Transduction Efficiency Research Articles

AAV vector transduction restriction and attenuated toxicity in hESCs via a rationally designed inverted terminal repeat.

Adeno-associated virus (AAV) inverted terminal repeats (ITRs) induce p53-dependent apoptosis in human embryonic stem cells (hESCs). To interrogate this phenomenon, a synthetic ITR (SynITR), harboring substitutions in putative p53 binding sites was generated and evaluated for vector production and gene delivery. Replication of SynITR flanked transgenic genome was similar compared to wildtype (wt)ITR, with a modest increase in vector titers. Packaged in the AAV2 capsid, wtITR and SynITR vectors demonstrated similar transduction efficiencies of human cells without toxicity. Following AAV2-wtITR vector infection of hESCs, rapid apoptosis was observed as reported. In contrast, infection by AAV2 vectors packaged with SynITRs attenuated the wtITR-induced hESC toxicity. While hESC particle entry and the abundance of double stranded circular episomes was similar for the ITR contexts, reporter expression was inhibited from transduced SynITR genomes. Mechanistically, infection of hESCs induced γH2AX in an ITR-independent manner, however, canonical activation of p53α was uncoupled using AAV-SynITR. Further investigations in hESCs revealed additional novel findings: (i) p53β is uniquely and constitutively activeand (ii) AAV vector infection, independent of the ITR sequence, induces activation of p53ψ. The data herein reveal an ITR-dependent AAV vector transduction restriction specific to hESCs and manipulation of the DNA damage response via ITR engineering.

Screening of a retinal-targeting Adeno-Associated Virus (AAV) via DNA shuffling.

Due to its unique physiological structure and functions, the eye has received considerable attention in the field of adeno-associated virus (AAV) gene therapy. Inherited retinal degenerative diseases, which arise from pathogenic mutations in mRNA transcripts expressed in the eye's photoreceptor cells or retinal pigment epithelium (RPE), are the most common cause of vision loss. However, current retinal gene therapy mostly involves subretinal injection of therapeutic genes, which treats a limited area, entails retinal detachment, and requires sophisticated techniques. Intravitreal (IVT) injection provides an alternative method with less invasion and more convenience for retinal gene therapy. In the present study, we performed a directed evolution via DNA shuffling in RHO-GFP mice and identified a novel recombinant AAV vector (AAV-M04) suitable for IVT injection in the gene delivery of retinal tissue. Compared with AAV2, AAV9, and AAV2.7m8, AAV-M04 vector exhibited higher transduction efficiency in retinal ganglion cell line-5 (RGC-5) cells as well as in human embryonic stem cell derived retinal organoids. Importantly, when delivered via IVT injection in mice, the AAV-M04 vector also showed better delivery efficiency of transgene as indicated by the red fluorescence protein mScarlet. The red fluorescence was distributed in a wider retinal area of AAV-M04 injected mice, suggesting the potent retinal targeting of AAV-M04 vector. In addition, AAV-M04 qualities including the packaging efficiency, the thermal stability, and the capsid integrity were superior to controls, which were important in drug manufacture. In summary, we screened a novel AAV-M04 vector with great retinal-targeting via IVT injection, which provides the potential of AAV-M04 for effective gene therapy of retinal diseases.

Integrated physiological, transcriptomic and metabolomic analyses reveal ROS regulatory mechanisms in two castor bean varieties under alkaline stress.

