.B, this AAV1-based vector does not penetrate the blood-brain barrier. Here we discuss the implications for AAV capsid engineering and the transfer of engineered activities between serotypes. IMPORTANCE Targeting AAV vectors to specific cellular receptors is a promising strategy for enhancing expression in target cells or tissues while reducing off-target transgene expression. The AAV9-PHP.B/Ly6a interaction provides a model system with a robust biological readout that can be interrogated to better understand the biology of AAV vectors' interactions with target receptors. In this work, we analyzed the sequence and structural features required to successfully transfer the Ly6a receptor-binding epitope from AAV9-PHP.B to another capsid of clinical interest, AAV1. We found that AAV1- and AAV9-based vectors targeted to the same receptor exhibited different brain-transduction profiles. Our work suggests that, in addition to attachment-receptor binding, the capsid context in which this binding occurs is important for a vector's performance.

Many tailed bacteriophages assemble ejection proteins and a portal-tail complex at a unique vertex of the capsid. The ejection proteins form a transenvelope channel extending the portal-tail channel for the delivery of genomic DNA in cell infection. Here, we report the structure of the mature bacteriophage T7, including the ejection proteins, as well as the structures of the full and empty T7 particles in complex with their cell receptor lipopolysaccharide. Our near-atomic-resolution reconstruction shows that the ejection proteins in the mature T7 assemble into a core, which comprises a fourfold gene product 16 (gp16) ring, an eightfold gp15 ring, and a putative eightfold gp14 ring. The gp15 and gp16 are mainly composed of helix bundles, and gp16 harbors a lytic transglycosylase domain for degrading the bacterial peptidoglycan layer. When interacting with the lipopolysaccharide, the T7 tail nozzle opens. Six copies of gp14 anchor to the tail nozzle, extending the nozzle across the lipopolysaccharide lipid bilayer. The structures of gp15 and gp16 in the mature T7 suggest that they should undergo remarkable conformational changes to form the transenvelope channel. Hydrophobic α-helices were observed in gp16 but not in gp15, suggesting that gp15 forms the channel in the hydrophilic periplasm and gp16 forms the channel in the cytoplasmic membrane., Competing Interests: The authors declare no competing interest.

In the era of genomic medicine, diagnoses of rare paediatric neurological diseases are increasing. Many are untreatable and life-limiting, leading to an exceptional increase in gene therapy development. It is estimated that 20 gene therapy products will have received approval from the US Food and Drug Administration by 2025. With viral gene therapy considered a potential single-dose cure for patients with spinal muscular atrophy type 1 as one example, and contemporaneously tragically resulting in the deaths of three male children with X-linked myotubular myopathy receiving high-dose gene therapy in 2020, what is the current state of gene therapy? What is behind the decades of hype around viral gene therapy and is it high impact, but high risk? In this review, we outline principles of viral gene therapy development and summarize the most recent clinical evidence for the therapeutic effect of gene therapy in paediatric neurological diseases. We discuss adeno-associated virus and lentiviral vectors, antisense oligonucleotides, emerging genetic editing approaches, and current limitations that the field still faces. What this paper adds Viral gene therapy development and clinically used transgenes, regulatory elements, capsids, dosage, and delivery routes are summarized. Viral gene therapy for 18 childhood neurological disorders involving over 600 children in 40 clinical trials are reviewed., (© 2021 The Authors. Developmental Medicine & Child Neurology published by John Wiley & Sons Ltd on behalf of Mac Keith Press.)

To better understand the pathogenesis of nasal polyps (NPs) and sinonasal inverted papillomas (SIPs), we aimed to establish cell lines from fresh tissues of NPs and SIPs and characterize them. Primary cell cultures were obtained from two NP tissues (NP2 and NP3) and one SIP tissue (IP4). All the cells were polygonal in shape, expressed cytokeratin 14, and had normal diploid chromosome status. HPV58 DNA was detected in NP3. To obtain immortal primary cells, NP2 and IP4 cells were transduced with a combination of mutant CDK4, cyclinD1 and TERT. These cells were thereafter named NP2/K4DT and IP4/K4DT, respectively. HPV58-positive NP3 cells were transduced with TERT alone, the resulting cells named NP3/T. Phenotypic and genotypic identity of original tissues and derived cells was investigated. All the cell cultures with transgenes were confirmed to be derived from their parental cells and primary tumor tissues by analysis of short tandem repeats (STR) and maintained in vitro growth, genetic profiles and gene expression characteristics of the primary cells. These virtually immortalized cells, as well as the primary cells, have potential as in vitro models for studying the pathogenesis of NPs and SIPs and for preclinical study to develop new therapeutic agents., (© 2021. The Author(s).)

