INVESTIGADORES
PODHAJCER Osvaldo Luis
congresos y reuniones científicas
Título:
Improving the lytic capacity of a conditionally replicative adenoviruses by incorporating promoter elements responsive to tumor environmental conditons.
Autor/es:
DIEGO L. VIALE; VERONICA M. LOPEZ; EDUARDO A. CAFFERATA; DAVID GOULD; YUTI CHERNAJOVSKY; OSVALDO L. PODHAJCER
Reunión:
Congreso; 13th Annual Meeting of the American Society of Gene & Cell Therapy; 2010
Resumen:
Human tumors are composed of a heterogeneous mass of
malignant cells that also include tumor-associated stromal cells that might
become a barrier for successful therapies. Conditionally Replicative
Adenoviruses (CRAds) are a new modality for cancer therapy. We have previously
reported that a new CRAd (Ad-F512) where E1A transcription is driven by a 0.5
Kb fragment of the SPARC promoter (SPPr) exhibited strong killing effect over established
human melanomas xenografted in nude mice. However, its oncolytic effect was strongly
reduced on established tumors made of a mix of melanoma cells and human fibroblasts
(Lopez MV et al, PLOS One, 2009).
In order to improve the therapeutic efficacy of Ad-F512
we hypothesized that addition of DNA sequences containing responsive elements
to different patho-physiological conditions that characterize tumor tissue,
such as hypoxia and inflammation, would potentiate its replication and,
therefore its oncolytic activity.
We constructed twelve different chimeric promoters containing
SPPr combined with a Hypoxia-Response Element (HRE), an NFkB-response element (NFkB) or both and
compared its transcriptional activity in luciferase expressing plasmids. Based
on their activity/specificity ratio in different malignant and stromal cells we
selected the chimeric promoter HRE-SPPr where HREs were placed upstream of SPPr
and a triple chimeric promoter NFkB-SP(HRE)Pr where the
NFkB responsive elements were placed upstream of SPPr and HRE was placed inside
SPPr. We further evaluated the transcriptional activity of the chimeric
promoters in an adenovirus backbone using luciferase as a reporter gene and observed 2-10
fold induction of luciferase activity under hypoxia of both chimeric promoters and
a 5-fold induction under TNFa treatment of NFkB-SP(HRE)Pr in
tumor cells.
Based on these results, we constructed two CRAds (Ad-HRE-SPPr
and Ad-NFkB-SP(HRE)Pr) where E1A transcription was
driven by the chimeric promoters and evaluated the cytopathic effect (CPE),
viability and replication rate. Compared with Ad-F512 we observed a slight
increase of oncolytic effect under normoxic conditions over melanoma cells. However,
the best replication rate was observed under hypoxic conditions where both
CRAds exhibited 2 to 10 fold increased lytic effect on melanoma and
fibroblasts.
Finally, nude mice harboring tumors made of
a mix of melanoma cells and fibroblasts that were resistant to three
administrations of Ad-F512 were treated intratumorally with five
administrations of 1010 vp/mouse with either of the CRAds. Ad-F512
treatment induced the elimination of tumors in 2 out of 4 mice, while the other
2 mice showed a slight tumor growth. However in mice treated with Ad-NFkBSP(HRE)Pr we observed a complete elimination of the tumor
in all mice that did not recur even after a follow up period of more than 100
days. These results indicate that addition of enhancer elementsresponsive to environmental
conditions improved the therapeutic effect of a CRAd and might overcome the
restriction imposed by the presence of stromal cells.