Saline-alkaline stress has caused severe ecological and environmental problems. Castor bean is a potential alkali-tolerant plant, however, its reactive oxygen species (ROS) regulatory mechanisms under alkaline stress remain unclear. This study investigated the physiological, transcriptomic, and metabolomic characteristics of two varieties (ZB8, alkaline-sensitive; JX22, alkaline-resistant) under alkaline stress. Results showed that under alkaline stress, JX22's root length was 1.66-fold greater than ZB8's, while its superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities were 1.25-, 1.41-, and 1.29-fold higher than ZB8's, respectively. The levels of superoxide anion (O2-) and malondialdehyde (MDA) in JX22 were 0.2- and 0.68-fold of those in ZB8, respectively. Integrated transcriptomic and metabolomic analyses revealed that regarding ROS generation, alkaline stress promoted the upregulation of ACX1 and RBOHD genes in JX22, enabling more efficient ROS signal transduction and subsequent stress response regulation. In terms of ROS signal transduction, alkaline stress induced significant upregulation of protein kinase-encoding genes including CPK4, CPK9, and CPK10 in JX22, which cooperated with RBOHD to regulate ROS production. Concerning ROS scavenging, significant upregulation of SODA, CAT2, and PRXⅡB genes ensured a more efficient enzymatic ROS scavenging system in JX22 under alkaline stress. In contrast, ZB8 could only rely on less efficient non-enzymatic systems, such as carotenoid antioxidants, to mitigate oxidative damage, where genes like CCD7, CYP897B and metabolites including lutein and zeaxanthin played crucial roles. These findings elucidate the ROS response mechanisms of castor bean under alkaline stress, paving new ways for breeding alkaline-resistant varieties.

Peri-Implantation Targeting Using Modified Adenoviral Vectors

The aim was to conduct a comparative morphological analysis of the effect of implantation and modified adenovirus Ad 5/3 on the efficiency of local gene delivery to cells of the peri-implantation niche in vivo. Material and methods. The experiments were carried out on laboratory rats, which were implanted with collagen membranes for a period of 2 weeks. Three and seven days after the introduction of adenoviruses (wt, Ad 5/3), the tissues were fixed for histological examination and analysis of transduced cells by in situ PCR. Results. The results showed that the presence of a collagen implant improved the biodistribution of viral particles, and the modification of Ad 5/3 significantly increased the efficiency of transduction of fibroblasts and macrophages in the peri-implantation zone. In the absence of an implant, the transduction efficiency decreased for both types of adenoviruses. Morphometric analysis revealed that the main transduced cells were fibroblasts of the connective tissue capsule surrounding the implant. Conclusion. Based on comparative morphological and molecular biological studies, an optimal protocol for local gene delivery using adenoviral vectors for peri-implantation targeting was determined. The effect of adenovirus modification on the tropism and efficiency of gene delivery to the cellular components of the PIN was characterized, and it was found that Ad 5/3 significantly exceeds AD wt in these parameters. The development and further application of the peri-implantation targeting method proposed by us can ensure local, effective and safe gene delivery, which will significantly expand the indications for gene therapy and its availability to doctors and patients. In addition, this method can be adapted for use in various organs and for various diseases.

Challenges in Humoral Immune Response to Adeno-Associated Viruses Determination.

Adeno-associated viruses (AAVs) are non-pathogenic, replication-deficient viruses that have gained widespread attention for their application as gene therapy vectors. While these vectors offer high transduction efficiency and long-term gene expression, the host immune response poses a significant challenge to their clinical success. This review focuses on the obstacles to evaluating the humoral response to AAVs. We discuss the problems with the validation of in vitro tests and the possible approaches to overcome them. Using published data on neutralizing titers of AAV serotypes, we built the first antigenic maps of AAVs in order to visualize the antigenic relationships between varying serotypes.

Heparan sulfate proteoglycan affinity of adeno-associated virus vectors: Implications for retinal gene delivery.

Adeno-associated virus (AAV)-based vectors have emerged as an effective and widely used technology for somatic gene therapy approaches, including those targeting the retina. A major advantage of the AAV technology is the availability of a large number of serotypes that have either been isolated from nature or produced in the laboratory. These serotypes have different properties in terms of sensitivity to neutralizing antibodies, cellular transduction profile and efficiency. The infectivity of AAV vectors depends on the affinity to certain molecules on the cell surface, in particular to cellular glycosaminoglycans (GAGs) such as heparan sulfate proteoglycans (HSPGs). Here, we tested how altering HSPG affinity in AAV vectors affects cellular tropism and transduction efficiency. The previously developed AAV2.GL variant was used as a starting variant to alter or disrupt HSPG affinity. The HSPG-independent AAV9 serotype was used to introduce different HSPG-binding sites. As an indicator of HSPG affinity, we measured the binding strength of the vector variant on a heparin chromatography column. We show that modification of capsid-exposed residues has a strong impact on HSPG affinity, cellular tropism and transduction efficiency in HeLa cells and in vivo in mouse retina. Our study shows that key properties of AAV vectors can be tailored in different directions and used to improve tropism and efficiency.

