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.

Development of new adeno-associated virus capsid variants for targeted gene delivery to human cardiomyocytes.

Recombinant adeno-associated viruses (rAAVs) have emerged as one of the most promising gene therapy vectors that have been successfully used in pre-clinical models of heart disease. However, this has not translated well to humans due to species differences in rAAV transduction efficiency. As a result, the search for human cardiotropic capsids is a major contemporary challenge. We used a capsid-shuffled rAAV library to perform directed evolution in human iPSC-derived cardiomyocytes (hiPSC-CMs). Five candidates emerged, with four presenting high sequence identity to AAV6, while a fifth divergent variant was related to AAV3b. Functional analysis of the variants was performed in vitro using hiPSC-CMs, cardiac organoids, human cardiac slices, non-human primate and porcine cardiac slices, as well as mouse heart and liver in vivo. We showed that cell entry was not the best predictor of transgene expression efficiency. The novel variant rAAV.KK04 was the best-performing vector in human-based screening platforms, exceeding the benchmark rAAV6. None of the novel capsids demonstrate a significant transduction of liver in vivo. The range of experimental models used revealed the value of testing for tropism differences under the conditions of human specificity, bona fide, myocardium and cell type of interest.

Direct optogenetic activation of upper airway muscles in an acute model of upper airway hypotonia mimicking sleep onset.

STUDY OBJECTIVES: Obstructive sleep apnea (OSA), where the upper airway collapses repeatedly during sleep due to inadequate dilator muscle tone, is challenging to treat as current therapies are poorly tolerated or have variable and unpredictable efficacy. We propose a novel, optogenetics-based therapy, that stimulates upper airway dilator muscle contractions in response to light. To determine the feasibility of a novel optogenetics-based OSA therapy, we developed a rodent model of human sleep-related upper airway muscle atonia. Using this model, we evaluated intralingual delivery of candidate optogenetic constructs, notably a muscle-targeted approach that will likely have a favorable safety profile. METHODS: rAAV serotype 9 viral vectors expressing a channelrhodopsin-2 variant, driven by a muscle-specific or nonspecific promoter were injected into rat tongues to compare strength and specificity of opsin expression. Light-evoked electromyographic responses were recorded in an acute, rodent model of OSA. Airway dilation was captured with ultrasound. RESULTS: The muscle-specific promoter produced sufficient opsin expression for light stimulation to restore and/or enhance electromyographic signals (linear mixed model, F = 140.0, p < 0.001) and induce visible tongue contraction and airway dilation. The muscle-specific promoter induced stronger (RM-ANOVA, F(1,8) = 10.0, p = 0.013) and more specific opsin expression than the nonspecific promoter in an otherwise equivalent construct. Viral DNA and RNA were robust in the tongue, but low or absent in all other tissues. CONCLUSIONS: Significant functional responses to direct optogenetic muscle activation were achieved following muscle-specific promoter-driven rAAV-mediated transduction, providing proof-of-concept for an optogenetic therapy for patients with inadequate dilator muscle activity during sleep.

Long-term ex situ normothermic perfusion of human split livers for more than 1 week.

Current machine perfusion technology permits livers to be preserved ex situ for short periods to assess viability prior to transplant. Long-term normothermic perfusion of livers is an emerging field with tremendous potential for the assessment, recovery, and modification of organs. In this study, we aimed to develop a long-term model of ex situ perfusion including a surgical split and simultaneous perfusion of both partial organs. Human livers declined for transplantation were perfused using a red blood cell-based perfusate under normothermic conditions (36 °C) and then split and simultaneously perfused on separate machines. Ten human livers were split, resulting in 20 partial livers. The median ex situ viability was 125 h, and the median ex situ survival was 165 h. Long-term survival was demonstrated by lactate clearance, bile production, Factor-V production, and storage of adenosine triphosphate. Here, we report the long-term ex situ perfusion of human livers and demonstrate the ability to split and perfuse these organs using a standardised protocol.

