Tailoring capsid-directed evolution technology for improved AAV-mediated CAR-T generation.

Chimeric antigen receptor (CAR) T cell (CAR-T) therapies present options for patients diagnosed with certain leukemias. Recent advances of the technology included a method to integrate the CAR into the T cell receptor alpha constant (TRAC) locus to take advantage of the endogenous promoter and regulatory elements for CAR expression. This method used adeno-associated viral (AAV) vectors based on AAV6 to deliver the donor template encoding the CAR construct.

Alzheimer's disease induced neurons bearing mutations exhibit reduced excitability.

Alzheimer's disease (AD) is a devastating neurodegenerative condition that affects memory and cognition, characterized by neuronal loss and currently lacking a cure. Mutations in (Presenilin 1) are among the most common causes of early-onset familial AD (fAD). While changes in neuronal excitability are believed to be early indicators of AD progression, the link between mutations and neuronal excitability remains to be fully elucidated.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 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.

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.

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.

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.

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.

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.

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 () population. The disease is caused by two independent devil facial tumours (DFT1 and DFT2). These transmissible cancers have a mortality rate of nearly 100 %.