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.

Engineering domain-inlaid SaCas9 adenine base editors with reduced RNA off-targets and increased on-target DNA editing.

Precision genome engineering has dramatically advanced with the development of CRISPR/Cas base editing systems that include cytosine base editors and adenine base editors (ABEs). Herein, we compare the editing profile of circularly permuted and domain-inlaid Cas9 base editors, and find that on-target editing is largely maintained following their intradomain insertion, but that structural permutation of the ABE can affect differing RNA off-target events. With this insight, structure-guided design was used to engineer an SaCas9 ABE variant (microABE I744) that has dramatically improved on-target editing efficiency and a reduced RNA-off target footprint compared to current N-terminal linked SaCas9 ABE variants. This represents one of the smallest AAV-deliverable Cas9-ABEs available, which has been optimized for robust on-target activity and RNA-fidelity based upon its stereochemistry.

Attenuation of Heparan Sulfate Proteoglycan Binding Enhances In Vivo Transduction of Human Primary Hepatocytes with AAV2.

Use of the prototypical adeno-associated virus type 2 (AAV2) capsid delivered unexpectedly modest efficacy in an early liver-targeted gene therapy trial for hemophilia B. This result is consistent with subsequent data generated in chimeric mouse-human livers showing that the AAV2 capsid transduces primary human hepatocytes in vivo with low efficiency. In contrast, novel variants generated by directed evolution in the same model, such as AAV-NP59, transduce primary human hepatocytes with high efficiency. While these empirical data have immense translational implications, the mechanisms underpinning this enhanced AAV capsid transduction performance in primary human hepatocytes are yet to be fully elucidated. Remarkably, AAV-NP59 differs from the prototypical AAV2 capsid by only 11 aa and can serve as a tool to study the correlation between capsid sequence/structure and vector function. Using two orthogonal vectorological approaches, we have determined that just 2 of the 11 changes present in AAV-NP59 (T503A and N596D) account for the enhanced transduction performance of this capsid variant in primary human hepatocytes in vivo, an effect that we have associated with attenuation of heparan sulfate proteoglycan (HSPG) binding affinity. In support of this hypothesis, we have identified, using directed evolution, two additional single amino acid substitution AAV2 variants, N496D and N582S, which are highly functional in vivo. Both substitution mutations reduce AAV2's affinity for HSPG. Finally, we have modulated the ability of AAV8, a highly murine-hepatotropic serotype, to interact with HSPG. The results support our hypothesis that enhanced HSPG binding can negatively affect the in vivo function of otherwise strongly hepatotropic variants and that modulation of the interaction with HSPG is critical to ensure maximum efficiency in vivo. The insights gained through this study can have powerful implications for studies into AAV biology and capsid development for preclinical and clinical applications targeting liver and other organs.

Efficient in vivo editing of OTC-deficient patient-derived primary human hepatocytes.

BACKGROUND & AIMS: Genome editing technology has immense therapeutic potential and is likely to rapidly supplant contemporary gene addition approaches. Key advantages include the capacity to directly repair mutant loci with resultant recovery of physiological gene expression and maintenance of durable therapeutic effects in replicating cells. In this study, we aimed to repair a disease-causing point mutation in the ornithine transcarbamylase (OTC) locus in patient-derived primary human hepatocytes in vivo at therapeutically relevant levels. METHODS: Editing reagents for precise CRISPR/SaCas9-mediated cleavage and homology-directed repair (HDR) of the human OTC locus were first evaluated against an OTC minigene cassette transposed into the mouse liver. The editing efficacy of these reagents was then tested on the native OTC locus in patient-derived primary human hepatocytes xenografted into the FRG (Fah -/- Rag2 -/- Il2rg -/-) mouse liver. A highly human hepatotropic capsid (NP59) was used for adeno-associated virus (AAV)-mediated gene transfer. Editing events were characterised using next-generation sequencing and restoration of OTC expression was evaluated using immunofluorescence. RESULTS: Following AAV-mediated delivery of editing reagents to patient-derived primary human hepatocytes in vivo, OTC locus-specific cleavage was achieved at efficiencies of up to 72%. Importantly, successful editing was observed in up to 29% of OTC alleles at clinically relevant vector doses. No off-target editing events were observed at the top 10 in silico-predicted sites in the genome. CONCLUSIONS: We report efficient single-nucleotide correction of a disease-causing mutation in the OTC locus in patient-derived primary human hepatocytes in vivo at levels that, if recapitulated in the clinic, would provide benefit for even the most therapeutically challenging liver disorders. Key challenges for clinical translation include the cell cycle dependence of classical HDR and mitigation of unintended on- and off-target editing events. LAY SUMMARY: The ability to efficiently and safely correct disease-causing mutations remains the holy grail of gene therapy. Herein, we demonstrate, for the first time, efficient in vivo correction of a patient-specific disease-causing mutation in the OTC gene in primary human hepatocytes, using therapeutically relevant vector doses. We also highlight the challenges that need to be overcome for this technology to be translated into clinical practice.

Systemic AAV8-mediated delivery of a functional copy of muscle glycogen phosphorylase (Pygm) ameliorates disease in a murine model of McArdle disease.

McArdle disease is a disorder of carbohydrate metabolism that causes painful skeletal muscle cramps and skeletal muscle damage leading to transient myoglobinuria and increased risk of kidney failure. McArdle disease is caused by recessive mutations in the muscle glycogen phosphorylase (PYGM) gene leading to absence of PYGM enzyme in skeletal muscle and preventing access to energy from muscle glycogen stores. There is currently no cure for McArdle disease. Using a preclinical animal model, we aimed to identify a clinically translatable and relevant therapy for McArdle disease. We evaluated the safety and efficacy of recombinant adeno-associated virus serotype 8 (rAAV8) to treat a murine model of McArdle disease via delivery of a functional copy of the disease-causing gene, Pygm. Intraperitoneal injection of rAAV8-Pygm at post-natal day 1-3 resulted in Pygm expression at 8 weeks of age, accompanied by improved skeletal muscle architecture, reduced accumulation of glycogen and restoration of voluntary running wheel activity to wild-type levels. We did not observe any adverse reaction to the treatment at 8 weeks post-injection. Thus, we have investigated a highly promising gene therapy for McArdle disease with a clear path to the ovine large animal model endemic to Western Australia and subsequently to patients.

Codon-Optimization of Wild-Type Adeno-Associated Virus Capsid Sequences Enhances DNA Family Shuffling while Conserving Functionality.

Adeno-associated virus (AAV) vectors have become one of the most widely used gene transfer tools in human gene therapy. Considerable effort is currently being focused on AAV capsid engineering strategies with the aim of developing novel variants with enhanced tropism for specific human cell types, decreased human seroreactivity, and increased manufacturability. Selection strategies based on directed evolution rely on the generation of highly variable AAV capsid libraries using methods such as DNA-family shuffling, a technique reliant on stretches of high DNA sequence identity between input parental capsid sequences. This identity dependence for reassembly of shuffled capsids is inherently limiting and results in decreased shuffling efficiency as the phylogenetic distance between parental AAV capsids increases. To overcome this limitation, we have developed a novel codon-optimization algorithm that exploits evolutionarily defined codon usage at each amino acid residue in the parental sequences. This method increases average sequence identity between capsids, while enhancing the probability of retaining capsid functionality, and facilitates incorporation of phylogenetically distant serotypes into the DNA-shuffled libraries. This technology will help accelerate the discovery of an increasingly powerful repertoire of AAV capsid variants for cell-type and disease-specific applications.