In Search of Adeno-Associated Virus Vectors With Enhanced Cardiac Tropism for Gene Therapy.

Globally, adeno-associated virus (AAV) vectors have been increasingly used for clinical gene therapy trials. In Australia, AAV-based gene therapy is available for hereditary diseases such as retinal dystrophy or spinal muscular atrophy 1 (SMA1). Many preclinical studies have used AAV vectors for gene therapy in models of cardiac disease with outcomes of varying translational potential. However, major barriers to effective and safe therapeutic gene delivery to the human heart remain to be overcome. These include tropism, efficient gene transfer, mitigating off-target gene delivery and avoidance of the host immune response. Developing such an enhanced AAV vector for cardiac gene therapy is of great interest to the field of advanced cardiac therapeutics. In this review, we provide an overview of the approaches currently being employed in the search for cardiac cell-specific AAV capsids, ranging from natural AAVs selected as a result of infection and latency in the heart, to the use of cutting-edge molecular techniques to engineer and select AAVs specific for cardiac cells with the use of high-throughput methods.

Assessment of Pre-Clinical Liver Models Based on Their Ability to Predict the Liver-Tropism of Adeno-Associated Virus Vectors.

The liver is a prime target for in vivo gene therapies using recombinant adeno-associated viral vectors. Multiple clinical trials have been undertaken for this target in the past 15 years; however, we are still to see market approval of the first liver-targeted adeno-associated virus (AAV)-based gene therapy. Inefficient expression of the therapeutic transgene, vector-induced liver toxicity and capsid, and/or transgene-mediated immune responses reported at high vector doses are the main challenges to date. One of the contributing factors to the insufficient clinical outcomes, despite highly encouraging preclinical data, is the lack of robust, biologically and clinically predictive preclinical models. To this end, this study reports findings of a functional evaluation of 6 AAV vectors in 12 preclinical models of the human liver, with the aim to uncover which combination of models is the most relevant for the identification of AAV capsid variant for safe and efficient transgene delivery to primary human hepatocytes. The results, generated by studies in models ranging from immortalized cells, iPSC-derived and primary hepatocytes, and primary human hepatic organoids to in vivo models, increased our understanding of the strengths and weaknesses of each system. This should allow the development of novel gene therapies targeting the human liver.

Liver splitting during normothermic machine perfusion: a novel method to combine the advantages of both in-situ and ex-vivo techniques.

BACKGROUND: Split liver transplantation permits the transplant of two recipients using a single donor liver. Liver splitting can be performed using the ex-vivo technique (more convenient), or the in-situ technique (shorter cold ischaemic time). We aimed to develop a technique for liver splitting during normothermic machine perfusion which combines the advantages of both techniques and permits graft assessment prior to transplant. METHODS: Human livers declined for transplantation were perfused at 36 °C using a modified-commercial perfusion machine. We developed a six-step method to split whole livers into left lateral segment grafts and extended right grafts. Both partial livers were then perfused on separate machines for individual assessment. RESULTS: Using our technique, 10 whole livers were successfully split during normothermic perfusion resulting in 20 partial grafts. Apart from a single graft which failed due to a technical error, all grafts survived for 24-h after splitting. Survival was demonstrated by lactate clearance, bile production and synthesis of coagulation factors. CONCLUSIONS: Liver splitting during normothermic machine perfusion has the potential to revolutionise split liver transplantation. We describe a novel technique that reliably achieves two grafts from a single donor liver. This raises the possibility of semi-elective transplantation, and sophisticated graft assessment prior to implant.

Molecular mechanisms of amyotrophic lateral sclerosis as broad therapeutic targets for gene therapy applications utilizing adeno-associated viral vectors.

