Evaluation of a rapid multi-attribute combinatorial high-throughput UV-Vis/DLS/SLS analytical platform for rAAV quantification and characterization.

Recombinant adeno-associated virus (rAAV)-based gene therapies are expanding in their application. Despite progress in manufacturing, current analytical methods for product quantification and characterization remain largely unchanged. Although critical for product and process development, in-process testing, and batch release, current analytical methods are labor-intensive, costly, and hampered by extended turnaround times and low throughput. The field requires more efficient, cost-effective analytical techniques capable of handling large sample quantities to accelerate product and process development. Here, we evaluated Stunner from Unchained Labs for quantifying and characterizing rAAVs and compared it with established analytical methods. Stunner is a combinatorial analytic technology platform that interpolates ultraviolet-visible (UV-Vis) absorption with static and dynamic light scattering (SLS/DLS) analysis to determine capsid and genomic titer, empty and full capsid ratio, and assess vector size and polydispersity. The platform offers empirical measurements with minimal sample requirements. Upon testing hundreds of rAAV vectors, comprising various serotypes and transgenes, the data show a strong correlation with established analytical methods and exhibit high reproducibility and repeatability. Some analyses can be applied to in-process samples from different purification stages and processes, fulfilling the demand for rapid, high-throughput analysis during development. In sum, the pipeline presented streamlines small- and large-batch analytics.

SMRT Sequencing Enables High-Throughput Identification of Novel AAVs from Capsid Shuffling and Directed Evolution.

The use of AAV capsid libraries coupled with various selection strategies has proven to be a remarkable approach for generating novel AAVs with enhanced and desired features. The inability to reliably sequence the complete capsid gene in a high-throughput manner has been the bottleneck of capsid engineering. As a result, many library strategies are confined to localized and modest alterations in the capsid, such as peptide insertions or single variable region (VR) alterations. The caveat of short reads by means of next-generation sequencing (NGS) hinders the diversity of capsid library construction, shifting the field away from whole-capsid modifications. We generated AAV capsid shuffled libraries of naturally occurring AAVs and applied directed evolution in both mice and non-human primates (NHPs), with the goal of yielding AAVs that are compatible across both species for translational applications. We recovered DNA from the tissues of injected animal and used single molecule real-time (SMRT) sequencing to identify variants enriched in the central nervous system (CNS). We provide insights and considerations for variant identification by comparing bulk tissue sequencing to that of isolated nuclei. Our work highlights the potential advantages of whole-capsid engineering, as well as indispensable methodological improvements for the analysis of recovered capsids, including the nuclei-enrichment step and SMRT sequencing.

Systemically targeted cancer immunotherapy and gene delivery using transmorphic particles.

Immunotherapy is a powerful tool for cancer treatment, but the pleiotropic nature of cytokines and immunological agents strongly limits clinical translation and safety. To address this unmet need, we designed and characterised a systemically targeted cytokine gene delivery system through transmorphic encapsidation of human recombinant adeno-associated virus DNA using coat proteins from a tumour-targeted bacteriophage (phage). We show that Transmorphic Phage/AAV (TPA) particles provide superior delivery of transgenes over current phage-derived vectors through greater diffusion across the extracellular space and improved intracellular trafficking. We used TPA to target the delivery of cytokine-encoding transgenes for interleukin-12 (IL12), and novel isoforms of IL15 and tumour necrosis factor alpha (TNF α ) for tumour immunotherapy. Our results demonstrate selective and efficient gene delivery and immunotherapy against solid tumours in vivo, without harming healthy organs. Our transmorphic particle system provides a promising modality for safe and effective gene delivery, and cancer immunotherapies through cross-species complementation of two commonly used viruses.

Hexon modification of human adenovirus type 5 vectors enables efficient transduction of human multipotent mesenchymal stromal cells.

