Engineering and In Vitro Selection of a Novel AAV3B Variant with High Hepatocyte Tropism and Reduced Seroreactivity

Abstract

Limitations to successful gene therapy with adeno-associated virus (AAV) can comprise pre-existing neutralizing antibodies to the vector capsid that can block cellular entry, or inefficient transduction of target cells that can lead to sub-optimal expression of the therapeutic transgene. Recombinant serotype 3 AAV (AAV3) is an emerging candidate for liver-directed gene therapy. In this study, we integrated rational design by using a combinatorial library derived from AAV3B capsids with directed evolution by in vitro selection for liver-targeted AAV variants. The AAV3B-DE5 variant described herein was undetectable in the original viral library but gained a selective advantage upon in vitro passaging in human hepatocarcinoma spheroid cultures. AAV3B-DE5 contains 24 capsid amino acid substitutions compared with AAV3B, distributed among all five variable regions, with strong selective pressure on VR-IV, VR-V, and VR-VII. In vivo, AAV3B-DE5 demonstrated improved human hepatocyte tropism in a liver chimeric mouse model. Importantly, this variant exhibited reduced seroreactivity to human intravenous immunoglobulin (i.v. Ig), as well as individual serum samples from 100 healthy human donors. Therefore, molecular evolution using a combinatorial library platform generated a viral capsid with high hepatocyte tropism and enhanced evasion of pre-existing AAV neutralizing antibodies.

Keywords: AAV3; capsid library; directed evolution; gene therapy; hepatocyte; liver tropism; neutralizing antibody; seroprevalence.

Graphical abstract

Figure 1

Library Construction Step 1: sub-libraries A, B, and C were first assembled, each with mutations in different regions (VR-I, VR-V, and VR-VI for A; VR-IV for B; VR-VII for C). Step 2: variable regions from the three sub-libraries (from DNA isolated from successfully assembled AAV particles) were recombined to generate the final library that has mutations in all five regions. Step 3: the plasmid library is packaged into the viral library.

Figure 2

NGS Analysis of Plasmid and Viral AAV Libraries (A) Copy number distribution; for each copy number (x axis), the number of distinct sequences present at that copy number is shown (y axis). In the plasmid library, almost all sequences have a single occurrence, while in the viral library there is a wider distribution, with more sequences having diverse levels of abundance; in both cases, the sequence with the highest abundance is WT AAV3B, the library parent, indicated with arrows. (B) Distribution of the number of mutations per sequence; the number of mutations for each sequence varies between 0 (WT AAV3B) and 34 (100% of the 34 variable positions mutated), with a peak at 24 and 23 mutations, respectively, for the plasmid and viral libraries, and an average of 21.56 and 23.26, respectively. (C) Euler diagram showing the overlap between the distinct sequences in the plasmid and the viral libraries.

Figure 3

Evolution of Sequence Frequencies with Selection Rounds Sequences present at more than 1% after at least one round of selection are displayed with a separate color (V1–V16). All other sequences are combined under a single color (Other). AAV3B-DE5 is shown as sequence V1.

Figure 4

AAV3-DE5 Mutations Color code: blue, red, yellow, green, and orange for VR-I, VR-IV, VR-V, VR-VI, and VR-VII, respectively. (A) Alignment between parental capsid AAV3B and evolved capsid AAV3B-DE5; only the four regions that were diversified in the library design are shown (the sequences are identical outside these regions); numbers on top designate the region position in the capsid (VP1 numbering); sequence identity is represented by a dot. (B) Positions of the 24 mutations displayed in AAV3B 3D structure. (C) Heatmap showing enrichment factors between rounds of selection for each mutation; the enrichment factor between two rounds of selection (for example a and b) for a mutation at a particular position is defined as the frequency of the corresponding amino acid at that position in the NGS sample for round b divided by the corresponding frequency for round a. (D) Enrichment scores of AAV3B-DE5 mutations; for each position the score is defined as the product of the four enrichment factors shown in (C).