Despite the devastating clinical outcome of the neurodegenerative disease, amyotrophic lateral sclerosis (ALS), its etiology remains mysterious. Approximately 90% of ALS is characterized as sporadic, signifying that the patient has no family history of the disease. The development of an impactful disease modifying therapy across the ALS spectrum has remained out of grasp, largely due to the poorly understood mechanisms of disease onset and progression. Currently, ALS is invariably fatal and rapidly progressive. It is hypothesized that multiple factors can lead to the development of ALS, however, treatments are often focused on targeting specific familial forms of the disease (10% of total cases). There is a strong need to develop disease modifying treatments for ALS that can be effective across the full ALS spectrum of familial and sporadic cases. Although the onset of disease varies significantly between patients, there are general disease mechanisms and progressions that can be seen broadly across ALS patients. Therefore, this review explores the targeting of these widespread disease mechanisms as possible areas for therapeutic intervention to treat ALS broadly. In particular, this review will focus on targeting mechanisms of defective protein homeostasis and RNA processing, which are both increasingly recognized as design principles of ALS pathogenesis. Additionally, this review will explore the benefits of gene therapy as an approach to treating ALS, specifically focusing on the use of adeno-associated virus (AAV) as a vector for gene delivery to the CNS and recent advances in the field.

Characterization of the humanized FRG mouse model and development of an AAV-LK03 variant with improved liver lobular biodistribution.

Recent clinical successes have intensified interest in using adeno-associated virus (AAV) vectors for therapeutic gene delivery. The liver is a key clinical target, given its critical physiological functions and involvement in a wide range of genetic diseases. In the present study, we first investigated the validity of a liver xenograft mouse model repopulated with primary hepatocytes using single-nucleus RNA sequencing (sn-RNA-seq) by studying the transcriptomic profile of human hepatocytes pre- and post-engraftment. Complementary immunofluorescence analyses performed in highly engrafted animals confirmed that the human hepatocytes organize and present appropriate patterns of zone-dependent enzyme expression in this model. Next, we tested a set of rationally designed HSPG de-targeted AAV-LK03 variants for relative transduction performance in human hepatocytes. We used immunofluorescence, next-generation sequencing, and single-nucleus transcriptomics data from highly engrafted FRG mice to demonstrate that the optimally HSPG de-targeted AAV-LK03 displayed a significantly improved lobular transduction profile in this model.

Adeno-associated virus serotype 2 capsid variants for improved liver-directed gene therapy.

BACKGROUND AND AIMS: Current liver-directed gene therapies look for adeno-associated virus (AAV) vectors with improved efficacy. With this background, capsid engineering is explored. Whereas shuffled capsid library screenings have resulted in potent liver targeting variants with one first vector in human clinical trials, modifying natural serotypes by peptide insertion has so far been less successful. Here, we now report on two capsid variants, MLIV.K and MLIV.A, both derived from a high-throughput in vivo AAV peptide display selection screen in mice. APPROACH AND RESULTS: The variants transduce primary murine and human hepatocytes at comparable efficiencies, a valuable feature in clinical development, and show significantly improved liver transduction efficacy, thereby allowing a dose reduction, and outperform parental AAV2 and AAV8 in targeting human hepatocytes in humanized mice. The natural heparan sulfate proteoglycan binding ability is markedly reduced, a feature that correlates with improved hepatocyte transduction. A further property that might contribute to the improved transduction efficacy is the lower capsid melting temperature. Peptide insertion also caused a moderate change in sensitivity to human sera containing anti-AAV2 neutralizing antibodies, revealing the impact of epitopes located at the basis of the AAV capsid protrusions. CONCLUSIONS: In conclusion, MLIV.K and MLIV.A are AAV peptide display variants selected in immunocompetent mice with improved hepatocyte tropism and transduction efficiency. Because these features are maintained across species, MLIV variants provide remarkable potential for translation of therapeutic approaches from mice to men.

The future of recombinant host defense peptides.

The antimicrobial resistance crisis calls for the discovery and production of new antimicrobials. Host defense peptides (HDPs) are small proteins with potent antibacterial and immunomodulatory activities that are attractive for translational applications, with several already under clinical trials. Traditionally, antimicrobial peptides have been produced by chemical synthesis, which is expensive and requires the use of toxic reagents, hindering the large-scale development of HDPs. Alternatively, HDPs can be produced recombinantly to overcome these limitations. Their antimicrobial nature, however, can make them toxic to the hosts of recombinant production. In this review we explore the different strategies that are used to fine-tune their activities, bioengineer them, and optimize the recombinant production of HDPs in various cell factories.

