ADEVO: Proof-of-concept of adenovirus-directed EVOlution by random peptide display on the fiber knob.

Directed evolution of viral vectors involves the generation of randomized libraries followed by artificial selection of improved variants. Directed evolution only yielded limited results in adenovirus (AdV) engineering until now, mainly due to insufficient complexities of randomized libraries. Meanwhile, clinical applications of AdVs as gene therapy or oncolytic vectors are still hampered by the predetermined tropism of natural types. To overcome this challenge, we hypothesized that randomized peptide insertions on the capsid surface can be incorporated into the AdV bioengineering toolbox for retargeting. Here we developed AdV-directed EVOlution protocols based on fiber knob peptide display. Human AdV-C5-derived libraries were constructed following three distinct protocols and selected on a panel of cancer cell lines, with the goal of identifying variants able to infect and lyse these tumor cells more efficiently. All protocols enabled the construction of high complexity libraries with up to 9.6 × 105 unique variants, an approximate 100-fold improvement compared with previously published AdV libraries. After selection, the most enriched variants, which were robustly selected in various cancer cell lines, did not display enhanced infectivity but rather more efficient replication and cell lysis. Selected inserts also conferred enhanced lysis ability to oncolytic AdVs restricted to telomerase-expressing cell lines.

Identifying adeno-associated virus (AAV) vectors that efficiently target high grade glioma cells, for in vitro monitoring of temporal cell responses.

To improve the translation of preclinical cancer research data to successful clinical effect, there is an increasing focus on the use of primary patient-derived cancer cells with limited growth in culture to reduce genetic and phenotype drift. However, these primary lines are less amenable to standardly used methods of exogenous DNA introduction. Adeno-associated viral (AAV) vectors display tropism for a wide range of human tissues, avidly infect primary cells and have a good safety profile. In the present study, we therefore used a next-generation sequencing (NGS) barcoded AAV screening method to assess transduction capability of a panel of 36 AAVs in primary cell lines representing high-grade glioma (HGG) brain tumours including glioblastoma (GBM) and diffuse intrinsic pontine glioma (DIPG)/diffuse midline glioma (DMG). As proof of principle, we created a reporter construct to analyse activity of the transcriptional co-activators yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ). Transcriptional activation was monitored by promoter-driven expression of the Timer fluorescent tag, a protein that fluoresces green immediately after transcription and transitions to red fluorescence over time. As expected, attempts to express the reporter in primary HGG cells from plasmid expression vectors were unsuccessful. Using the top candidate from the AAV screen, we demonstrate successful AAV-mediated transduction of HGG cells with the YAP/TAZ dynamic activity reporter. In summary, the NGS-screening approach facilitated screening of many potential AAVs, identifying vectors that can be used to study the biology of primary HGG cells.

Enhanced AAV transduction across preclinical CNS models: A comparative study in human brain organoids with cross-species evaluations.

Viral vectors based on recombinant adeno-associated virus (rAAV) have become the most widely used system for therapeutic gene delivery in the central nervous system (CNS). Despite clinical safety and efficacy in neurological applications, a barrier to adoption of the current generation of vectors lies in their limited efficiency, resulting in limited transduction of CNS target cells. To address this limitation, researchers have bioengineered fit-for-purpose AAVs with improved CNS tropism and tissue penetration. While the preclinical assessment of these novel AAVs is primarily conducted in animal models, human induced pluripotent stem cell (hiPSC)-derived organoids offer a unique opportunity to functionally evaluate novel AAV variants in a human context. In this study, we performed a comprehensive and unbiased evaluation of a large number of wild-type and bioengineered AAV capsids for their transduction efficiency in hiPSC-derived brain organoids. We demonstrate that efficient AAV transduction observed in organoids was recapitulated in vivo in both mouse and non-human primate models after cerebrospinal fluid (CSF) delivery. In summary, our study showcases the use of brain organoid systems for the pre-screening of novel AAV vectors. Additionally, we report data for novel AAV variants that exhibit improved CNS transduction efficiency when delivered via the CSF in in vivo preclinical models.

