Human Gene Therapy - April 2023 - 295

ENGINEERED HEPATOTROPIC NAB EVASIVE AAV3B VARIANT
295
Figure 3. Comparison of in vitro transduction efficiency. (A) Transduction efficiencies of AAV3B, AAV3B-DE5, AAV3B-V04, and AAV3B-V05 at varying MOIs
on HUH-7 adherent cells, using RLuc relative luminescence units at 48 h as a readout. (B) Schema and fluorescence images of mpPHH cells transduced with
2.5 · 103 MOI of AAV3B, AAV3B-DE5, or AAV3B-V04 expressing the mScarlet reporter transgene product. AAV3B, AAV3B-DE5, and AAV3B-V04 MOIs
corresponding to 50% mScarlet transduction in mpPHH cells as quantified on Cytation 7 are indicated. Data are an average of at least two replicates per group
per test condition and are the best representative of at least three independent experiments. Statistical analysis is performed by two-way ANOVA witha
Sidak's multiple comparison test for A. Data are represented as mean- SEM. A p-value <0.05 is considered statistically significant and indicated as follows:
*p< 0.05, **p< 0.01, and ****p< 0.0001. MOI, multiplicity of infection; RLuc, Renilla luciferase; SEM, standard error of the mean.
capitulating the findings in HUH-7 cells, AAV3BV04
transduced mpPHH monolayers at higher
frequencies compared to AAV3B and AAV3BDE5
at most of the MOIs tested (Fig. 3B).
We further compared AAV3B and AAV3B-V04
transduction of human cell lines derived from different
tissues. Although AAV3B-V04 transduced HUH-7 cells
more efficiently than AAV3B, we observed that both
AAV3B and AAV3B-V04 exhibited similarly high
transduction of osteosarcoma cells (U2-OS and SA-OS),
and low transduction of pancreatic (CCD-13LU) or neuronal
cells (Neuro-2C) cells, indicating that tropism of
AAV3B-V04 did not appear to be altered (Supplementary
Fig. S1).
Improved in vivo hepatocyte transduction
by AAV3B-V04
huFNRG mice humanized with mpPHH were generated
as described.53,55 High engraftment ofPHH was confirmed
by high levels (median 10.5 mg/mL, ranging from 7.6 to
29.2 mg/mL) of human albumin in mouse serum, which
correlates with *90% humanization.56-58 We compared
transduction efficiencies of PHH in animals receiving
1 · 1011 vg of AAV3B-ST, AAV3B-DE5, and AAV3BV04
expressing the mScarlet transgene, administered intravenously
(Fig. 1, Step 3). We selected these two vectors
for comparison because they were previously shown to
transduce huFNRG livers with high efficiency.35 AAV3BST,
an engineered AAV3B variant with two amino acid

Human Gene Therapy - April 2023

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Human Gene Therapy - April 2023 - CT1
Human Gene Therapy - April 2023 - CT2
Human Gene Therapy - April 2023 - Cover1
Human Gene Therapy - April 2023 - Cover2
Human Gene Therapy - April 2023 - 239
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Human Gene Therapy - April 2023 - Contents
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Human Gene Therapy - April 2023 - Cover3
Human Gene Therapy - April 2023 - Cover4
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