Proteome-wide systems genetics identifies UFMylation as a regulator of skeletal muscle function.

Improving muscle function has great potential to improve the quality of life. To identify novel regulators of skeletal muscle metabolism and function, we performed a proteomic analysis of gastrocnemius muscle from 73 genetically distinct inbred mouse strains, and integrated the data with previously acquired genomics and >300 molecular/phenotypic traits via quantitative trait loci mapping and correlation network analysis. These data identified thousands of associations between protein abundance and phenotypes and can be accessed online (https://muscle.coffeeprot.com/) to identify regulators of muscle function. We used this resource to prioritize targets for a functional genomic screen in human bioengineered skeletal muscle. This identified several negative regulators of muscle function including UFC1, an E2 ligase for protein UFMylation. We show UFMylation is up-regulated in a mouse model of amyotrophic lateral sclerosis, a disease that involves muscle atrophy. Furthermore, in vivo knockdown of UFMylation increased contraction force, implicating its role as a negative regulator of skeletal muscle function.

A human adenovirus encoding IFN-γ can transduce Tasmanian devil facial tumour cells and upregulate MHC-I.

The devil facial tumour disease (DFTD) has led to a massive decline in the wild Tasmanian devil (Sarcophilus harrisii) population. The disease is caused by two independent devil facial tumours (DFT1 and DFT2). These transmissible cancers have a mortality rate of nearly 100 %. An adenoviral vector-based vaccine has been proposed as a conservation strategy for the Tasmanian devil. This study aimed to determine if a human adenovirus serotype 5 could express functional transgenes in devil cells. As DFT1 cells do not constitutively express major histocompatibility complex class I (MHC-I), we developed a replication-deficient adenoviral vector that encodes devil interferon gamma (IFN-γ) fused to a fluorescent protein reporter. Our results show that adenoviral-expressed IFN-γ was able to stimulate upregulation of beta-2 microglobulin, a component of MHC-I, on DFT1, DFT2 and devil fibroblast cell lines. This work suggests that human adenoviruses can serve as a vaccine platform for devils and potentially other marsupials.

Optogenetic restoration of high sensitivity vision with bReaChES, a red-shifted channelrhodopsin.

The common final pathway to blindness in many forms of retinal degeneration is the death of the light-sensitive primary retinal neurons. However, the normally light-insensitive second- and third-order neurons persist optogenetic gene therapy aims to restore sight by rendering such neurons light-sensitive. Here, we investigate whether bReaChES, a newly described high sensitivity Type I opsin with peak sensitivity to long-wavelength visible light, can restore vision in a murine model of severe early-onset retinal degeneration. Intravitreal injection of an adeno-associated viral vector carrying the sequence for bReaChES downstream of the calcium calmodulin kinase IIα promoter resulted in sustained retinal expression of bReaChES. Retinal ganglion cells (RGCs) expressing bReaChES generated action potentials at light levels consistent with bright indoor lighting (from 13.6 log photons cm-2 s-1). They could also detect flicker at up to 50 Hz, which approaches the upper temporal limit of human photopic vision. Topological response maps of bReaChES-expressing RGCs suggest that optogenetically activated RGCs may demonstrate similar topographical responses to RGCs stimulated by photoreceptor activation. Furthermore, treated dystrophic mice displayed restored cortical neuronal activity in response to light and rescued behavioral responses to a looming stimulus that simulated an aerial predator. Finally, human surgical retinal explants exposed to the bReaChES treatment vector demonstrated transduction. Together, these findings suggest that intravitreal gene therapy to deliver bReaChES to the retina may restore vision in human retinal degeneration in vivo at ecologically relevant light levels with spectral and temporal response characteristics approaching those of normal human photopic vision.