Novel AAV variants with improved tropism for human Schwann cells.

Gene therapies and associated technologies are transforming biomedical research and enabling novel therapeutic options for patients living with debilitating and incurable genetic disorders. The vector system based on recombinant adeno-associated viral vectors (AAVs) has shown great promise in recent clinical trials for genetic diseases of multiple organs, such as the liver and the nervous system. Despite recent successes toward the development of novel bioengineered AAV variants for improved transduction of primary human tissues and cells, vectors that can efficiently transduce human Schwann cells (hSCs) have yet to be identified. Here, we report the application of the functional transduction-RNA selection method in primary hSCs for the development of AAV variants for specific and efficient transgene delivery to hSCs. The two identified capsid variants, Pep2hSC1 and Pep2hSC2, show conserved potency for delivery across various in vitro, in vivo, and ex vivo models of hSCs. These novel AAV capsids will serve as valuable research tools, forming the basis for therapeutic solutions for both SC-related disorders or peripheral nervous system injury.

Harnessing whole human liver ex situ normothermic perfusion for preclinical AAV vector evaluation.

Developing clinically predictive model systems for evaluating gene transfer and gene editing technologies has become increasingly important in the era of personalized medicine. Liver-directed gene therapies present a unique challenge due to the complexity of the human liver. In this work, we describe the application of whole human liver explants in an ex situ normothermic perfusion system to evaluate a set of fourteen natural and bioengineered adeno-associated viral (AAV) vectors directly in human liver, in the presence and absence of neutralizing human sera. Under non-neutralizing conditions, the recently developed AAV variants, AAV-SYD12 and AAV-LK03, emerged as the most functional variants in terms of cellular uptake and transgene expression. However, when assessed in the presence of human plasma containing anti-AAV neutralizing antibodies (NAbs), vectors of human origin, specifically those derived from AAV2/AAV3b, were extensively neutralized, whereas AAV8- derived variants performed efficiently. This study demonstrates the potential of using normothermic liver perfusion as a model for early-stage testing of liver-focused gene therapies. The results offer preliminary insights that could help inform the development of more effective translational strategies.

A transient protein folding response targets aggregation in the early phase of TDP-43-mediated neurodegeneration.

Understanding the mechanisms that drive TDP-43 pathology is integral to combating amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD) and other neurodegenerative diseases. Here we generated a longitudinal quantitative proteomic map of the cortex from the cytoplasmic TDP-43 rNLS8 mouse model of ALS and FTLD, and developed a complementary open-access webtool, TDP-map ( https://shiny.rcc.uq.edu.au/TDP-map/ ). We identified distinct protein subsets enriched for diverse biological pathways with temporal alterations in protein abundance, including increases in protein folding factors prior to disease onset. This included increased levels of DnaJ homolog subfamily B member 5, DNAJB5, which also co-localized with TDP-43 pathology in diseased human motor cortex. DNAJB5 over-expression decreased TDP-43 aggregation in cell and cortical neuron cultures, and knockout of Dnajb5 exacerbated motor impairments caused by AAV-mediated cytoplasmic TDP-43 expression in mice. Together, these findings reveal molecular mechanisms at distinct stages of ALS and FTLD progression and suggest that protein folding factors could be protective in neurodegenerative diseases.

A hepatic network of dendritic cells mediates CD4 T cell help outside lymphoid organs.

While CD4+ T cells are a prerequisite for CD8+ T cell-mediated protection against intracellular hepatotropic pathogens, the mechanisms facilitating the transfer of CD4-help to intrahepatic CD8+ T cells are unknown. Here, we developed an experimental system to investigate cognate CD4+ and CD8+ T cell responses to a model-antigen expressed de novo in hepatocytes and reveal that after initial priming, effector CD4+ and CD8+ T cells migrate into portal tracts and peri-central vein regions of the liver where they cluster with type-1 conventional dendritic cells. These dendritic cells are locally licensed by CD4+ T cells and expand the number of CD8+ T cells in situ, resulting in larger effector and memory CD8+ T cell pools. These findings reveal that CD4+ T cells promote intrahepatic immunity by amplifying the CD8+ T cell response via peripheral licensing of hepatic type-1 conventional dendritic cells and identify intrahepatic perivascular compartments specialized in facilitating effector T cell-dendritic cell interactions.

