Publications by authors named "Sophia H Y Liao"

8 Publications

  • Page 1 of 1

Single amino acid insertion allows functional transduction of murine hepatocytes with human liver tropic AAV capsids.

Mol Ther Methods Clin Dev 2021 Jun 24;21:607-620. Epub 2021 Apr 24.

Translational Vectorology Research Unit, Children's Medical Research Institute, The University of Sydney, Westmead, NSW 2145, Australia.

Recent successes in clinical gene therapy applications have intensified the interest in using adeno-associated viruses (AAVs) as vectors for gene delivery into human liver. An inherent intriguing characteristic of AAVs is that vector variants vary substantially in their ability to transduce hepatocytes from different species. This has historically limited the value of preclinical studies using rodent models for predicting the efficiency of AAV vectors in liver-targeted gene therapy clinical studies. In this work, we aimed to investigate the key determinants of the observed differential interspecies transduction abilities among AAV variants. We took advantage of domain swapping strategies between AAV-KP1, a newly identified variant with enhanced murine liver tropism, and AAV3b, which functions poorly in mice. The systematic comparison of AAV3b/AAV-KP1 chimeric variants allowed us to identify a threonine insertion at position 265 within variable region I (VR-I) as the key residue that confers murine hepatic transduction to human-derived clade B (AAV2-like) and clade C (AAV3b-like) variants. We propose to use this insertion to generate phylogenetically related AAV surrogates in support of toxicology and dosing studies in the murine liver model.
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http://dx.doi.org/10.1016/j.omtm.2021.04.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8142051PMC
June 2021

Restoring the natural tropism of AAV2 vectors for human liver.

Sci Transl Med 2020 09;12(560)

Translational Vectorology Research Unit, Children's Medical Research Institute, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW 2145, Australia.

Recent clinical successes in gene therapy applications have intensified interest in using adeno-associated viruses (AAVs) as vectors for therapeutic gene delivery. Although prototypical AAV2 shows robust in vitro transduction of human hepatocyte-derived cell lines, it has not translated into an effective vector for liver-directed gene therapy in vivo. This is consistent with observations made in (FRG) mice with humanized livers, showing that AAV2 functions poorly in this xenograft model. Here, we derived naturally hepatotropic AAV capsid sequences from primary human liver samples. We demonstrated that capsid mutations, likely acquired as an unintentional consequence of tissue culture propagation, attenuated the intrinsic human hepatic tropism of natural AAV2 and related human liver AAV isolates. These mutations resulted in amino acid changes that increased binding to heparan sulfate proteoglycan (HSPG), which has been regarded as the primary cellular receptor mediating AAV2 infection of human hepatocytes. Propagation of natural AAV variants in vitro showed tissue culture adaptation with resulting loss of tropism for human hepatocytes. In vivo readaptation of the prototypical AAV2 in FRG mice with a humanized liver resulted in restoration of the intrinsic hepatic tropism of AAV2 through decreased binding to HSPG. Our results challenge the notion that high affinity for HSPG is essential for AAV2 entry into human hepatocytes and suggest that natural AAV capsids of human liver origin are likely to be more effective for liver-targeted gene therapy applications than culture-adapted AAV2.
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http://dx.doi.org/10.1126/scitranslmed.aba3312DOI Listing
September 2020

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

Mol Ther Methods Clin Dev 2020 Jun 13;17:1139-1154. Epub 2020 May 13.

Translational Vectorology Research Unit, Children's Medical Research Institute, The University of Sydney, Westmead, NSW 2145, Australia.

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 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 , 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 . 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 function of otherwise strongly hepatotropic variants and that modulation of the interaction with HSPG is critical to ensure maximum efficiency . 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.
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http://dx.doi.org/10.1016/j.omtm.2020.05.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7260615PMC
June 2020

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

JHEP Rep 2020 Feb 27;2(1):100065. Epub 2019 Dec 27.

Gene Therapy Research Unit, Children's Medical Research Institute, Faculty of Medicine and Health, The University of Sydney and Sydney Children's Hospitals Network, Westmead, Australia.

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 () locus in patient-derived primary human hepatocytes at therapeutically relevant levels.

Methods: Editing reagents for precise CRISPR/SaCas9-mediated cleavage and homology-directed repair (HDR) of the human locus were first evaluated against an minigene cassette transposed into the mouse liver. The editing efficacy of these reagents was then tested on the native locus in patient-derived primary human hepatocytes xenografted into the FRG ( ) 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 , locus-specific cleavage was achieved at efficiencies of up to 72%. Importantly, successful editing was observed in up to 29% of alleles at clinically relevant vector doses. No off-target editing events were observed at the top 10 -predicted sites in the genome.

Conclusions: We report efficient single-nucleotide correction of a disease-causing mutation in the locus in patient-derived primary human hepatocytes 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 correction of a patient-specific disease-causing mutation in the 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.
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http://dx.doi.org/10.1016/j.jhepr.2019.100065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7005564PMC
February 2020

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

Mol Ther Methods Clin Dev 2019 Mar 1;12:71-84. Epub 2018 Nov 1.

Translational Vectorology Group, Children's Medical Research Institute, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW 2006, Australia.