In adenovirus type 5 (HAdV-5)-derived viral vectors, the fiber protein has been the preferred locale for modifications to alter the natural viral tropism. Hexon, the most abundant capsid protein, has rarely been used for retargeting purposes, likely because the insertion of larger targeting peptides into Hexon often interferes with the assembly of the viral capsid. We previously observed that positively charged molecules enhance the transduction of human multipotent mesenchymal stromal cells (hMSCs)-a cell type of significant interest for clinical development but inefficiently transduced by unmodified HAdV-5-based vectors. As efficient HAdV-5-mediated gene transfer would greatly increase the therapeutic potential of hMSCs, we tested the hypothesis that introducing positively charged amino acids into Hexon might enhance the transduction of hMSCs, enabling efficient expression of selected transgenes. From the constructs that could be rescued as functional virions, one (HAdV-5-HexPos3) showed striking transduction of hMSCs with up to 500-fold increased efficiency. Evaluation of the underlying mechanism identified heparan sulfate proteoglycans (HSPGs) to be essential for virus uptake by the cells. The ease and efficiency of transduction of hMSCs with this vector will facilitate the development of genetically modified hMSCs as therapeutic vehicles in different disciplines, including oncology or regenerative medicine.

AAV-p40 Bioengineering Platform for Variant Selection Based on Transgene Expression.

The power of adeno-associated viral (AAV)-directed evolution for identifying novel vector variants with improved properties is well established, as evidenced by numerous publications reporting novel AAV variants. However, most capsid variants reported to date have been identified using either replication-competent (RC) selection platforms or polymerase chain reaction-based capsid DNA recovery methods, which can bias the selection toward efficient replication or unproductive intracellular trafficking, respectively. A central objective of this study was to validate a functional transduction (FT)-based method for rapid identification of novel AAV variants based on AAV capsid mRNA expression in target cells. We performed a comparison of the FT platform with existing RC strategies. Based on the selection kinetics and function of novel capsids identified in an in vivo screen in a xenograft model of human hepatocytes, we identified the mRNA-based FT selection as the most optimal AAV selection method. Lastly, to gain insight into the mRNA-based selection mechanism driven by the native AAV-p40 promoter, we studied its activity in a range of in vitro and in vivo targets. We found AAV-p40 to be a ubiquitously active promoter that can be modified for cell-type-specific expression by incorporating binding sites for silencing transcription factors, allowing for cell-type-specific library selection.

Phloem Metabolites of Prunus Sp. Rather than Infection with Candidatus Phytoplasma Prunorum Influence Feeding Behavior of Cacopsylla pruni Nymphs.

Phytoplasmas are specialized small bacteria restricted to the phloem tissue and spread by hemipterans feeding on plant sieve tube elements. As for many other plant pathogens, it is known that phytoplasmas alter the chemistry of their hosts. Most research on phytoplasma-plant interactions focused on the induction of plant volatiles and phytohormones. Little is known about the influence of phytoplasma infections on the nutritional composition of phloem and consequences on vector behavior and development. The plum psyllid Cacopsylla pruni transmits 'Candidatus Phytoplasma prunorum', the causing agent of European Stone Fruit Yellows (ESFY). While several Prunus species are susceptible for psyllid feeding, they show different responses to the pathogen. We studied the possible modulation of plant-insect interactions by bacteria-induced changes in phloem sap chemistry. Therefore, we sampled phloem sap from phytoplasma-infected and non-infected Prunus persica and Prunus insititia plants, which differ in their susceptibility to ESFY and psyllid feeding. Furthermore, the feeding behavior and development of C. pruni nymphs was compared on infected and non-infected P. persica and P. insititia plants. Phytoplasma infection did not affect phloem consumption by C. pruni nymphs nor their development time. In contrast, the study revealed significant differences between P. insititia and P. persica in terms of both phloem chemistry and feeding behavior of C. pruni nymphs. Phloem feeding phases were four times longer on P. insititia than on P. persica, resulting in a decreased development time and higher mortality of vector insects on P. persica plants. These findings explain the low infestation rates of peach cultivars with plum psyllids commonly found in field surveys.

Efficient genome editing of wild strawberry genes, vector development and validation.

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system is an effective genome editing tool for plant and animal genomes. However, there are still few reports on the successful application of CRISPR-Cas9 to horticultural plants, especially with regard to germ-line transmission of targeted mutations. Here, we report high-efficiency genome editing in the wild strawberry Fragaria vesca and its successful application to mutate the auxin biosynthesis gene TAA1 and auxin response factor 8 (ARF8). In our CRISPR system, the Arabidopsis U6 promoter AtU6-26 and the wild strawberry U6 promoter FveU6-2 were each used to drive the expression of sgRNA, and both promoters were shown to lead to high-efficiency genome editing in strawberry. To test germ-line transmission of the edited mutations and new mutations induced in the next generation, the progeny of the primary (T0) transgenic plants carrying the CRISPR construct was analysed. New mutations were detected in the progeny plants at a high efficiency, including large deletions between the two PAM sites. Further, T1 plants harbouring arf8 homozygous knockout mutations grew considerably faster than wild-type plants. The results indicate that our CRISPR vectors can be used to edit the wild strawberry genome at a high efficiency and that both sgRNA design and appropriate U6 promoters contribute to the success of genomic editing. Our results open up exciting opportunities for engineering strawberry and related horticultural crops to improve traits of economic importance.