Figure 5

Comparative Transduction of Human Hepatocellular Carcinoma Cell Lines In Vitro (A) Fluorescence images of HUH-7 and HEPG2 spheroid cultures transduced with WT AAV3B and AAV3B-DE5 vectors, expressing the mApple reporter transgene. (B) Graphical representation of (A) transduction efficiencies (% mApple⁺ cells), mApple MFI, and FLuc activity levels for HUH-7 adherent cells transduced with either WT AAV3B or AAV3B-DE5 at different MOIs. (C) Kinetics of transduction efficiencies (% mApple⁺) and AAV genome copy/cell of HUH-7 adherent cells transduced with an MOI of 5 × 10⁵ vg/cell of either WT AAV3B or AAV3B-DE5. (D) GFP transgene expression analysis of primary human hepatocytes, primary monkey hepatocytes, or primary mouse hepatocytes transduced with different MOIs of either AAV8 or AAV3B-DE5 in vitro. Transgene expression was normalized to GAPDH expression levels. Statistical analysis was performed by two-way ANOVA with a Sidak’s multiple comparison test for (B) and (C). Data are represented as mean ± SEM. A p value <0.05 was considered statistically significant (*).

Figure 6

In Vitro Assays for Neutralization of Transduction (A) Determination of reciprocal NAb titers to WT AAV3B or AAV3B-DE5 by using increasing concentrations of pooled i.v. Ig (100–1,000 μg/mL). The i.v. Ig concentration at which 50% mApple expression is reduced as compared to no i.v. Ig control is indicated with the red dotted lines. % mApple⁺ cells are quantified by flow cytometry. (B) Determination of reciprocal NAb titers to WT AAV3B or AAV3B-DE5 by using increasing concentrations of pooled i.v. Ig (100–6,400 μg/mL). The i.v. Ig concentration at which 50% FLuc expression is reduced, as compared to no i.v. Ig control, is indicated with the red dotted lines. (C) Average reciprocal NAb titers for WT AAV3B and AAV3BDE5, determined using the FLuc-based in vitro neutralizing assay. Data are the average of 10 independent experiments. Data are represented as mean ± SD for (A), with statistical significance between WT AAV3B and AAV3B-DE5 conducted by two-way ANOVA with a Sidak’s multiple comparison test. Data are represented as mean ± SEM for (C), with statistical significance between WT AAV3B and AAV3B-DE5 conducted by an unpaired t test with Welch’s correction. A p value <0.05 was considered statistically significant (*).

Figure 7

Neutralization of Individual Serum Samples (A) Reciprocal NAb titers for WT AAV3B and AAV3B-DE5 obtained from 100 donor serum samples using the in vitro FLuc-based neutralizing assay. (B) Determination of seroprevalence for WT AAV3B and AAV3B-DE5 from (A) using either 1:2 or 1:5 serum dilution as the cutoff. (C) 48 samples with detectable titers were analyzed for fold difference between WT AAV3B and AAV3B-DE5 reciprocal NAb titers. 35% and 40% of these 48 patient samples have 2- to 16-fold and 1.5- to 2-fold higher titers, respectively, for WT AAV3B as compared to AAV3B-DE5. 19% and 6% of patient samples have 1- to 1.5-fold and 0.7- to 1-fold higher titers, respectively, for WT AAV3B as compared to AAV3B-DE5. Statistical analysis for (A) was performed using a two-tailed Mann-Whitney test following a normality distribution determination, and for (B) was conducted by Fisher’s exact test two-tailed test using a 2 × 2 contingency table. A p value <0.05 was considered statistically significant (*).

Figure 8

In Vivo Transduction Efficiency in Human Liver Chimeric Mice (A) Schematic representing steps in the generation of the human liver chimeric mouse. Fah^−/− NOD Rag1^−/−Il2rg^null (FNRG) mice were humanized with mouse-passaged primary human hepatocytes (huFNRG mice). After transplantation, mice were cycled off NTBC to expand the human graft. (B) Chimerism was determined by human albumin (hAlb) quantification in mouse serum, which correlates well with humanization. Once mice had reached peak hAlb levels they were challenged 1 day after restarting NTBC. (C) Flow gating strategy to differentiate between human and mouse hepatocytes, using human HLA-1 and mouse CD81 antibodies, and to quantify the frequencies of GFP-expressing cells. (D) Comparison of transduction frequencies of AAV3-ST, AAV3B-DE5, and LK03 by quantifying percent GFP⁺ human hepatocytes by flow cytometry. Data for (B) and (D) are shown as median ± SEM.