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.
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http://dx.doi.org/10.1016/j.omtm.2018.10.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6279885PMC
March 2019

Identification of liver-specific enhancer-promoter activity in the 3' untranslated region of the wild-type AAV2 genome.

Nat Genet 2017 Aug 19;49(8):1267-1273. Epub 2017 Jun 19.

Gene Therapy Research Unit, Children's Medical Research Institute and Sydney Children's Hospitals Network, University of Sydney, Sydney, New South Wales, Australia.

Vectors based on adeno-associated virus type 2 (AAV2) are powerful tools for gene transfer and genome editing applications. The level of interest in this system has recently surged in response to reports of therapeutic efficacy in human clinical trials, most notably for those in patients with hemophilia B (ref. 3). Understandably, a recent report drawing an association between AAV2 integration events and human hepatocellular carcinoma (HCC) has generated controversy about the causal or incidental nature of this association and the implications for AAV vector safety. Here we describe and functionally characterize a previously unknown liver-specific enhancer-promoter element in the wild-type AAV2 genome that is found between the stop codon of the cap gene, which encodes proteins that form the capsid, and the right-hand inverted terminal repeat. This 124-nt sequence is within the 163-nt common insertion region of the AAV genome, which has been implicated in the dysregulation of known HCC driver genes and thus offers added insight into the possible link between AAV integration events and the multifactorial pathogenesis of HCC.
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http://dx.doi.org/10.1038/ng.3893DOI Listing
August 2017

Limiting Thymic Precursor Supply Increases the Risk of Lymphoid Malignancy in Murine X-Linked Severe Combined Immunodeficiency.

Mol Ther Nucleic Acids 2017 Mar 10;6:1-14. Epub 2016 Dec 10.

Gene Therapy Research Unit, Children's Medical Research Institute, The University of Sydney and The Sydney Children's Hospitals Network, Westmead, NSW 2145, Australia; Discipline of Child and Adolescent Health, The University of Sydney, Westmead, NSW 2145, Australia. Electronic address:

In early gene therapy trials for SCID-X1, using γ-retroviral vectors, T cell leukemias developed in a subset of patients secondary to insertional proto-oncogene activation. In contrast, we have reported development of T cell leukemias in SCID-X1 mice following lentivirus-mediated gene therapy independent of insertional mutagenesis. A distinguishing feature in our study was that only a proportion of transplanted γc-deficient progenitors were transduced and therefore competent for reconstitution. We hypothesized that reconstitution of SCID-X1 mice with limiting numbers of hematopoietic progenitors might be a risk factor for lymphoid malignancy. To test this hypothesis, in the absence of transduction, SCID-X1 mice were reconstituted with serially fewer wild-type hematopoietic progenitors. A robust inverse correlation between hematopoietic progenitor cell dose and T-lymphoid malignancy was observed, with earlier disease onset at lower cell doses. Malignancies were of donor origin and carried activating Notch1 mutations. These findings align with emerging evidence that thymocyte self-renewal induced by progenitor deprivation carries an oncogenic risk that is modulated by intra-thymic competition from differentiation-committed cells. Although insertional proto-oncogene activation is required for the development of malignancy in humans, failure of γc-deficient thymocytes to effectively compete with this at-risk cell population may have also contributed to oncogenesis observed in early SCID-X1 trials.
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http://dx.doi.org/10.1016/j.omtn.2016.11.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5363493PMC
March 2017

Lymphomagenesis in SCID-X1 mice following lentivirus-mediated phenotype correction independent of insertional mutagenesis and gammac overexpression.

Mol Ther 2010 May 30;18(5):965-76. Epub 2010 Mar 30.

Gene Therapy Research Unit of the Children's Medical Research Institute and The Children's Hospital at Westmead, Westmead, New South Wales, Australia.

The development of leukemia as a consequence of vector-mediated genotoxicity in gene therapy trials for X-linked severe combined immunodeficiency (SCID-X1) has prompted substantial research effort into the design and safety testing of integrating vectors. An important element of vector design is the selection and evaluation of promoter-enhancer elements with sufficient strength to drive reliable immune reconstitution, but minimal propensity for enhancer-mediated insertional mutagenesis. In this study, we set out to explore the effect of promoter-enhancer selection on the efficacy and safety of human immunodeficiency virus-1-derived lentiviral vectors in gammac-deficient mice. We observed incomplete or absent T- and B-cell development in mice transplanted with progenitors expressing gammac from the phosphoglycerate kinase (PGK) and Wiscott-Aldrich syndrome (WAS) promoters, respectively. In contrast, functional T- and B-cell compartments were restored in mice receiving an equivalent vector containing the elongation factor-1-alpha (EF1alpha) promoter; however, 4 of 14 mice reconstituted with this vector subsequently developed lymphoma. Extensive analyses failed to implicate insertional mutagenesis or gammac overexpression as the underlying mechanism. These findings highlight the need for detailed mechanistic analysis of tumor readouts in preclinical animal models assessing vector safety, and suggest the existence of other ill-defined risk factors for oncogenesis, including replicative stress, in gene therapy protocols targeting the hematopoietic compartment.
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http://dx.doi.org/10.1038/mt.2010.50DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2890120PMC
May 2010
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