Transduction of primary T cells has become prominent with the introduction of chimeric antigen receptor T-cell therapy. Although there are many protocols for the transduction of human T cells, it remains a challenge to transduce murine T cells. We present an optimized protocol for the retroviral transduction of murine CD4 T cells, which overcomes major challenges including large-scale production and long-term culturing of transduced cells. The optimized protocol combines high transduction efficiency with a low rate of cell death. For complete details on the use and execution of this protocol, please refer to Eremenko et al., 2019., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Lentiviral vectors are the most frequently used tool to stably transfer and express genes in the context of gene therapy for monogenic diseases. The vast majority of clinical applications involves an ex vivo modality whereby lentiviral vectors are used to transduce autologous somatic cells, obtained from patients and re-delivered to patients after transduction. Examples are hematopoietic stem cells used in gene therapy for hematological or neurometabolic diseases or T cells for immunotherapy of cancer. We review the design and use of lentiviral vectors in gene therapy of monogenic diseases, with a focus on controlling gene expression by transcriptional or post-transcriptional mechanisms in the context of vectors that have already entered a clinical development phase.

Cell line development from shrimp is not a novel venture as researchers across the globe have been trying to have crustacean cell lines over 30 years. The reason for not attaining a crustacean or precisely a shrimp cell line is believed to be the replicative senescence and the inability to maintain telomere length in vitro. Moreover, spontaneous in vitro transformations do not happen in shrimp cells. Oncogenic induction in primary cell culture is one of the ways to attain in vitro transformation by way of disrupting the mechanisms which involve cellular senescence. In this context, a recombinant baculovirus with shrimp viral promoter IHHNV-P2 was used for the transduction aimed at immortalization. An oncogene, H-ras, was successfully amplified and cloned in to the baculoviral vector, downstream to shrimp viral promoter IHHNV-P2 and upstream to GFP. Recombinant baculovirus with H-ras was generated and used for transduction into shrimp lymphoid cells during early dividing stage. Accordingly, fibroblast-like primary cell culture got developed, and H-ras and GFP expression could be confirmed. The study suggests that the simple method of incubating recombinant baculovirus with minced tissue enables in vitro transduction during early dividing stage of the cells, and the transduction efficiency gets enhanced by adding 5 mM sodium butyrate to the culture medium., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Despite several studies on the effect of adeno-associated virus (AAV)-based therapeutics on osteoarthritis (OA), information on the transduction efficiency and applicable profiles of different AAV serotypes to chondrocytes in hard cartilage tissue is still limited. Moreover, the recent discovery of additional AAV serotypes makes it necessary to screen for more suitable AAV serotypes for specific tissues. Here, we compared the transduction efficiencies of 14 conventional AAV serotypes in human chondrocytes, mouse OA models, and human cartilage explants obtained from OA patients. Methods: To compare the transduction efficiency of individual AAV serotypes, green fluorescent protein (GFP) expression was detected by fluorescence microscopy or western blotting. Likewise, to compare the transduction efficiencies of individual AAV serotypes in cartilage tissues, GFP expression was determined using fluorescence microscopy or immunohistochemistry, and GFP-positive cells were counted. Results: Only AAV2, 5, 6, and 6.2 exhibited substantial transduction efficiencies in both normal and OA chondrocytes. All AAV serotypes except AAV6 and rh43 could effectively transduce human bone marrow mesenchymal stem cells. In human and mouse OA cartilage tissues, AAV2, AAV5, AAV6.2, AAV8, and AAV rh39 showed excellent tissue specificity based on transduction efficiency. These results indicate the differences in transduction efficiencies of AAV serotypes between cellular and tissue models. Conclusions: Our findings indicate that AAV2 and AAV6.2 may be the best choices for AAV-mediated gene delivery into intra-articular cartilage tissue. These AAV vectors hold the potential to be of use in clinical applications to prevent OA progression if appropriate therapeutic genes are inserted into the vector., Competing Interests: Competing interests: G.G. and J.H.S. are co-founders of AAVAA Therapeutics. G.G. is a scientific co-founder of Voyager Therapeutics and Aspa Therapeutics Inc. and an inventor on patents with potential royalties licensed to other biopharmaceutical companies. They hold equity in these companies. Other authors declare no competing interests., (© The author(s).)