AAVone: A Cost-Effective, Single-Plasmid Solution for Efficient AAV Production with Reduced DNA Impurities.

Currently, the most common approach for manufacturing GMP-grade adeno-associated virus (AAV) vectors involves transiently transfecting mammalian cells with three plasmids that carry the essential components for production. The requirement for all three plasmids to be transfected into a single cell and the necessity for high quantities of input plasmid DNA, limits AAV production efficiency, introduces variability between production batches, and increases time and labor costs. Here, we developed an all-in-one, single-plasmid AAV production system, called AAVone. In this system, the adenovirus helper genes ( E2A , E4orf6 , and VA RNA ), packaging genes ( rep and cap ), and the vector transgene cassette are consolidated into a single compact plasmid with a 13-kb backbone. The AAVone system achieves a two- to four-fold increase in yields compared to the traditional triple-plasmid system. Furthermore, the AAVone system exhibits low batch-to-batch variation and eliminates the need for fine-tuning the ratios of the three plasmids, simplifying the production process. In terms of vector quality, AAVs generated by the AAVone system show similar in vitro and in vivo transduction efficiency, but a substantial reduction in sequences attributed to plasmid backbones and a marked reduction in non-functional snap-back genomes. In Summary, the AAVone platform is a straightforward, cost-effective, and highly consistent AAV production system - making it particularly suitable for GMP-grade AAV vectors.

Effects of an initial anti-CD19 CAR T-cell therapy on subsequent anti-CD22 CAR T-cell manufacturing and clinical outcomes in patients with r/r LBCL.

Patients with large B-cell lymphoma (LBCL) progressing after anti-CD19 CAR T-cell (CAR19) therapy have poor outcomes. Subsequent CAR T-cell therapy shows promise, but the impact of residual CAR19 and early relapse remains unclear. We evaluated 37 CAR19-refractory LBCL patients who received anti-CD22 CAR T-cell (CAR22) in a phase 1b trial (NCT04088890). Residual CAR19 was unquantifiable in 17 of 33 evaluable patients post-CAR22 infusion. Single-cell RNA sequencing revealed minimal CAR19/CAR22 co-transduction. Peak CAR19 transgene levels did not affect CAR22 efficacy or toxicities. CAR22 products from patients undergoing leukapheresis > 6 months post-CAR19 had higher CD4+ naïve T and CD4+/CD8+ T- central memory (TCM) cells, with lower CD4+ T-effector memory cells. High and low percentages of CAR22 TCM and TEM, respectively, were correlated with CAR22 transduction efficiency and achieving complete response. Residual CAR19 and leukapheresis timing did not significantly affect outcomes, while CAR22 product composition was significantly correlated with treatment response.

AAVR Expression is Essential for AAV Vector Transduction in Sensory Hair Cells.

Adeno-associated virus (AAV) vectors are a leading platform for gene therapy. Recently, AAV-mediated gene therapy in the inner ear has progressed from laboratory use to clinical trials, but the lower transduction rates in outer hair cells (OHCs) in the organ of Corti and in vestibular hair cells in adult mice still pose a challenge. OHCs are particularly vulnerable to inner ear insults. In this study, we demonstratedthat expression of a key AAV receptor (AAVR, Kiaa0319l, or Au040320) in OHCs and vestibular hair cells decreases significantly in mature mice and AAV particles directly interact with AAVR by forming complexes. Consequently, antibody blockage of AAVR significantly inhibits AAV transduction in sensory hair cells in cochlear explants. Moreover, use of AAVR knockout mice confirms inhibition of AAV transduction in sensory hair cells in vivo. Finally, conditional overexpression of AAVR in sensory hair cells of adult mice successfully restores AAV transduction efficiency in OHCs and vestibular hair cells. In conclusion, this strong evidence that AAVR is essential for AAV transduction in sensory hair cells will help to increase the efficacy of future gene therapy in inner ear.

