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RANA ET AL.
In vivo transduction
C57BL/6 mice were injected via tail vein at 1 · 1011 vg/
mouse (n = 4/group) with AAV3B, AAV3B-V04, or
AAV8 vectors encoding either the mScarlet or GFP fluorescent
reporter transgene. Transduced hepatocytes were
quantified at 2 weeks by flow cytometry.
Human liver cell suspensions of 0.5-1· 106 were injected
into the spleens of FNRG mice under anesthesia.35
Starting on the day of transplantation, mice were cycled off
the liver protective drug 2-nitro-4-trifluoromethylbenzoyl1,3-cyclohexanedione
(NTBC or nitisinone; Yecuris,
Tualatin, OR)52
to stimulate repopulation with human
hepatocytes. Human albumin levels in mouse sera were
measured by ELISA (Bethyl Laboratories, Montgomery,
TX). All AAV vectors packaged a self-complementary
genome encoding the mScarlet fluorescent reporter transgene
flanked by AAV2 ITRs. One day before AAV injection,
mice were placed back onNTBC using the ''efficient''
protocol termed previously.53 AAV3B-ST, AAV3B-DE5,
AAV3B-V04, or vehicle control were injected through the
tail vein at 1 ·1011 vg/mouse (n = 4-9/group). Two weeks
later, human-chimeric mouse livers were isolated and processed.
In vivo transduction efficiency was determined for
each group by quantifying frequencies of mScarlet+ HLA
class I+ human hepatocytes by flow cytometry on day 14.
Real-time PCR
C57BL/6 mice were euthanized 2 weeks after AAV3B,
AAV3B-V04, or AAV8 tail vein injection. Genomic DNA
was isolated from liver, kidney, heart, spleen, muscle, and
brain using the genomic DNA isolation kit (Qiagen, Germantown,
MD) according to the manufacturer's instructions.
One hundred nanograms of input genomic DNA was
used to quantitate AAV vector genome copies using
transgene-specific primers. A PCR reaction was performed
using the 2 ·SYBR Green supermix (Bio-Rad,
Hercules, CA), using the following cycling conditions:
50C for 2 min, 95C for 10 min, 35 cycles at 95C for
15 s, 60C for 15 s, and 72C for 30 s, using a CFX96
Touch real-time PCR detection system (Bio-Rad).
In vitro neutralization assay
1 · 103 vg/cell MOI of AAV3B, AAV3B-DE5, or
AAV3B-V04 expressing the RLuc transgene were incubated
with serial twofold dilutions of pooled human IVIg
(Privigen; CSL Behring, Bradley, IL). Further, 1 · 103 vg/
cell MOI of AAV3B, AAV3B-DE5, or AAV3B-V04 were
preincubated with 30 individual healthy donor serum
samples (Innovative Research, Inc., Novi, MI) for 2 h at
37C. HUH-7 cell monolayers in 96-well plates were
transduced as described with the vector: serum mix. RLuc
expression was quantified after 24 h using the Renilla-Glo
luciferase assay system (Promega) and measured on an
ELISA reader with luminescence detection capacity (Synergy
HTX; BioTek, Winooski, VT).
Neutralization titer for each sample is defined as the
serum dilution at which RLuc expression is reduced by
50% compared to naive sera.
In a separate experiment, 1 · 104 vg/cell MOI of RLuc
transgene expressing AAV3B, AAV3B-V04, or AAV5
were incubated with twofold dilutions of pooled human
IVIg and overlayed on 2V6.11 cell monolayers (expressing
the adenovirus E4 ORF gene product) as described.54
RLuc expression was quantified after 24 h.
Statistical analysis
Data are normally distributed (Shapiro-Wilk test) unless
indicated otherwise. For neutralization assays, reciprocal
NAb titers were extrapolated by nonlinear curve
fitting methods and analyzed using four- to five parameter
half-maximal inhibitory concentration (IC50) tests or
fourth-order polynomial curve fitting in GraphPad Prism
8. Comparison between multiple groups was performed
using one-way or two-way ANOVA with Tukey's or
Dunnett's post hoc analysis.
RESULTS
Directed evolution
The AAV3B combinatorial library used to identify the
new capsid variants was described earlier.35 In brief,
surface-exposed residues in VR-I to VR-VII were diversified
by PCR using degenerate oligonucleotides. Library
construction was incremental to maximize the likelihood of
combining mutations compatible with capsid assembly.
Three sublibraries were first constructed, each with mutations
in one or more different regions. Their viral DNA was
then used to generate the final library that included mutations
in all five VRs, therefore filtering out sequences that
failed to result in functional viral particles (Fig. 1, Step 1).
Viral complexity was estimated to be in the order of1 · 10.7
The directed evolution process aimed at selecting variants
with hepatocyte tropism was also described previously.35
In brief, five iterative selections in 3D human
hepatocellular carcinoma (HUH-7) spheroid cultures were
performed, using an initial MOI of 1 and subsequent MOIs
of 0.01. Superinfection with human adenovirus 5 (Ad5)
was performed at each round of selection to facilitate AAV
replication.34
To minimize the potential bias caused by low MOI, use
of Ad5, and the large number of selection cycles,49 we
focused on the results from the first three rounds of selection
(Fig. 1, Step 2). In addition, we reasoned that diversification
of VR-I had introduced unnecessary
complexity into the analysis because we observed no
significant enrichment for any mutations in this region.
Therefore, we elected to treat the VR-I region as wt to
simplify data analysis.
The resulting amino acid sequences were sorted by their
maximal frequency across all rounds of selection. The
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