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. 2023 Apr;34(7-8):273-288.
doi: 10.1089/hum.2022.188.

Assessment of Pre-Clinical Liver Models Based on Their Ability to Predict the Liver-Tropism of Adeno-Associated Virus Vectors

Adrian Westhaus  1   2 , Marti Cabanes-Creus  1 , Kimberley L Dilworth  1 , Erhua Zhu  3 , David Salas Gómez  4 , Renina G Navarro  1 , Anais K Amaya  3 , Suzanne Scott  1   3   5 , Magdalena Kwiatek  6 , Alexandra L McCorkindale  7 , Tara E Hayman  7 , Silke Frahm  8 , Dany P Perocheau  9   10   11 , Bang Manh Tran  12 , Elizabeth Vincan  12   13   14 , Sharon L Wong  15   16 , Shafagh A Waters  15   16   17 , Georgina E Riddiough  12   18 , Marcos V Perini  18 , Laurence O W Wilson  5   19 , Julien Baruteau  9   10   11 , Sebastian Diecke  8 , Gloria González-Aseguinolaza  4 , Giorgia Santilli  2 , Adrian J Thrasher  2 , Ian E Alexander  3   20 , Leszek Lisowski  1   21   22
Affiliations

Assessment of Pre-Clinical Liver Models Based on Their Ability to Predict the Liver-Tropism of Adeno-Associated Virus Vectors

Adrian Westhaus et al. Hum Gene Ther. 2023 Apr.

Erratum in

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.

Keywords: AAV; hepatocyte; liver gene therapy; nonhuman primate; xenograft model.

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Conflict of interest statement

L.L., I.E.A. and A.J.T. have commercial affiliations. L.L. and I.A.E. have consulted on technologies discussed in this article. L.L. and I.A.E. have stock and/or equity in companies with technologies broadly related to this study. L.L. is a co-inventor of, and receives licensing royalties from, several AAV variants used in the study. A.L.M. and T.E.H. are employees, shareholders, and/or optionees of Inventia Life Science Pty. Ltd. Inventia has an interest in commercializing the 3D bioprinting technology. All other authors declare no competing financial interests.

Figures

Figure 1.
Figure 1.
Ex vivo results in human and NHP liver models. (a) Schematic of transduction of indicated in vitro and ex vivo models. (b) NGS read contribution (%) for each AAV from extracted DNA. (c) NGS read contribution (%) for each AAV from mRNA-derived complementary DNA. AAV, adeno-associated virus; NGS, next-generation sequencing; NHP, nonhuman primate.
Figure 2.
Figure 2.
NHP and human xenograft in vivo results. (a) Schematic of transduction of engrafted FRG mice. (b) NGS read contribution (%) for each AAV from extracted xenograft bulk DNA. Cells were sorted for xenograft species only. (c) NGS read contribution (%) for each AAV from extracted eGFP-positive xenograft hepatocyte DNA. Cells were sorted for xenograft species as well as eGFP expression where indicated. (d) NGS read contribution (%) for each AAV from xenograft mRNA-derived complementary DNA. Cells were sorted for xenograft species as well as eGFP. eGFP, enhanced green fluorescent protein; FRG, Fah-/-/Rag2-/-/Il2rg-/-.
Figure 3.
Figure 3.
In vivo results cynomolgus monkey liver. (a) Schematic of column-based antibody depletion followed by NHP transduction. (b) NGS read contribution (%) for each AAV from whole tissue extracted DNA. (c) NGS read contribution (%) for each AAV from whole tissue mRNA-derived complementary DNA.
Figure 4.
Figure 4.
AAV-treated NHP serum analysis. (a) Schematic of serum collection before apheresis (antibody depletion), before AAV injection, 1 and 24 h after injection as well as at killing 1 week after injection. (b) NAb titers in collected serum at indicated time points for the indicated AAV variants. (c) AAV copy number per microliter and AAV variant from collected serum at indicated time points. NAb, neutralizing antibody.
Figure 5.
Figure 5.
In vivo transduction with NHP preinjection serum-incubated AAV mix. (a) Schematic of transduction of engrafted FRG mice with coincubated AAV mix and antibodies from the postapheresis/preinjection step of the experiment using Macaca fascicularis NHP. (b) NGS read contribution (%) for each AAV from extracted xenograft DNA in absence or presence of serum in indicated dilutions. Cells were sorted for xenograft species. (c) NGS read contribution (%) for each AAV from mouse extracted DNA in absence or presence of serum in indicated dilutions. Cells were sorted for mouse origin. All data shown as “Rh/Cy/hFRG no sera” are also used for Fig. 2b and are shown again for ease of comparability. hFRG, humanized FRG.
Figure 6.
Figure 6.
Seroprevalence and titers of NAbs of liver-tropic capsids. (a) Seroprevalence per AAV serotype. (b) Seroprevalence according to age. (c) Neutralizing titer per serotype. Each dot represents a seropositive sample. The line represents the median. (d) Cross-reactivity by AAV serotype. Shown are the percentage of samples that are positive for AAV-X in samples that were positive for AAV-Y.
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