Anais Amaya

All Publications

• Assessment of Pre-Clinical Liver Models Based on Their Ability to Predict the Liver-Tropism of Adeno-Associated Virus Vectors. Human gene therapy Westhaus, A., Cabanes-Creus, M., Dilworth, K. L., Zhu, E., Salas Gomez, D., Navarro, R. G., Amaya, A. K., Scott, S., Kwiatek, M., McCorkindale, A. L., Hayman, T. E., Frahm, S., Perocheau, D. P., Tran, B. M., Vincan, E., Wong, S. L., Waters, S. A., Riddiough, G. E., Perini, M. V., Wilson, L. O., Baruteau, J., Diecke, S., Gonzalez-Aseguinolaza, G., Santilli, G., Thrasher, A. J., Alexander, I. E., Lisowski, L. 2023; 34 (7-8): 273-288

  Abstract

  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.

  View details for DOI 10.1089/hum.2022.188

  View details for PubMedID 36927149

• Novel human liver-tropic AAV variants define transferable domains that markedly enhance the human tropism of AAV7 and AAV8. Molecular therapy. Methods & clinical development Cabanes-Creus, M., Navarro, R. G., Zhu, E., Baltazar, G., Liao, S. H., Drouyer, M., Amaya, A. K., Scott, S., Nguyen, L. H., Westhaus, A., Hebben, M., Wilson, L. O., Thrasher, A. J., Alexander, I. E., Lisowski, L. 2022; 24: 88-101

  Abstract

  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. Here, we report the bioengineering of a set of next-generation AAV vectors, named AAV-SYDs (where "SYD" stands for Sydney, Australia), with increased human hepato-tropism in a liver xenograft mouse model repopulated with primary human hepatocytes. We followed a two-step process that staggered directed evolution and domain-swapping approaches. Using DNA-family shuffling, we first mapped key AAV capsid regions responsible for efficient human hepatocyte transduction in vivo. Focusing on these regions, we next applied domain-swapping strategies to identify and study key capsid residues that enhance primary human hepatocyte uptake and transgene expression. Our findings underscore the potential of AAV-SYDs as liver gene therapy vectors and provide insights into the mechanism responsible for their enhanced transduction profile.

  View details for DOI 10.1016/j.omtm.2021.11.011

  View details for PubMedID 34977275

  View details for PubMedCentralID PMC8693155

• Attenuation of Heparan Sulfate Proteoglycan Binding Enhances In Vivo Transduction of Human Primary Hepatocytes with AAV2 MOLECULAR THERAPY-METHODS & CLINICAL DEVELOPMENT Cabanes-Creus, M., Westhaus, A., Navarro, R., Baltazar, G., Zhu, E., Amaya, A. K., Liao, S. Y., Scott, S., Sallard, E., Dilworth, K. L., Rybicki, A., Drouyer, M., Hallwirth, C. V., Bennett, A., Santilli, G., Thrasher, A. J., Agbandje-McKenna, M., Alexander, I. E., Lisowski, L. 2020; 17: 1139-1154

  Abstract

  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.

  View details for DOI 10.1016/j.omtm.2020.05.004

  View details for Web of Science ID 000540906400102

  View details for PubMedID 32490035

  View details for PubMedCentralID PMC7260615

• Efficient in vivo editing of OTC-deficient patient-derived primary human hepatocytes JHEP REPORTS Ginn, S. L., Amaya, A. K., Liao, S. Y., Zhu, E., Cunningham, S. C., Lee, M., Hallwirth, C., Logan, G. J., Tay, S. S., Cesare, A. J., Pickett, H. A., Grompe, M., Dilworth, K., Lisowski, L., Alexander, I. E. 2020; 2 (1): 100065

  Abstract

  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.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.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.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.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.

  View details for DOI 10.1016/j.jhepr.2019.100065

  View details for Web of Science ID 000648931200006

  View details for PubMedID 32039406

  View details for PubMedCentralID PMC7005564

• Inhibition of proliferation in primary human hepatocytes following in vivo AAV-mediated genome editing Amaya, A. K., Ginn, S. L., Liao, S. Y., Zhu, C., Smyth, C. M., Logan, G. J., Lisowski, L., Alexander, I. E. MARY ANN LIEBERT, INC. 2019: A18

  View details for Web of Science ID 000495173100049

• Efficient in vivo editing of patient-derived primary human hepatocytes Ginn, S. L., Amaya, A. K., Liao, S. Y., Zhu, E., Cunningham, S. C., Hallwirth, C. V., Tay, S. S., Lee, M., Pickett, H. A., Grompe, M., Dilworth, K., Lisowski, L., Alexander, I. E. MARY ANN LIEBERT, INC. 2019: A2-A3

  View details for Web of Science ID 000481913400004

• Codon-Optimization of Wild-Type Adeno-Associated Virus Capsid Sequences Enhances DNA Family Shuffling while Conserving Functionality MOLECULAR THERAPY-METHODS & CLINICAL DEVELOPMENT Cabanes-Creus, M., Ginn, S. L., Amaya, A. K., Liao, S. Y., Westhaus, A., Hallwirth, C. V., Wilmott, P., Ward, J., Dilworth, K. L., Santilli, G., Rybicki, A., Nakai, H., Thrasher, A. J., Filip, A. C., Alexander, I. E., Lisowski, L. 2019; 12: 71-84

  Abstract

  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.

