Using a barcoded AAV capsid library to select for clinically relevant gene therapy vectors
Katja Pekrun, Gustavo De Alencastro, Qing-Jun Luo, Jun Liu, Youngjin Kim, Sean Nygaard, Feorillo Galivo, Feijie Zhang, Ren Song, Matthew R. Tiffany, Jianpeng Xu, Matthias Hebrok, Markus Grompe, Mark A. Kay
Katja Pekrun, Gustavo De Alencastro, Qing-Jun Luo, Jun Liu, Youngjin Kim, Sean Nygaard, Feorillo Galivo, Feijie Zhang, Ren Song, Matthew R. Tiffany, Jianpeng Xu, Matthias Hebrok, Markus Grompe, Mark A. Kay
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Resource and Technical Advance Therapeutics

Using a barcoded AAV capsid library to select for clinically relevant gene therapy vectors

Abstract

While gene transfer using recombinant adeno-associated viral (rAAV) vectors has shown success in some clinical trials, there remain many tissues that are not well transduced. Because of the recent success in reprogramming islet-derived cells into functional β cells in animal models, we constructed 2 highly complex barcoded replication competent capsid shuffled libraries and selected for high-transducing variants on primary human islets. We describe the generation of a chimeric AAV capsid (AAV-KP1) that facilitates transduction of primary human islet cells and human embryonic stem cell–derived β cells with up to 10-fold higher efficiency compared with previously studied best-in-class AAV vectors. Remarkably, this chimeric capsid also enabled transduction of both mouse and human hepatocytes at very high levels in a humanized chimeric mouse model, thus providing a versatile vector that has the potential to be used in both preclinical testing and human clinical trials for liver-based diseases and diabetes.

Authors

Katja Pekrun, Gustavo De Alencastro, Qing-Jun Luo, Jun Liu, Youngjin Kim, Sean Nygaard, Feorillo Galivo, Feijie Zhang, Ren Song, Matthew R. Tiffany, Jianpeng Xu, Matthias Hebrok, Markus Grompe, Mark A. Kay

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Figure 6

In vitro transduction experiments using rAAV-FLuc vectors.

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In vitro transduction experiments using rAAV-FLuc vectors. (A) Transduct...
(A) Transduction efficiency of the capsids, as well as AAV-DJ and AAV-LK03 capsids, on a variety of human- and nonhuman-derived cell types. Cells were transduced with the different capsid containing rAAVs that were packaging a FLuc expression cassette at a MOI of 1,000 in triplicate (with the exception of islet cells), and cell lysates were analyzed 48 hours after transduction in a luciferase activity assay. For primary human islet cells, results of 1 experiment that had been performed twice are shown. Two-fold dilutions of recombinant FLuc enzyme were used to prepare a standard curve, and raw luminescence units were calculated into luciferase molecules based on the standard curve. (B) Neutralization assay of rAAVs packaged with different capsids using dilutions of 2 different batches of pooled human immunoglobulin (IVIG). Huh-7 cells were transduced at a MOI of 100 with FLuc-expressing rAAVs that had been preincubated with different concentrations of IVIG for 1 hour at 37°C. Luciferase activity in cell lysates was measured 24 hours after transduction. Mean values of 5 replicates (obtained in 2 independent experiments) with SDs are shown for each sample. Experimental values were assessed via 2-way ANOVA using Tukey’s multiple comparisons test. Only statistically significant differences are indicated in the legend below the graph. ***P < 0.001, ****P < 0.0001.