Development of CNS tropic AAV1-like variants with reduced liver-targeting following systemic administration in mice.

Directed evolution of natural AAV9 using peptide display libraries have been widely used in the search for an optimal recombinant AAV (rAAV) for transgene delivery across the blood-brain barrier (BBB) to the CNS following intravenous ( IV) injection. In this study, we used a different approach by creating a shuffled rAAV capsid library based on parental AAV serotypes 1 through 12. Following selection in mice, 3 novel variants closely related to AAV1, AAV-BBB6, AAV-BBB28, and AAV-BBB31, emerged as top candidates. In direct comparisons with AAV9, our novel variants demonstrated an over 270-fold improvement in CNS transduction and exhibited a clear bias toward neuronal cells. Intriguingly, our AAV-BBB variants relied on the LY6A cellular receptor for CNS entry, similar to AAV9 peptide variants AAV-PHP.eB and AAV.CAP-B10, despite the different bioengineering methods used and parental backgrounds. The variants also showed reduced transduction of both mouse liver and human primary hepatocytes in vivo. To increase clinical translatability, we enhanced the immune escape properties of our new variants by introducing additional modifications based on rational design. Overall, our study highlights the potential of AAV1-like vectors for efficient CNS transduction with reduced liver tropism, offering promising prospects for CNS gene therapies.

Neuronal Replenishment via Hydrogel-Rationed Delivery of Reprogramming Factors.

The central nervous system's limited capacity for regeneration often leads to permanent neuronal loss following injury. Reprogramming resident reactive astrocytes into induced neurons at the site of injury is a promising strategy for neural repair, but challenges persist in stabilizing and accurately targeting viral vectors for transgene expression. In this study, we employed a bioinspired self-assembling peptide (SAP) hydrogel for the precise and controlled release of a hybrid adeno-associated virus (AAV) vector, AAVDJ, carrying the NeuroD1 neural reprogramming transgene. This method effectively mitigates the issues of high viral dosage at the target site, off-target delivery, and immunogenic reactions, enhancing the vector's targeting and reprogramming efficiency. In vitro, this vector successfully induced neuron formation, as confirmed by morphological, histochemical, and electrophysiological analyses. In vivo, SAP-mediated delivery of AAVDJ-NeuroD1 facilitated the trans-differentiation of reactive host astrocytes into induced neurons, concurrently reducing glial scarring. Our findings introduce a safe and effective method for treating central nervous system injuries, marking a significant advancement in regenerative neuroscience.

AAV capsid bioengineering in primary human retina models.

Adeno-associated viral (AAV) vector-mediated retinal gene therapy is an active field of both pre-clinical as well as clinical research. As with other gene therapy clinical targets, novel bioengineered AAV variants developed by directed evolution or rational design to possess unique desirable properties, are entering retinal gene therapy translational programs. However, it is becoming increasingly evident that predictive preclinical models are required to develop and functionally validate these novel AAVs prior to clinical studies. To investigate if, and to what extent, primary retinal explant culture could be used for AAV capsid development, this study performed a large high-throughput screen of 51 existing AAV capsids in primary human retina explants and other models of the human retina. Furthermore, we applied transgene expression-based directed evolution to develop novel capsids for more efficient transduction of primary human retina cells and compared the top variants to the strongest existing benchmarks identified in the screening described above. A direct side-by-side comparison of the newly developed capsids in four different in vitro and ex vivo model systems of the human retina allowed us to identify novel AAV variants capable of high transgene expression in primary human retina cells.