Glioblastoma is the most malignant and most common form of brain tumor, still today associated with a poor 14-months median survival from diagnosis. Protein kinase A, particularly its regulatory subunit R2Alpha, presents a typical intracellular distribution in glioblastoma cells compared to the healthy brain parenchyma and this peculiarity might be exploited in a therapeutic setting. In the present study, a third-generation lentiviral system for delivery of shRNA targeting the regulatory subunit R2Alpha of protein kinase A was developed. Generated lentiviral vectors are able to induce an efficient and stable downregulation of R2Alpha in different cellular models, including non-stem and stem-like glioblastoma cells. In addition, our data suggest a potential correlation between silencing of the regulatory subunit of protein kinase A and reduced viability of tumor cells, apparently due to a reduction in replication rate. Thus, our findings support the role of protein kinase A as a promising target for novel anti-glioma therapies.

Adoptive T cell therapy with T cell receptor (TCR)-modified T cells has shown promise in treating metastatic melanoma and other malignancies. However, studies are needed to improve the efficacy and durability of responses of TCR-modified T cells. Standard protocols for generating TCR-modified T cells involve activating T cells through CD3 stimulation to allow for the efficient transfer of tumor-reactive receptors with viral vectors. T cell activation results in terminal differentiation and shortening of telomeres, which are likely suboptimal for therapy. In these studies, we demonstrate efficient T cell transduction with the melanoma-reactive TIL1383I TCR through culturing with interleukin 7 (IL-7) in the absence of CD3 activation. The TIL1383I TCR-modified T cells generated following IL-7 culture were enriched with naïve (T N ) and memory stem cell populations (T SCM ) while maintaining longer telomere lengths. Furthermore, we demonstrated melanoma-reactivity of TIL1383I TCR-modified cells generated following IL-7 culture using in vitro assays and a superior response in an in vivo melanoma model. These results suggest that utilizing IL-7 to generate TCR-modified T cells in the absence of activation is a feasible strategy to improve adoptive T cell therapies for melanoma and other malignancies.

Bacterial transduction particles were critical to early advances in molecular biology and are currently experiencing a resurgence in interest within the diagnostic and therapeutic fields. The difficulty of developing a robust and specific transduction reagent capable of delivering a genetic payload to the diversity of strains constituting a given bacterial species or genus is a major impediment to their expanded utility as commercial products. While recent advances in engineering the reactivity of these reagents have made them more attractive for product development, considerable improvements are still needed. Here, we demonstrate a synthetic biology platform derived from bacteriophage P1 as a chassis to target transduction reagents against four clinically prevalent species within the Enterobacterales order. Bacteriophage P1 requires only a single receptor binding protein to enable attachment and injection into a target bacterium. By engineering and screening particles displaying a diverse array of chimeric receptor binding proteins, we generated a potential transduction reagent for a future rapid phenotypic carbapenem-resistant Enterobacterales diagnostic assay.

Human multipotent mesenchymal stromal cells (hMSCs) are currently developed as cell therapeutics for different applications, including regenerative medicine, immune modulation, and cancer treatment. The biological properties of hMSCs can be further modulated by genetic engineering. Viral vectors based on human adenovirus type 5 (HAdV-5) belong to the most frequently used vector types for genetic modification of human cells in vitro and in vivo. However, due to a lack of the primary attachment receptor coxsackievirus and adenovirus receptor (CAR) in hMSCs, HAdV-5 vectors are currently not suitable for transduction of this cell type without capsid modification. Here we present several transduction enhancers that strongly enhance HAdV-5-mediated gene transfer into both bone marrow- and adipose tissue-derived hMSCs. Polybrene, poly-l-lysine, human lactoferrin, human blood coagulation factor X, spermine, and spermidine enabled high eGFP expression levels in hMSCs. Importantly, hMSCs treated with enhancers were not affected in their migration behavior, which is a key requisite for many therapeutic applications. Exemplary, strongly increased expression of tumor necrosis factor (TNF)-stimulated gene 6 (TSG-6) (a secreted model therapeutic protein) was achieved by enhancer-facilitated HAdV-5 transduction. Thus, enhancer-mediated HAdV-5 vector transduction is a valuable method for the engineering of hMSCs, which can be further exploited for the development of innovative hMSC therapeutics.

