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Gene Therapy Where We Are: and Where To Go?
Gene Therapy Where We Are: and Where To Go?
Where we are
GT in TUMORS: TARGETS
Tumor cells
Immune system
Hematopoietic stem cells
(HSCs)
Inhibit
tumor cell
proliferation
Induce
tumor-cell
specific toxicity
(suicide genes)
Virus mutants
selectively replicate
and lyse
tumor cells
Anti-cancer
Vaccination
(DNA vaccines)
Increasing
the cytotoxic
response
against tumor-cell
Genetic
modifications
of T cells
(redirecting)
Target 3: HSCs
Transferring genes conferring their resistance to
high dose chemotherapy (HDC) into
hematopoietic stem cells
Supramaximal chemotherapy therapy possible in
clinical trials concerning
breast
ovary
testicle
small cell lung cancer because of specific drug prophile
1) ABC proteins (as MDR-1)
2) genes of antioxidant enzymes (GS-transferase)
are considered
Inhibit
tumor cell
proliferation
Induce
tumor-cell
specific toxicity
(suicide genes)
Virus mutants
selectively replicate
and lyse
tumor cells
Supplementary (Substitutive) GT
Clinical trial examples
Gene
Development
(clinical
phase)
Application
III
P53 (Ad5CMV-p53)
III
Allovectin (HLAB7/microglobulin 2)
III
TNF- (TNFerade)
I, II
Cycline G
IL-2
II
Suppressory GT of tumor
Triple helix target:
IGF-I, survivine
DNA
mRNA
Aptamers, target:
target
protein
VEGF, MUC1,
Antisense therapy 1
triple-helix anti-IGF-I
Watsona-Crick bonds
Hoogsteen bonds
Suppressory therapy 1
Development
I, III
I,II,III
Bcl-2 (Genasense)
Application
SC lung cancer; melanoma,
myeloma multiplex
II
C-myb
Breast cancer
Suppressory therapy 1
Development
I, III
I,II,III
Bcl-2 (Genasense)
Application
SC lung cancer; melanoma,
myeloma multiplex
II
C-myb
Breast cancer
Suppressory therapy 2
Target
Application
I-III
Bcl-2 (Genasense,
SPC2996)
I, II
X-IAP (AEG35156103)
II
HIF-1 (EZN-2968)
prostate cancer
Different tumors
Suppressory therapy 3
Ribozymes (bcr-abl; ras)
siRNA features
Native siRNA, products of nucleolytic dsRNA degradation driven by
ribonuclease Dicer have charakteristic composition.,,
2nt unpaired
2nt unpaired
RNAi phases
I siRNA duplex generation
II RISC silencing complex
formation
III
RISC activation and
recognition of mRNA
complementary to antisense
siRNA strand
IVFinal mRNA degradation
Consequence:
Gene silencing (no protein
expression)
Limitations of RNAi
RNAi efficiency is limited by ability of synthetic siRNA
molecules to bind mRNA and inhibit the target gene.
Different siRNA molecules directed against the same gene
fragment are characterized by different silencing potential.
Thats why efficiency of each siRNA type should be
evaluated separately.
It was also found, that RNAi specifity depends on the amount
of siRNA used in the assay. Its excess induces expression
of many genes associated with cell response to the stress /
viral RNA.
SELEX
Apoptosis
Apoptosis
PROBLEMS
cell stress,
cytotoxic chemotherapy,
radiation therapy
and hormone therapy
and has been shown to inhibit cell death.
Drug: Docetaxel
Late effects
Vascularisation
inhibition
VEGF
P
P
P
P
VEGFR-1
P
P
P
P
VEGFR-2
Endothelial Cell
Anti-VEGF Strategies
MAbs
TKIs
Ligand
conjugates
Ligand
Ligand
Ligand
K K
K K
Cell
death
Protein
synthesis
Antisense
oligonucleotides
Ligand
K K
Signal
transduction
K K TKI
Signal
transduction
Anti-cancer
vaccination
Increasing
the cytotoxic
response
against tumor-cell
Genetic
modifications
of T cells
(redirecting)
TUMOR
CELL
Irradiated tumor
cells
Dendritic
cells
Plasmids
Peptides
Viral vectors
coding neo-antigens
TUMOR
CELL
Irradiated tumor
cells
Dendritic
cells
Plasmids
Peptides
Viral vectors
coding neo-antigens
TUMOR
CELL
Irradiated tumor
cells
Dendritic
cells
Plasmids
Peptides
Viral vectors
coding neo-antigens
TCR
CD8 / CD4
CD80 / CD86
CD28
Fas (CD95)
ICAM-1 (-2)
LFA-3
CD40
FasL (CD178)
LFA-1
CD2
CD40L
TCR
CD8 / CD4
CD80 / CD86
CD28
Fas (CD95)
ICAM-1 (-2)
LFA-3
CD40
FasL (CD178)
LFA-1
CD2
CD40L
TCR
CD8 / CD4
CD80 / CD86
CD28
Fas (CD95)
ICAM-1 (-2)
LFA-3
CD40
FasL (CD178)
LFA-1
CD2
CD40L
TCR
CD8 / CD4
CD80 / CD86
CD28
Fas (CD95)
ICAM-1 (-2)
LFA-3
CD40
FasL (CD178)
LFA-1
CD2
CD40L
2
3
TCR
CD8 / CD4
CD28
Fas Ligand (CD178)
LFA-1
CD2
CD40L
Metastases generation
Przerzut
metastasis
metastasis
Przerzut
Przerzut
metastasis
TUMOR Progression
DNA Vaccines 1
Phase of
clinicla trial
Commercial
name
II
1650-G
II
Ad-CCL23
II
Mel
Cancer Vac
II
INGN-225
DNA Vaccines 2
Phase of
clinicla
trial
Commercial
name
III
BLP-25/
START
III
TG4010
FowlpoxCEA/
TRICOM
*
**
Carcinoembryonal antigen
TRICOM = B7.1 (CD80) + ICAM-1 + LFA-3
Anti-cancer
vaccination
Increasing
the cytotoxic
response
against tumor-cell
Genetic
modifications
of T cells
(redirecting)
Anti-cancer
vaccination
Increasing
the cytotoxic
response
against tumor-cell
Genetic
modifications
of T cells
(redirecting)
Generations of CARs
Limitations of GT 1
Short term character of GT
DNA and/or RNA introduced to cells must be active for a
longer time
Transfected cells should live and function normally
However, in supplementation GT transgene integrates with
genome with difficulties; the site of integration is
completely unpredictable
Cell divisions often eliminate effects of transfection patients need the repetitive therapies
Limitations of GT 2
Immune response
In GT in vivo patient exposition to foreign
antigens (DNA, protein career) may
induce immune reaction
In supplementation GT, immune system
response is more and increased with
consecutive trangene exposition
Limitations of GT 3
Troubles with viral vectors
Shortcomings:
toxicity,
Immune and inflammatory response,
Infection of unwanted cells,
problems with control of transgene expression.
Limitations of GT 4
Multifactor ethiopathogenesis of
diseases
In some cases only, the disease could be cured
with approach directed again one single gene
GT systems based on multi-gene simultaneous
correction in the same cell are, up to date, not
sufficient enough
Where we go?
Perspectives of GT in tumors
Gene Therapy + Immune therapy = immune gene
therapy?
GT vaccinations as adjuvant therapy?
GT as chemotherapy assistance?
go in all?
Improved vectors and transfection systems?
Can we really mimic full succesful anti-tumor immune
responses?
Turning point or step by step progress?