Underwater low-power electromagnetic transducers with Central permanent magnet integration.

High-efficiency electromagnetic transducers are crucial for enabling the self-sustained operation of underwater electromagnetic sound sources under power-constrained conditions as noted by Hao, Xie, and Ma [Proceedings of the 2019 Western China Acoustics Academic Conference, Guangzhou, China (November 5-9, 2019)]. This paper proposes a permanent magnet drive technology to enhance the electromechanical conversion efficiency of can-type electromagnetic transducers under low-power driving conditions. The can-type transducers consist of coils, an armature, and a cylindrical magnetic core with a central pillar, similar to the pot core proposed by Cui, Xu, Xu, and Shui [Electr. Eng. 101, 911-919 (2019)]. The proposed driving technology involves adding a permanent magnet to the central pillar to introduce a direct current excitation bias, thereby adjusting the static operating point and the electromechanical conversion efficiency of the transducer. In this paper, a theoretical model of the electromagnetic driving force is established, and finite element simulations show that adding a permanent magnet increases the electromechanical efficiency of can-type transducers from less than 1% to 84.43% under low-power conditions. Furthermore, prototypes of underwater can-type electromagnetic transducers with and without a permanent magnet at the core center were fabricated in this study. The experimental results confirmed that the permanent magnet drive technology significantly improves electromechanical conversion efficiency by adding a permanent magnet to the core center.

High-Resolution Respirometry Methodology for Bioenergetic and Metabolic Studies in Intact Brain Slices.

The brain is critically dependent on energetic substrates as it consumes circa 20% of glucose and oxygen under normal physiological conditions. Although different cell types and at different locations might experience particular specificities in the utilization of these substrates, overall, mitochondrial oxidative phosphorylation supports the most efficient energy transduction process, enabling the complete oxidation of glucose to CO2 coupled to ATP synthesis in the presence of O2. Impairment of mitochondrial bioenergetics has been identified as an early event in many brain diseases and aging. Thus, novel methodologies to readily assess mitochondrial respiration in brain tissue, while preserving cellular and mitochondrial architecture and overcoming the serious drawbacks of studies using isolated mitochondrial preparations, are needed. Here we describe a methodology for studying functional parameters defining tissue metabolic respiration in brain hippocampal slices. The methodology can be used for physiological, pharmacological, and toxicological studies.

Investigation of electromagnetic acoustic transducer efficiency with composite magnetisation direction magnets

Investigation of electromagnetic acoustic transducer efficiency with composite magnetisation direction magnets

Research Status and Applications of Adeno-Associated Virus.

Adeno-associated virus (AAV) has emerged as a powerful and effective tool for the delivery of exogenous genes into various cells or tissues. To improve the gene delivery efficiency, as well as the safety and specificity of AAV's cell-targeting capabilities, extensive investigations have been conducted into its molecular biological characteristics, including capsid structure, cellular tropism, and the mechanisms underlying its entry, replication, DNA packaging, and capsid assembly. Significant differences exist between human and non-human primate AAVs regarding tissue targeting and transduction efficiency. These differences are primarily attributed to the amino acid sequences of AAV capsid proteins, the structural characteristics of these proteins, and the interactions of AAV with surface factors on host cells, such as cell surface receptors, signaling molecules, and associated proteins. This review primarily focuses on several key aspects of AAV, including its genome, coat proteins and their structures, genome replication, virus assembly, and the role of helper viruses. Additionally, it examines the utilization of recombinant adeno-associated viruses (rAAV), detailing their production methods, mechanisms of cell entry and trafficking, and various serotypes. The review further interprets the role of rAAV by analyzing its current applications in research and therapy.