  View details for DOI 10.1016/j.omtm.2018.10.016

  View details for Web of Science ID 000461055600006

  View details for PubMedID 30534580

  View details for PubMedCentralID PMC6279885

• Successful in vivo editing of patient-derived primary human hepatocytes Amaya, A. K., Ginn, S. L., Liao, S. Y., Zhu, C., Lee, M., Pickett, H. A., Hallwirth, C. V., Cunningham, S. C., Lisowski, L., Alexander, I. E. MARY ANN LIEBERT, INC. 2018: A26-A27

  View details for Web of Science ID 000453707700099

• Gene therapy clinical trials worldwide to 2017: An update JOURNAL OF GENE MEDICINE Ginn, S. L., Amaya, A. K., Alexander, I. E., Edelstein, M., Abedi, M. R. 2018; 20 (5): e3015

  Abstract

  To date, almost 2600 gene therapy clinical trials have been completed, are ongoing or have been approved worldwide. Our database brings together global information on gene therapy clinical activity from trial databases, official agency sources, published literature, conference presentations and posters kindly provided to us by individual investigators or trial sponsors. This review presents our analysis of clinical trials that, to the best of our knowledge, have been or are being performed worldwide. As of our November 2017 update, we have entries on 2597 trials undertaken in 38 countries. We have analysed the geographical distribution of trials, the disease indications (or other reasons) for trials, the proportions to which different vector types are used, and the genes that have been transferred. Details of the analyses presented, and our searchable database are available via The Journal of Gene Medicine Gene Therapy Clinical Trials Worldwide website at: http://www.wiley.co.uk/genmed/clinical. We also provide an overview of the progress being made in gene therapy clinical trials around the world, and discuss key trends since the previous review, namely the use of chimeric antigen receptor T cells for the treatment of cancer and advancements in genome editing technologies, which have the potential to transform the field moving forward.

  View details for DOI 10.1002/jgm.3015

  View details for Web of Science ID 000433596500001

  View details for PubMedID 29575374

• Evaluation of Recombinant Adeno-Associated Virus-Based Genome Editing Reagents for Homology-Directed Repair to Target a Human Liver Locus In Vivo Ginn, S. L., Amaya, A. K., Liao, S. Y., Zhu, C., Hallwirth, C. V., Tay, S. S., Cunningham, S. C., Logan, G. J., Dilworth, K., Lisowski, L., Alexander, I. CELL PRESS. 2018: 113

  View details for Web of Science ID 000435342201112

• Successful In Vivo Editing of the OTC Locus in Primary Human Hepatocytes Xenografted into the FRG Mouse Liver Amaya, A. K., Ginn, S. L., Liao, S. Y., Zhu, C., Lee, M., Pickett, H. A., Hallwirth, C. V., Cunningham, S. C., Logan, G. J., Dilworth, K., Lisowski, L., Alexander, I. E. CELL PRESS. 2018: 448-449

  View details for Web of Science ID 000435342205195

• TREATMENT OF GENETIC LIVER DISEASE BY AAV-MEDIATED GENOME EDITING AND SELECTIVE EXPANSION OF REPAIRED HEPATOCYTES Amaya, A. K., Zhu, C., Ginn, S. L., Hallwirth, C. V., Tay, S. S., Cunningham, S. C., Logan, G. J., Alexander, I. E. WILEY. 2018

  View details for Web of Science ID 000426528300043

• DISCOVERY OF A LIVER-SPECIFIC ENHANCER-PROMOTER ELEMENT IN THE 3 ' UTR OF THE WILD-TYPE AAV2 GENOME PROVIDES NOVEL INSIGHTS INTO AAV VECTOR SAFETY IN THE HUMAN LIVER Logan, G. J., Dane, A. P., Hallwirth, C. V., Smyth, C. M., Wilkie, E. E., Amaya, A. K., Zhu, E., Khandekar, N., Ginn, S. L., Liao, S., Cunningham, S. C., Sasaki, N., Tam, P. L., Russell, D. W., Lisowski, L., Alexander, I. E. WILEY. 2018

  View details for Web of Science ID 000426528300029

• Identification of liver-specific enhancer-promoter activity in the 3 ' untranslated region of the wild-type AAV2 genome NATURE GENETICS Logan, G. J., Dane, A. P., Hallwirth, C. V., Smyth, C. M., Wilkie, E. E., Amaya, A. K., Zhu, E., Khandekar, N., Ginn, S. L., Liao, S. Y., Cunningham, S. C., Sasaki, N., Cabanes-Creus, M., Tam, P. L., Russell, D. W., Lisowski, L., Alexander, I. E. 2017; 49 (8): 1267-+

  Abstract

  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.

  View details for DOI 10.1038/ng.3893

  View details for Web of Science ID 000406397900018

  View details for PubMedID 28628105