The lentivirus-short hairpin RNA (shRNA) system is a widely used tool for RNA interference. Multiple factors may affect the RNA interference efficiency during lentivirus production and transduction procedures. Thus, an optimized protocol is required to achieve high-titer lentivirus and efficient gene delivery. In the present study, lentivirus was produced by transfecting lentiviral transfer and packaging plasmids into HEK 293T cells. The factors affecting lentiviral titer were assessed, including lentiviral plasmid ratio, lentiviral transfer plasmid type, serum type for cell culture, transfection reagent-plasmid mixture incubation time, and the inoculation density of 293T cells for transfection. The high-titer lentivirus was achieved when plasmids were transfected at a molar ratio of 1:1:1:2, and the transfection reagent-plasmid mixture was replaced 6-8 h after transfection. The pLVX-shRNA2 lentiviral transfer plasmid was associated with the highest lentiviral titer, while both pLVX-shRNA2 and psi-LVRU6GP plasmids were associated with efficient RNA interference in target cells. The serum type for 293T cell culture affected the lentiviral titer significantly, while the inoculation density of 293T cells showed no influence on transfection efficiency or lentiviral titer. Moreover, the human primary fibroblasts infected with lentivirus, using the centrifugation method, achieved higher transduction efficiency than those infected with the non-centrifugation method. In conclusion, this study helped optimize lentiviral production and transduction procedures for more efficient gene delivery., (© 2021 Wiley-VCH GmbH.)

Recombinant adeno-associated virus (rAAV) platforms hold promise for in vivo gene therapy but are undermined by the undesirable transduction of antigen presenting cells (APCs), which in turn can trigger host immunity towards rAAV-expressed transgene products. In light of recent adverse events in patients receiving high systemic AAV vector doses that were speculated to be related to host immune responses, development of strategies to mute innate and adaptive immunity is imperative. The use of miRNA binding sites (miR-BSs) to confer endogenous miRNA-mediated regulation to detarget transgene expression from APCs has shown promise for reducing transgene immunity. Studies have shown that designing miR-142BSs into rAAV1 vectors were able to repress costimulatory signals in dendritic cells (DCs), blunt the cytotoxic T cell response, and attenuate clearance of transduced muscle cells in mice to allow sustained transgene expression in myofibers with negligible anti-transgene IgG production. In this study, we screened individual and combinatorial miR-BS designs against 26 miRNAs that are abundantly expressed in APCs, but not in skeletal muscle. The highly immunogenic ovalbumin (OVA) transgene was used as a proxy for foreign antigens. In vitro screening in myoblasts, mouse DCs, and macrophages revealed that the combination of miR-142BS and miR-652-5pBS strongly mutes transgene expression in APCs but maintains high myoblast and myocyte expression. Importantly, rAAV1 vectors carrying this novel miR-142/652-5pBS cassette achieve higher transgene levels following intramuscular injections in mice than previous detargeting designs. The cassette strongly inhibits cytotoxic CTL activation and suppresses the Th17 response in vivo . Our approach, thus, advances the efficiency of miRNA-mediated detargeting to achieve synergistic reduction of transgene-specific immune responses and the development of safe and efficient delivery vehicles for gene therapy., Competing Interests: GG is a scientific co-founder of Voyager Therapeutics, Adrenas Therapeutics, and Aspa Therapeutics, and holds equity in these companies. GG is an inventor on patents with potential royalties licensed to Voyager Therapeutics, Aspa Therapeutics, and other biopharmaceutical companies. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Muhuri, Zhan, Maeda, Li, Lotun, Chen, Sylvia, Dasgupta, Arjomandnejad, Nixon, Keeler, Manokaran, He, Su, Tai and Gao.)

(1) Adeno-associated viruses (AAV) are safe and efficient gene therapy vectors with promising results in the treatment of several diseases. Extracellular vesicles (EV) are phospholipid bilayer-surrounded structures carrying several types of lipids, proteins, and nucleic acids with the ability to cross biological barriers. EV-associated AAVs might serve as new and efficient gene therapy vectors considering that they carry the benefits of both AAVs and EVs. (2) We tested vesicle-associated AAVs and vesicles mixed with AAVs on two major cell types of the central nervous system: a neural cell line (N2A) and primary astrocyte cells. (3) In contrast to previously published in vivo observations, the extracellular vesicle packaging did not improve but, in the case of primary astrocyte cells, even inhibited the infection capacity of the AAV particles. The observed effect was not due to the inhibitory effects of the vesicles themselves, since mixing the AAVs with extracellular vesicles did not change the effectiveness. (4) Our results suggest that improvement of the in vivo efficacy of the EV-associated AAV particles is not due to the enhanced interaction between the AAV and the target cells, but most likely to the improved delivery of the AAVs through tissue barriers and to the shielding of AAVs from neutralizing antibodies.

