Human Gene Therapy - April 2023 - 276

276
WESTHAUS ET AL.
patocytes were seeded 400,000 cells/well in complete
hepatocyte plating media (HCM kit; Cat. No. CC-3198;
Lonza). After 4 h of incubation at 37C and 5% CO2,
media were removed and gently covered with hepatocyte
maintaining media (HCM kit; Cat. No. CC-3198; Lonza)
containing 0.25 mg/mL Matrigel (Cat. No. 354234;
Corning) and incubated at 37C for 90 min. The AAV mix
was added diluted in hepatocyte basal media (HBM; Cat.
No. CC-3199; Lonza). Media were changed daily for
3 days, until harvest using Cell Recovery Solution (Cat.
No. 354253; BD) and processing for NGS.
The 3D printed hydrogels containing human and rhesus
hepatocytes were generated using a Rastrum Cell Printer
(Inventia, Sydney, Australia). The cell printing followed
the manufacturer's instructions and previously published
protocols.38,39 In brief, hepatocytes were thawed, resuspended
in crosslinker solution (Inventia), and printed in
96-well plates at 8,000 cells/well. Cells were maintained
in HBM and AAVs were added as described previously.
Cells were harvested as whole printed gels and processed
for NGS.
Primary human and simian hepatocytes
engrafted into FRG mice
Cynomolgus and rhesus macaque primary hepatocytes
were purchased from Lonza (Cat. No. MKC118 and Cat.
No. MKR103). Human hepatocytes were also purchased
from Lonza (Cat. No. HUM181971) and corresponded to a
15-month-old donor.
The engraftment procedure was performed as previously
published.28,40 Mouse studies were supported by the
BioResources Core Facility at Children's Medical Research
Institute. All animal care and experimental procedures
were approved by the joint Children's Medical
Research Institute and The Children's Hospital at Westmead
Animal Care and Ethics Committee. The FRG mice
were housed, treated, and killed following previously
published methods.40
Liver organoid transduction
Human liver organoids were generated as previously
described41 and used for research purposes with approval
from the human ethics committee of School of Biosciences,
University of Melbourne (Ethics No. 1851272).
To carry out transduction with AAV, the Matrigelsupported
planar infection method was adapted.42 In brief,
mature liver organoids embedded in Matrigel domes were
isolated by dispersing Matrigel with cold basal media.
After centrifugation, the medium was discarded, and the
organoid pellet was suspended with expansion medium
containing 10 lM Y-27632 (Cat. No. S1049; Selleckchem).
The AAV cocktail was added to the medium at
the described dose, and then the organoid-AAV mixture
was transferred to a 24-well plate precoated with 80 lLof
75% Matrigel.
The organoid-AAV mixtures were incubated at 37C
with 5% CO2 for 12 h in a planar manner after which
organoids on the Matrigel surface were collected, transferred
to a tube, centrifuged, and supernatant discarded.
The organoids were then washed with cold basal media
followed by centrifugation. After the final wash and centrifugation,
the organoids were returned to 3D culture by
resuspending in Matrigel and seeding at 50 lL per well
into 24-well plates. Once the Matrigel had set, 450 lLof
expansion culture medium (Supplementary Table S1) was
added and then replaced every other day for 7 days. To
harvest, the organoids were suspended in cold basal media
after the medium was discarded, pelleted by centrifugation,
and then snap frozen until processing for DNA and
RNA extraction. All centrifugations were carried out at
400 g at 4C for 5 min.
Polymerase chain reaction
Standard and Illumina amplicon-seq NGS polymerase
chain reactions (PCRs) were performed using Q5 (Cat. No.
M0491; NEB), dNTPs (Cat. No. N0447; NEB), and
primers (all Sigma-Aldrich; Supplementary Table S2) and
were performed strictly following previously published
protocols.36
AAV production
All AAV capsids used in this study were produced
using polyethylenimine transfection of the LSP-GFPbarcode
(LSP-BC) transgene cassette,31,37,43,44 as well as
adenovirus and Rep2-Cap2/3/5/8/LK03/NP59 helper plasmids
using previously described methods.37 The resulting
cell lysates and purified media were either purified using
iodixanol gradients (all experiments apart from NHP) or
CsCl ultracentrifugation (for NHP vectors) following the
previously published protocols, respectively.26,36 AAV titers
were established using quantitative PCR and enhanced
green fluorescent protein (eGFP) primers following previously
published methods.36,45 The capsids were then mixed
at an equimolar ratio as previously described.36
Neutralization assays
For experiments using the NHP, neutralization assays
determining the anti-capsid antibodies for the different
AAVs for all indicated timepoints were performed after
the following protocol. At day 1, HEK293T cells were
seeded into a 96-well plate at a density of104 cells/well. At
day 2, NHP sera were diluted in DMEM supplemented
with 2% FBS in a total volume of 100 lL, beginning with a
1:5 dilution followed by dilution series of 1:3 and mixed
with a dose of 104 vg/cell of the corresponding AAV serotype
coding for luciferase that were incubated for 2 h at
37C. The mix was subsequently used to transduce target
cells. Each serum dilution was tested in duplicate.
Negative controls of nontransduced cells, as well as
positive controls of cells transduced without AAVs not
Human Gene Therapy - April 2023

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