Development of AAV Variants with Human Hepatocyte Tropism and Neutralizing Antibody Escape Capacity

Figure 1. Schematic Diagram for Isolation of AAV Mutants from Hepatocytes of Humanized Mice Humanized mice were first pretreated by injecting 30 mg IVIG via retro-orbital vein and then administered 2 × 1011 particles of AAV capsid shuffling library viruses. Three days later, adenovirus dl309 was injected into the mice, and at day 5, hepatocytes in the mice were harvested and lysed. The AAV in the cell lysate from humanized liver was re-injected into another group of IVIG-pretreated mice for the next round of AAV amplification as described above. The AAV amplification was repeated three times more for a total of four rounds of selection. After the last round of AAV amplification, DNA was extracted from the humanized liver, and the AAV capsid DNA was amplified using capsid specific primers and then reconstructed into the pXR backbone for sequencing and AAV vector production.

Figure 2. Schematic Capsid Diagram for AAV Mutants Isolated from the Livers of Humanized Mice Different colors are used to indicate the composition of capsids from different AAV serotypes, and point mutations are indicated on the top of each clone diagram. The frequency representation indicates the number of capsids isolated from a total of 25 available clones.

Figure 3. AAV Packaging and Transduction Efficiency of Isolated AAV Capsid Mutants (A) Packaging efficiency of individual AAV mutants. The production efficiency of mutants and wild-type AAV vectors was normalized to AAV2. (B) Transduction efficiency in Huh 7 cells from mutant AAV vectors. Huh7 cells were infected with 1 × 104 particles per cell of AAV vectors encoding the luciferase gene in 96-well plates, and 48 h later, the transduction efficiency was measured by luciferase assay.

Figure 4. AAV Neutralization Analysis of AAV Mutants against Human IVIG 1 × 109 particles of AAV/luc vectors were pre-incubated with different concentrations of IVIG for 1 h at 4°C, and then the mixtures were added into the cell culture medium of Huh7 in a 48-well plate. Forty-eight hours later, the luciferase expression was measured, and the Nab titer was determined.

Figure 5. The Prevalence and Titer of Nabs against AAV Mutants in Healthy Subjects and Hemophilia Patients (A) The AAV Nab prevalence in healthy human subjects. (B) The comparison of Nab titers for mutant AAV LP2-10 and naturally occurring serotypes in healthy individuals. (C) The AAV Nab prevalence in hemophilia patients. (D) The comparison of Nab titers for mutant AAV LP2-10 and naturally occurring serotypes in hemophilia patients. ∗p < 0.05 when compared to other serotypes for Nab titer of the mutant AAV LP2-10. NS, no significance.

Figure 6. The Transduction Profile of Mutant AAV LP2-10 in Human Hepatocytes of Chimeric Mice 1 × 1011 particles of scAAV/GFP (AAV8 or mutant AAV LP2-10) were injected into human hepatocyte xenografted mice via retro-orbital vein, and 3 weeks later, the liver was harvested and analyzed by immunohistochemistry. The antibody against human albumin was used to identify human hepatocytes (Red), and GFP expression was stained in green.

Figure 7. Phylogeny of AAV Mutants and Structural Modeling of Mutant AAV LP2-10 (A) A phylogenetic tree was used to analyze the genetic distance between wild-type AAVs and Nab-escaping AAV mutants isolated from the livers of humanized mice based on DNA sequence. (B) The fragments and point mutations on LP2-10 capsid subunit were structurally analyzed. Capsid domains derived from different AAV serotypes in the VP3 subunit of the mutant AAV LP2-10 and point mutations are highlighted as follows (according to VP1 numbering): yellow (AAV6), green (AAV8), and red (point mutations at residues 531, 725, and 735 of AAV8). (C) The fully assembled mutant AAV LP2-10 virion (60-mer) was displayed based on the AAV8 VP3 subunit from (B).