The neurotrophin nerve growth factor (NGF) modulates the growth of human gliomas and is able to induce cell differentiation through the engagement of tropomyosin receptor kinase A (TrkA) receptor, although the role played in controlling glioma survival has proved controversial. Unfortunately, the slow growth rate of low-grade gliomas (LGG) has made it difficult to investigate NGF effects on these tumors in preclinical models. In fact, patient-derived low-grade human astrocytoma cells duplicate only a limited number of times in culture before undergoing senescence. Nevertheless, replicative senescence can be counteracted by overexpression of hTERT, the catalytic subunit of telomerase, which potentially increases the proliferative potential of human cells without inducing cancer-associated changes. We have extended, by hTERT transduction, the proliferative in vitro potential of a human LGG cell line derived from a pediatric pilocytic astrocytoma (PA) surgical sample. Remarkably, the hTERT-transduced LGG cells showed a behavior similar to that of the parental line in terms of biological responses to NGF treatment, including molecular events associated with induction of NGF-related differentiation. Therefore, transduction of LGG cells with hTERT can provide a valid approach to increase the in vitro life-span of patient-derived astrocytoma primary cultures, characterized by a finite proliferative potential., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Franzese, Di Francesco, Meco, Graziani, Cusano, Levati, Riccardi and Ruggiero.)

Adeno-associated virus (AAV) vector, an excellent gene therapy vector, has been widely used in the treatment of various central nervous system (CNS) diseases. Due to the presence of the blood-brain barrier (BBB), early attempts at AAV-based CNS diseases treatment were mainly performed through intracranial injections. Subsequently, systemic injections of AAV9, the first AAV that was shown to have BBB-crossing ability in newborn and adult mice, were assessed in clinical trials for multiple CNS diseases. However, the development of systemic AAV injections to treat CNS diseases is still associated with many challenges, such as the efficiency of AAV in crossing the BBB, the peripheral toxicity caused by the expression of AAV-delivered genes, and the immune barrier against AAV in the blood. In this review, we will introduce the biology of the AAV vector and the advantages of systemic AAV injections to treat CNS diseases. Most importantly, we will introduce the challenges associated with systemic injection of therapeutic AAV in treating CNS diseases and suggest feasible solutions., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The cation channel transient receptor potential melastatin 4 (TRPM4) is a calcium-activated non-selective cation channel and acts in cardiomyocytes as a negative modulator of the L-type Ca 2+ influx. Global deletion of TRPM4 in the mouse led to increased cardiac contractility under β-adrenergic stimulation. Consequently, cardiomyocyte-specific inactivation of the TRPM4 function appears to be a promising strategy to improve cardiac contractility in heart failure patients. The aim of this study was to develop a gene therapy approach in mice that specifically silences the expression of TRPM4 in cardiomyocytes. First, short hairpin RNA miR30 (shRNA miR30 ) sequences against the TRPM4 mRNA were screened in vitro using lentiviral transduction for a stable expression of the shRNA cassettes. Western blot analysis identified three efficient shRNA miR30 sequences out of six, which reduced the endogenous TRPM4 protein level by up to 90 ± 6%. Subsequently, the most efficient shRNA miR30 sequences were delivered into cardiomyocytes of adult mice using adeno-associated virus serotype 9 (AAV9)-mediated gene transfer. Initially, the AAV9 vector particles were administered via the lateral tail vein, which resulted in a downregulation of TRPM4 by 46 ± 2%. Next, various optimization steps were carried out to improve knockdown efficiency in vivo. First, the design of the expression cassette was streamlined for integration in a self-complementary AAV vector backbone for a faster expression. Compared to the application via the lateral tail vein, intravenous application via the retro-orbital sinus has the advantage that the vector solution reaches the heart directly and in a high concentration, and eventually a TRPM4 knockdown efficiency of 90 ± 7% in the heart was accomplished by this approach. By optimization of the shRNA miR30 constructs and expression cassette as well as the route of AAV9 vector application, a 90% reduction of TRPM4 expression was achieved in the adult mouse heart. In the future, AAV9-RNAi-mediated inactivation of TRPM4 could be a promising strategy to increase cardiac contractility in preclinical animal models of acute and chronic forms of cardiac contractile failure.