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

Cabanes‐Creus, Marti; Navarro, Renina Gale; Liao, Sophia H.Y.; Scott, Suzanne; Carlessi, Rodrigo; Roca-Pinilla, Ramon; Knight, Maddison; Baltazar, Grober; Zhu, Erhua; Jones, Matthew; Denisenko, Elena; Forrest, Alistair R.R.; Alexander, Ian E.; Tirnitz–Parker, Janina E.E.; Lisowski, Leszek

doi:10.1016/j.omtm.2022.12.014

Cited by 9 publications

(6 citation statements)

References 56 publications

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“…However, we noted high discrepancies in transduction between the hFRG and human liver explant models for AAVhu.Lvr06 19 and AAV-LK03-REDH. 18 Despite performing well in the humanized mouse model, these variants did not achieve the same level of performance in the liver explant model. This is specially intriguing for AAV-hu.Lvr06, a variant that was isolated directly from a human liver.…”

Section: Discussionmentioning

confidence: 98%

“…AAV-LK03's performance varied depending on the degree of humanization, due to its high affinity for heparan sulfate proteoglycan (HSPG), a property contributing to a distinct periportal transduction profile. 18 In low engrafted FRG mice, AAV-LK03 underperformed as fewer human hepatocyte clusters resided within the periportal zone. On the other hand, the relative performance of this capsid increased in highly engrafted mice where more human cells were randomly present within this zone.…”

Section: Discussionmentioning

confidence: 99%

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Harnessing the power of whole human liver ex situ normothermic perfusion for preclinical AAV vector evaluation

Cabanes‐Creus

Liao

Navarro

et al. 2023

Preprint

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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 establishes the use of normothermic liver perfusion as an invaluable preclinical model for evaluating liver-targeted gene therapies and providing guidance for making essential decisions that promote the most effective translational programs.

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Section: Discussionmentioning

confidence: 98%

Section: Discussionmentioning

confidence: 99%

Harnessing the power of whole human liver ex situ normothermic perfusion for preclinical AAV vector evaluation

Cabanes‐Creus

Liao

Navarro

et al. 2023

Preprint

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“…Nevertheless, it is well-established that the transduction efficiency of AAV8 in mice is significantly higher than in human hepatocytes, as evidenced by several independent studies (Lisowski et al, 2014 ; Cabanes-Creus et al, 2022 ). Thus, for this strategy to be translated to clinical applications, it would be essential to explore novel vectors with a strong affinity for human hepatocytes (Lisowski et al, 2014 ; Cabanes-Creus et al, 2022 , 2023 ). Furthermore, one limitation of AAV-delivered genome editing therapies is the durability of transgene expression, potentially leading to long-term toxicities.…”

Section: Discussionmentioning

confidence: 99%

Efficient and safe therapeutic use of paired Cas9-nickases for primary hyperoxaluria type 1

Torella,

Klermund,

Bilbao-Arribas

et al. 2024

EMBO Mol Med

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The therapeutic use of adeno-associated viral vector (AAV)-mediated gene disruption using CRISPR-Cas9 is limited by potential off-target modifications and the risk of uncontrolled integration of vector genomes into CRISPR-mediated double-strand breaks. To address these concerns, we explored the use of AAV-delivered paired Staphylococcus aureus nickases (D10ASaCas9) to target the Hao1 gene for the treatment of primary hyperoxaluria type 1 (PH1). Our study demonstrated effective Hao1 gene disruption, a significant decrease in glycolate oxidase expression, and a therapeutic effect in PH1 mice. The assessment of undesired genetic modifications through CIRCLE-seq and CAST-Seq analyses revealed neither off-target activity nor chromosomal translocations. Importantly, the use of paired-D10ASaCas9 resulted in a significant reduction in AAV integration at the target site compared to SaCas9 nuclease. In addition, our study highlights the limitations of current analytical tools in characterizing modifications introduced by paired D10ASaCas9, necessitating the development of a custom pipeline for more accurate characterization. These results describe a positive advance towards a safe and effective potential long-term treatment for PH1 patients.

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“…Early efforts to address this challenge are only just appearing in the gene transfer literature. 36 Yet another important challenge, most notably in the paediatric context, is liver growth. This is a particularly important consideration when using episomal vector systems as hepatocellular replication results in dilution and loss of vector genomes over time with an associated decline in therapeutic effect.…”

Section: Understanding the Therapeutic Challengementioning

confidence: 99%

“…As a consequence, gene therapy interventions for UCDs should ideally preferentially target this region of the hepatic lobule, as gene delivery/editing events near the central vein will contribute relatively little to the restoration of ureagenic activity. Early efforts to address this challenge are only just appearing in the gene transfer literature 36 …”

Section: Understanding the Therapeutic Challengementioning

confidence: 99%

Gene therapy for urea cycle defects: An update from historical perspectives to future prospects

Duff

Alexander

Baruteau

2023

J of Inher Metab Disea

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Urea cycle defects (UCDs) are severe inherited metabolic diseases with high unmet needs which present a permanent risk of hyperammonaemic decompensation and subsequent acute death or neurological sequelae, when treated with conventional dietetic and medical therapies. Liver transplantation is currently the only curative option, but has the potential to be supplanted by highly effective gene therapy interventions without the attendant need for life‐long immunosuppression or limitations imposed by donor liver supply. Over the last three decades, pioneering genetic technologies have been explored to circumvent the consequences of UCDs, improve quality of life and long‐term outcomes: adenoviral vectors, adeno‐associated viral vectors, gene editing, genome integration and non‐viral technology with messenger RNA. In this review, we present a summarised view of this historical path, which includes some seminal milestones of the gene therapy's epic. We provide an update about the state of the art of gene therapy technologies for UCDs and the current advantages and pitfalls driving future directions for research and development.

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

Cited by 9 publications

References 56 publications

Harnessing the power of whole human liver ex situ normothermic perfusion for preclinical AAV vector evaluation

Harnessing the power of whole human liver ex situ normothermic perfusion for preclinical AAV vector evaluation

Efficient and safe therapeutic use of paired Cas9-nickases for primary hyperoxaluria type 1

Gene therapy for urea cycle defects: An update from historical perspectives to future prospects

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