IIBBA   05544
INSTITUTO DE INVESTIGACIONES BIOQUIMICAS DE BUENOS AIRES
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Improving the lytic capacity of a conditionally replicative adenovirus by incorporating promoter elements responsive to tumor enviromental conditons
Autor/es:
DIEGO L. VIALE; LÓPEZ, M.V; CAFFERATA, E.G.A; DAVID T CURIEL; DAVID GOULD; YUTI CHERNAJOVSKY; OSVALDO L PODHAJCER
Lugar:
Seattle
Reunión:
Congreso; 14th Annual Meeting of the American Society of Gene & Cell Therapy; 2011
Institución organizadora:
American Society of Gene & Cell Therapy
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 regulated 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 increase its replication and, therefore
its oncolytic activity.
We constructed twelve different chimeric promoters containing
SPPr combined with a Hypoxia-Response Element (HRE), a NFkB-response element (NFkB) or both and
compared its transcriptional activity in luciferase expressing plasmids. Based
on their activity/specificity ratio in different cell types 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 response elements
were placed upstream of SPPr and HREs were placed inside SPPr. We further evaluated
the transcriptional activity of the chimeric promoters in an adenovirus
backbone using luciferase as a reporter gene. We 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 malignant
cells.
Based on these results, we constructed two CRAds (Ad-HRE-SPPr
and Ad-NFkB-SP(HRE)Pr) where E1A transcription was regulated
by the chimeric promoters. Both CRAds exhibited 2 to 10 fold increased lytic
effect under hypoxia on melanoma cells and fibroblasts compared to the parental
Ad-F512.
Finally, nude mice harboring tumors made of
a mix of melanoma cells and fibroblasts that were resistant to three
administrations of Ad-F512 (Lopez et al, PlosONE 2009) 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-NFkB-SP(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 elements responsive to tumor-environmental
conditions improved the therapeutic effect of a CRAd and might overcome the
restriction imposed by the presence of stromal cells.