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MENDIOLA PLA ET AL.
(Sakura, Torrance, CA), or formalin fixation. Biopsies of
the left anterior descending artery, lungs, liver, spleen, and
psoas muscle were also collected and preserved similarly.
Tissue lysate preparation
Tissue samples (50-150 mg) underwent mechanical
homogenization using a Dounce homogenizer with passive
lysis buffer (Promega). The lysate was then incubated
on ice for 30 min before centrifugation at 12,000g for
10 min at 4C. Protein concentration of the resulting supernatant
was determined using the Pierce BCA Protein
Assay Kit (Thermo Fisher Scientific) and a biokinetics
reader (EL-340; BioTek Instruments, Winooski, VT).
Equivalent protein amounts (400 lg) of the supernatant
were used in the luciferase activity assay.
Concentration of OCS perfusate
Samples of the OCS perfusate were collected at 15-30min
intervals during the perfusion period and were concentrated
before use in cell-based assays by centrifugation
at 800g for 10 min to remove red blood cells. The supernatant
was concentrated using Amicon Ultra-4 50 kDa
Centrifugal Columns (MilliporeSigma) until there was
*1mL of perfusate. This concentrated perfusate was then
used for qPCR and luciferase activity assay analysis.
Luciferase enzymatic activity assay
All luminometry was performed with a Veritas luminometer
(Turner Biosystems, Sunnyvale, CA). Luciferase
assay reagent (Promega) was added to each well (50 lL)
and light emission per well was measured in relative light
units (RLU).
Histology and immunofluorescent staining
Tissue samples of the allograft and native heart were
fixed in either 10% neutral buffered formalin and embedded
in paraffin or embedded in OCT compound. Paraffinembedded
sections of5 lmwere stained with Hematoxylin
and Eosin and assessed for structural tissue changes and
evidence of rejection. OCT-embedded frozen sections of
10lm were stained for luciferase expression. This was
done using a primary mouse anti-luciferase monoclonal
antibody (Sigma-Aldrich, Cat no. L2164, St. Louis, MO)
diluted 1:150, and a goat anti-mouse secondary polyclonal
antibody conjugated to Alexa Fluor 594 (Abcam, Cat no.
ab150116, Cambridge, UK) diluted 1:300. Imaging was
done using a Zeiss 780 upright confocal microscope (Carl
Zeiss Microscopy, White Plains, NY).
qPCR analysis
Total DNA was isolated using the ReliaPrep gDNA
Tissue Miniprep Kit (Promega). DNA purity and concentration
were assessed using a NanoDrop Spectrophotometer
(Thermo Fisher Scientific). qPCR was then performed for
the firefly luciferase gene using the SYBR Green Supermix
(Bio-Rad, Hercules, CA) and the CFX Connect Real-Time
PCR Detection System (Bio-Rad). The primers used for
luciferase gene amplification were Forward-5¢-CTCACTG
AGACTACATCAGC-3 and Reverse-5¢-TCCAGATCCA
CAACCTTCGC-3. A standard curve generated using serial
dilution of the CMV-Luciferase plasmid was used to calculate
the total number of VGCs in solutions and tissues.
Statistical analyses
Statistical methods are detailed in the figure legends.
Normality was assessed using the Shapiro-Wilk test.
Parametric data were compared using Student's t-test.
Nonparametric data were compared using Mann-Whitney
Utest. Multiple comparisons were assessed using two-way
analysis of variance (ANOVA) followed by Dunnett's
multiple comparisons test. All analyses were performed
using GraphPad Prism version 9.4.1 (861) for Windows
(GraphPad Software, San Diego, CA). Statistical notations
used in the figures: p> 0.05, not significant (ns); p< 0.05,
*; p< 0.01, **; p< 0.001, ***; and p< 0.0001, ****.
RESULTS
Assessment of transduction efficiency
of rAAV in OCS perfusate
We evaluated the transduction efficiency of rAAV serotypes
1, 2, 4, 5, 6, 7, 8, and 9 and the AAV3b capsid
‰
Figure 2. Assessment of transduction efficacy among various rAAV serotypes and perfusate additives. (A) Transduction efficacy was compared through
luciferase enzymatic cell-based assay using HeLa cells incubating in either PBS or perfusate solution during the transduction period. SASTG was observed to have
the highest efficiency of the rAAV serotypes in both PBS and perfusate. AMann-Whitney Utest was used to compare SASTG activity in P versus PBS. A Student's
t-test was used to compare AAV1 and AAV9 activity in P versus PBS, respectively. Results are shown as mean-SD; n= 3 for each group. (B) Select rAAV serotypes
were then assessed for transduction efficacy using RNCMs incubating in either PBS, perfusate, methylprednisolone (0.373mg/mL), or calcium gluconate (1.04mg/
mL). A two-way ANOVA with multiple comparisons was used to compare luciferase activity from each rAAV against AAV1 and AAV9. Results are shown as
mean-SD; n= 3 for each group. SASTG also outperformed other rAAV serotypes when incubated in perfusate or perfusate additives during transduction. Effects of
individual perfusate components on SASTG transduction were assessed using different cell lines (HeLa, 911, RNCM). (C) In HeLa cells, SASTG transduction was
suppressed with priming solution alone, but enhanced in the presence of the perfusate solution and each individual perfusate additive. (D) In RNCM cells, SASTG
transduction was suppressed in the presence of priming solution alone, but significantly enhanced in the presence of methylprednisolone and calcium gluconate.
(E) In 911 cells, SASTG transduction was enhanced in the presence of all components of the perfusate solution, especially in heparin, multivitamin, methylprednisolone,
and calciumgluconate. A two-way ANOVA withmultiple comparisons was used to compare luciferase activity in each additive against activity in PBS.
Results are shown as mean-SD; n= 3 for each group. ANOVA, analysis of variance; CG, calcium gluconate; Hep, heparin; MP, methylprednisolone; MV,
multivitamin; P, perfusate solution; PBS, phosphate-buffered saline; PS, priming solution; RNCM, rat neonatal cardiomyocyte; SASTG; SD, standard deviation.

Human Gene Therapy - April 2023

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