Human Gene Therapy - April 2023 - 298

298
RANA ET AL.
Figure 5. In vitro neutralization assays. (A) IVIg: Inhibition of HUH-7 adherent cell transduction by AAV3B, AAV3B-DE5, AAV3B-V04, or AAV3B-V05 using
increasing concentrations of pooled IVIg (20-1,500 lg/mL). The IVIg concentration corresponding to 50% reduction in RLuc expression (RLU) for each group,
compared to a no IVIg control, is indicated with dashed lines. (B) Determination of mean reciprocal NAb titers to AAV3B, AAV3B-DE5, and AAV3B-V04 for 30
seropositive samples from individual healthy donors using an in vitro RLuc-based assay. Samples that can inhibit transduction by 50% at ‡1:5 dilution are
considered seropositive. (C) Distribution of NAb titers from (B). Frequencies of serum samples with NAb titers <1:20, <1:100, and >1:100 to AAV3B, AAV3B-DE5,
and AAV3B-V04 are indicated. (D) Eight individual serum samples without detectable (<1:5) NAb to AAV3B are evaluated for NAb to AAV3B-DE5 and AAV3BV04.
Data are represented as mean-SEM are an average of at least two replicates per group per test condition and are the best representative of at least two
independent experiments. IC50 is determined for (A, B, D) using a four-parameter curve fit test. Statistical analysis is performed by two-way ANOVA with a
Dunnett's comparison test for (A) and a one-way ANOVA with a Tukey's multiple comparison test for (B, D).A p-value <0.05 is considered statistically
significant and indicated as follows: *p< 0.05, **p< 0.01, ***p< 0.001, and ****p< 0.0001. IC50, half-maximal inhibitory concentration.
Capsid engineering can alleviate this by improving or
modifying tissue tropism, thereby reducing the overall
vector burden. Lower capsid titers can also reduce the
formation of capsid antigen-antibody complexes, thus
evading complement activation and deleterious innate
immune responses.72 Engineered capsids have not yet
been widely adopted in clinical settings, although this is
expected to increase, with several being explored in preclinical
studies.
The most common capsid engineering strategy is capsid
shuffling followed by directed evolution in the cell or
tissue of interest.40,48,73,74 This has resulted in the isolation
of promising candidates such as LK03, currently used in
clinical trials for liver gene transfer (NCT05092685,
NCT05092685, etc.).8,34 Interestingly, although LK03 is a
shuffled capsid, it shares the most identity with AAV3B.
Therefore, our approach was to introduce rational mutagenesis
into surface-exposed residues in the VR loops of
AAV3B, which led to the recent isolation of the AAV3BDE5
variant,35 and to our current candidate, AAV3B-V04.
Recent work by the Srivastava laboratory and others
revealed the ability of AAV3B to transduce human and
nonhuman primate hepatocytes in vitro and in vivo,19,43,59
likely owing to the use of human hepatocyte growth factor
receptor for cellular entry.75 Performance of the AAV3
capsid was further improved by eliminating one serine and
one threonine residue (AAV3-ST containing mutations
S663V and T492V). Here, we show that AAV3B-V04

Human Gene Therapy - April 2023

Table of Contents for the Digital Edition of Human Gene Therapy - April 2023