Plasmid DNA Manufacturing for Indirect and Direct Clinical Applications.

Plasmid DNA is currently gaining increasing importance for clinical research applications in gene therapy and genetic vaccination. For direct gene transfer into humans, good manufacturing practice (GMP)-grade plasmid DNA is mandatory. The same holds true if the drug substance contains a genetically modified cell, for example chimeric antigen receptor (CAR) T cells, where these cells as well as the contained plasmids are used. According to the responsible regulatory agencies, they have to be produced under full GMP. On the other hand, for GMP production of, for example, mRNA or viral vectors (lentiviral vectors, adeno-associated virus vectors, etc.), in many cases, High Quality Grade plasmid DNA is accepted as a starting material. The manufacturing process passes through different production steps. To ensure the right conditions are used for the plasmid, a pilot run must be conducted at the beginning. In this step, a followed upscaling with respect to reproducibility and influences on product quality is performed. Subsequently, a cell bank of the transformed productions strain is established and characterized. This cell bank is used for the cultivation process. After cell harvesting and lysis, several chromatography steps are conducted to receive a pure plasmid product. Depending on the respective required quality grade, the plasmid product is subject to several quality controls. The last step consists of formulation and filling of the product.

Vector development and vitellogenin determine the transovarial transmission of begomoviruses.

The majority of plant viruses are transmitted by insect vectors between hosts, and transovarial transmission of viruses from vector parents to offspring has great significance to their epidemiology. Begomoviruses are transmitted by the whitefly Bemisia tabaci in a circulative manner and are maintained through a plant-insect-plant cycle. Other routes of begomovirus transmission are not clearly known. Here, we report that transovarial transmission from female whiteflies to offspring often happens for one begomovirus, Tomato yellow leaf curl virus (TYLCV), and may have contributed significantly to its global spread. We found that TYLCV entry of the reproductive organ of its vector mainly depended on the developmental stage of the whitefly ovary, and the transovarial transmission of TYLCV to offspring increased with whitefly adult age. The specific interaction between virus coat protein (CP) and whitefly vitellogenin (Vg) was vital for virus entry into whitefly ovary. When knocking down the expression of Vg, the entry of TYLCV into ovary was inhibited and the transovarial transmission efficiency decreased. In contrast, another begomovirus, Papaya leaf curl China virus (PaLCuCNV), CP did not interact with whitefly Vg, and PaLCuCNV could not be transovarially transmitted by whiteflies. We further showed that TYLCV could be maintained for at least two generations in the absence of virus-infected plants, and the adult progenies were able to infect healthy plants in both the laboratory and field. This study reports the transovarial transmission mechanism of begomoviruses, and it may help to explain the evolution and global spread of some begomoviruses.

Determining Culex annulirostris larval densities and control efforts across a coastal wetland, Northern Territory, Australia.

The Darwin coastal wetlands provide suitable breeding conditions for Culex annulirostris, which is abundant between December and August each year. This species is the principal vector for arboviruses, including Ross River virus and Murray Valley encephalitis, and is an appreciable pest species. Aerial control is conducted when routine larval surveys for this species predict high numbers of emergent adults. We sought to determine the most productive vegetation categories and seasonal aspects associated with Cx. annulirostris breeding and control operations in these wetlands. By applying a generalized linear model to compare larval densities and aerial control efforts for each vegetation category, we found that Schoenoplectus reeds were the most productive vegetation type in May and June and were associated with the greatest amount of control required. Other vegetation categories associated with tidal mangroves and lower topographic elevation were also productive during these months for extended periods, while rain-affected reticulate areas and grassland floodplains were most productive in January and April. In addition, areas associated with nutrient rich organic matter appeared to initiate Cx. annulirostris breeding and were highly productive seasonally. This study has highlighted the vegetation categories most significantly associated with Cx. annulirostris breeding in a Darwin wetland. This knowledge can be applied to current control efforts to improve aerial control efficiency for this species and could be applicable in other areas of northern Australia.