Conventional and Tropism-Modified High-Capacity Adenoviral Vectors Exhibit Similar Transduction Profiles in Human iPSC-Derived Retinal Organoids

Viral vector delivery of gene therapy represents a promising approach for the treatment of numerous retinal diseases. Adeno-associated viral vectors (AAV) constitute the primary gene delivery platform; however, their limited cargo capacity restricts the delivery of several clinically relevant retinal genes. In this study, we explore the feasibility of employing high-capacity adenoviral vectors (HC-AdVs) as alternative delivery vehicles, which, with a capacity of up to 36 kb, can potentially accommodate all known retinal gene coding sequences. We utilized HC-AdVs based on the classical adenoviral type 5 (AdV5) and on a fiber-modified AdV5.F50 version, both engineered to deliver a 29.6 kb vector genome encoding a fluorescent reporter construct. The tropism of these HC-AdVs was evaluated in an induced pluripotent stem cell (iPSC)-derived human retinal organoid model. Both vector types demonstrated robust transduction efficiency, with sustained transgene expression observed for up to 110 days post-transduction. Moreover, we found efficient transduction of photoreceptors and Müller glial cells, without evidence of reactive gliosis or loss of photoreceptor cell nuclei. However, an increase in the thickness of the photoreceptor outer nuclear layer was observed at 110 days post-transduction, suggesting potential unfavorable effects on Müller glial or photoreceptor cells associated with HC-AdV transduction and/or long-term reporter overexpression. These findings suggest that while HC-AdVs show promise for large retinal gene delivery, further investigations are required to assess their long-term safety and efficacy.

Multimodal Loudspeaker Based on a Dielectric Elastomer Roll Actuator

A novel loudspeaker design integrating lightweight and efficient acoustic transducers derived from dielectric elastomer films is presented. This innovation centers on core-free dielectric elastomer roll actuators, functioning akin to artificial muscles, altering shape upon the application of an electric field. The actuators serve two roles: propelling acoustically radiating surfaces and emitting sound themselves. The paper provides a model that describes the axial vibration behavior via electromechanical and acoustic networks, facilitating understanding and comparability with electrodynamic loudspeaker systems without the need for extensive background knowledge of dielectric elastomer technology. Based on established methodologies, the design process and adaptability to diverse applications is evident. A demonstrator developed as a proof of concept with the aim of creating a broadband speaker for speech reproduction was designed, constructed, and examined utilizing the established methodology. It shows promising practicality and, in certain aspects, technological superiority over electrodynamic counterparts. Notable features, such as increased efficiency, significant weight reduction, and wide radiation pattern make this speaker viable and particularly attractive for mobile PA applications such as those found in trains and aircraft. Furthermore, the underlying principle and modeling framework allow the future creation and adaptation to a broad spectrum of applications, promising further innovation in the field.

The vectors went in two-by-two: Transduction efficiency and tolerability of dual and triple rAAV vector delivery following intravitreal injection for genome-editing applications.

Genome or prime editing has become a promising tool for the treatment of hereditary disorders affecting the inner retina, such as dominant optic neuropathies. In vivo delivery of gene editors, such as Cas9, is typically achieved using recombinant adeno-associated virus (rAAV) vectors, which have a broad range of cellular tropisms and are well tolerated following intravitreal administration. Owing to the large size of gene editing constructs and the limited carrying capacity of rAAV (<5.1kb) it is unfortunately usually necessary to split therapeutic transgene cassettes across multiple co-administered vector genomes. While the efficiency with which multiple vector genomes recombine following cellular entry has been studied extensively, another potentially limiting factor is the likelihood of target cells (e.g. retinal ganglion cells) receiving two or more vectors containing genomes that correspond to the full-length expression cassette when recombined. In this study we examine the efficiency with which two or more vector genomes transduce various retinal cell types following intravitreal administration. rAAV2/2[MAX] vectors expressing individual fluorescent reporters (GFP, BFP or mCherry) were co-injected intravitreally singly or in combination (dual or triple), allowing the extent of co-transduction to be assessed through multimodal in vivo imaging, electroretinography, flow cytometry and post-mortem histology. We find that intravitreal co-administration of vectors containing multiple genomes is well tolerated - with no observed alterations in retinal thickness or ERG amplitudes - but that co-transduction efficiency decreases significantly with increasing genome number. As such co-transduction of multiple vectors may be a major bottleneck limiting gene editing of inherited disorders affecting the inner retina.

PD1-Targeted Transgene Delivery to Treg Cells.

