Human Gene Therapy - April 2023 - 309

EX VIVO GENE DELIVERY TO PORCINE HEARTS USING AAV
309
Figure 3. PCR and Luciferase enzymatic assay assessment of rAAV at serial perfusion time points. rAAVs isolated from the perfusate solution at serial time
points were (A) assessed by PCR for percent of VGCs detectable and by (B) luciferase enzymatic cell-based assay using HeLa cells as a measure of overall
vector presence. Results are shown as mean- SD; n= 3 for each group. PCR, polymerase chain reaction; VGCs, viral genome copies.
right (2 · 1013 VGC: 51.5 VGC/pg ofDNA; 8 · 1013 VGC:
44,441.6 VGC/pg of DNA; 1· 1014 VGC: 20,353.8 VGC/
pg of DNA) ventricle of the allografts, while vector DNA
was consistently absent in the native heart ventricles and in
the recipient's liver. Notably, a dose-dependent response
was observed such that the lowest titer of SASTG administered
had the lowest amount ofSASTGDNAdetectable in
the allograft, whereas the higher titers led to higher DNA
amounts (left ventricle [LV] fold-change between lowest
and highest titer: 6,354.2-fold; right ventricle [RV] foldchange
between lowest and highest titer: 395.3-fold).
The expression of the luciferase transgene was assessed
by enzymatic assay (Fig. 5). Again, there was luciferase
enzymatic activity throughout the right ventricle (2 · 1013
VGC: 1,074-1,258 RLU/mg of protein; 8 · 1013 VGC:
3,764-63,456 RLU/mg of protein; 1 · 1014 VGC: 10,429-
26,884 RLU/mg of protein), interventricular septum
(2 · 1013 VGC: 32-2,664 RLU/mg of protein; 8 · 1013
VGC: 19,054-28,038 RLU/mg of protein; 1 · 1014 VGC:
14,469-55,629 RLU/mg of protein), and left ventricle
(2 · 1013 VGC: 471-9,852 RLU/mg of protein; 8 · 1013
VGC: 43,550-94,975 RLU/mg of protein; 1 · 1014 VGC:
112,348-278,052 RLU/mg of protein) of the allografts.
Therewasno significant luciferase activity noted in anyofthe
native recipient hearts. Adose-dependent response was again
observed such that the lowest titer of SASTG-luciferase administered
achieved the lowest amount of luciferase activity
detectable in the allograft, whereas the higher titers led to
higher amounts of enzymatic activity (average fold difference
in LV between lowest and highest titer: 37.8-fold;
average fold difference in RV between lowest and highest
titer: 16.0-fold; average fold difference in interventricular
septum between lowest and highest titer: 26.0-fold).
Noncardiac tissues were also examined (lung, spleen,
liver, and psoas muscle) and no luciferase activity was
detectable. Finally, immunofluorescent staining using
anti-Luciferase antibodies was assessed to understand the
histologic distribution pattern of luciferase in the transduced
allografts and compared against the respective
nontransduced native recipient hearts (Fig. 6). The results
demonstrated positive staining for luciferase in the allograft
hearts transduced at higher VGC doses. The luciferase
was homogeneously distributed in these heart
sections, whereas in contrast, there was no luciferase expression
in any of the native hearts.
Figure 4. Results of quantitative PCR analysis of the cardiac allograft,
native recipient heart, and native recipient liver. Measured VGCs in the
allografts demonstrated dose-dependent DNA levels. No VGCs were detected
in the native recipient hearts or liver. Results are shown as
mean- SD; n= 3 for each group.
DISCUSSION
The concept of developing gene therapy to enhance
transplant outcomes has been long envisioned. Early ex

Human Gene Therapy - April 2023

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