Achieving the precise targeting of lentiviral vectors (LVs) to specific cell populations is crucial for effective gene therapy, particularly in cancer treatment where the modulation of the tumor microenvironment can enhance anti-tumor immunity. Programmed cell death protein 1 (PD-1) is overexpressed on activated tumor-infiltrating T lymphocytes, including regulatory T cells that suppress immune responses via FOXP3 expression. We developed PD1-targeted LVs by incorporating the anti-PD1 nanobody nb102c3 into receptor-blinded measles virus H and VSV-Gmut glycoproteins. We assessed the retargeting potential of nb102c3 and evaluated transduction efficiency in activated T lymphocytes. FOXP3 expression was suppressed using shRNA delivered by these LVs. Our results demonstrate that PD1-targeted LVs exerted pronounced tropism towards PD1+ cells, enabling the selective transduction of activated T lymphocytes while sparing naive T cells. The suppression of FOXP3 in Tregs reduced their suppressive activity. PD1-targeted glycoprotein H provided greater specificity, whereas the VSV-Gmut, together with the anti-PD1 pseudoreceptor, achieved higher viral titers but was less selective. Our study demonstrates that PD1-targeted LVs may offer a novel strategy to modulate immune responses within the tumor microenvironment with the potential for developing new therapeutic strategies aimed at enhancing anti-tumor immunity.

AAV library screening identifies novel vector for efficient transduction of human aorta.

Targeted gene delivery to vascular smooth muscle cells (VSMCs) could prevent or improve a variety of diseases affecting the vasculature and particularly the aorta. Thus, we aimed to develop a delivery vector that efficiently targets VSMCs. We selected engineered adeno-associated virus (AAV) capsids from a random AAV capsid library and tested the top enriched motifs in parallel screening through individual barcoding. This approach allowed us to distinguish capsids that only transduce cells based on genomic DNA (gDNA) from those also mediating transgene expression based on transcribed cDNA reads. After three rounds of selection on primary murine VSMCs (mVSMCs), we identified a novel targeting motif (RFTEKPA) that significantly improved transduction and gene expression efficiency over AAV9-wild type(WT)and increased expression in mVSMCsby70% compared to thepreviously identified SLRSPPS peptide. Further analysis showed that the novel motif also improved expression in human aortic smooth muscle cells (HAoSMCs) and human aortic tissue ex vivo up to threefold compared to SLRSPPS and approximately 70-fold to AAV9-WT. This high cross-species transduction efficiency makes the novel capsid motif a potential candidate for future clinical application in vascular diseases.

Rotavirus-Inspired Nanointerface Engineered Biosensors for All-in-One Cancer Diagnosis.

Ultrasensitive and population-scale cancer screening technologies are critical to reducing cancer mortality. However, the current qRT-PCR falls short in high-throughput screening of multiple cancers. Here, a rotavirus-inspired multicancer diagnosis system (RMDS) is developed via nanointerface engineering. RMDS employs Y-shaped DNA (YDNA) probes to encircle the graphene quantum dots (GQDs) for nanointerface modification. The biotransduction mechanisms at the nanointerface are systematically investigated. RMDS greatly enhances the transduction efficiency of biological analytes by optimizing the probe density and configuration. RMDS realizes ultrasensitive detection of the lung cancer KARS G12D mutation with a limit of detection (LoD) of 5.7 aM and the breast cancer-related AKT2 gene (LoD: 3.0 aM). The multiple chambers enable simultaneous diagnosis of multiple cancers and determination of cancer progression. Clinical validation shows RMDS can be a practical solution, which could complement or replace qRT-PCR and become the next-generation all-in-one tool for large-scale population cancer screening.

Adenoviral Vectors for Gene Therapy of Hereditary Diseases.

Adenoviral vectors (AdVs) are effective vectors for gene therapy due to their broad tropism, high capacity, and high transduction efficiency, which makes them actively used as oncolytic vectors and for creating vector vaccines. However, despite their numerous advantages, AdVs have not yet found their place in gene therapy for hereditary diseases. This review provides an overview of AdVs, their features, and clinical trials using them for gene replacement therapy in monogenic diseases and analyzes the reasons for the failures of these studies. Additionally, current research on the modification of AdVs to reduce immune responses and target delivery is discussed.