Adenoviruses:

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Adenoviruses applications in cancer gene therapy
M.A.I. Abou EL Hassan, Ph.D. Cancer Biology
Department NCI, Cairo University
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(No Transcript)
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What can we realize?

• Gene replacement
• Gene correction
• Gene augmentation
• (Friedman, Science, 1997)

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What can we realize?

• Gene replacement
• Gene correction
• Gene augmentation
• (Friedman, Science, 1997)

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What can we realize?

• Removal of a mutant gene and its replacement with
  a correct gene
• (Friedman, Science, 1997)

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What can we realize?

• Gene replacement
• Gene correction
• Gene augmentation
• (Friedman, Science, 1997)

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What can we realize?

• Correction of a defective sequence of a gene
  without any additional changes in the target
  genome
• Homologous recombination
• (Friedman, Science, 1997)

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What can we realize?

• Correction of a mutant gene sequence without any
  additional changes in the target genome
• Homologous recombination
• (Friedman, Science, 1997)

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What can we realize?

• Gene replacement
• Gene correction
• Gene augmentation
• (Friedman, Science, 1997)

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Gene augmentation/therapy

• Introduction of foreign normal genetic sequences
  to compensate for a defect in a mutant gene
• (Friedman, Science, 1997)

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Gene therapy

• Non-viral vectors
• Viral vectors

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Gene therapy- non-viral vectors

• Non-viral vectors
• Viral vectors

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Gene therapy- non-viral vectors

• Cationic liposomes lipid molecules used as
  vehicles for nucleic acid (e.g. DOTMA and DOTAP)
• Molecular conjugates lipid molecules can be
  conjugated to cancer cells specific ligand

Cell
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Gene therapy- non-viral vectors

• Cationic liposomes lipid molecules used as
  vehicles for nucleic acid (e.g. DOTMA and DOTAP)
• Molecular conjugates lipid molecules can be
  conjugated to cancer cells specific ligand

Cell
Nucleus
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Gene therapy

• Non-viral vectors
• Viral vectors

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Viral vectors- viral vectors

• Retroviruses
• Lentiviruses
• Herpes simplex virus (HSV)
• Adeno-associated viruses (AAV)
• Adenoviruses (Ad)

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Viral vectors-RNA viruses
Envelope
Capsid
Cell
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Viral vectors-RNA viruses
Cell
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Viral vectors-RNA viruses
Cell
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Viral vectors-RNA viruses
Reverse transcription
Cell
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Viral vectors-RNA viruses
Integration
Cell
Provirus
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Viral vectors-DNA viruses
Capsid
Cell
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Viral vectors-DNA viruses
Cell
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Viral vectors-DNA viruses
Cell
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Viral vectors-DNA viruses
Episomal localization
Cell
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Viral vectors-Ad

• Linear double-stranded DNA
• At least 52 human serotypes (2 and 5)
• Genome size 36 Kb

( Adenoviruses Basic Biology to Gene therapy by
P Seth, 1999)
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Viral vectors-Ad

• Icosahedral capsid (hexon, penton, fiber)
• Genes E and L
• Life-cycle is about 40 h PI

Advantage
Disadvantage

• large transgene capacity
• Efficient infectivity to a wide range of cells

• Targeting

(Nakai et al., JVI, 1999)
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Adenovirus-life-cycle
av integrins
CAR
Endosome disruption
Endosome
nucleus
(Rots et al., JCR, 2003)
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Adenovirus-Ad gene expression
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Adenovirus-Ad gene expression
L
E3
E1 (A/B)
ITR
ITR
E2
E4
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Adenovirus-Ad proteins function

• E1A region encodes E1A 9, 12 and 13S
• Functions
• Activation of downstream genes (E1B, E2 and E3)
• Binds to RB TSG

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Adenovirus-Ad proteins function
2-binds to RB TSG
E1A
E2F
RB
In uninfected cells the release of E2F is done by
Cdk, which phosphorylate RB during G1 phase
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Adenovirus-Ad proteins function

• E1B region encodes E1B19Kda and E1B55Kda
• Functions
• E1B19Kda is Bcl2 homologue and prevents immature
  cell death
• E1B55Kda plays an important role in p53
  proteolytic degradation

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Adenovirus-Ad proteins function

• What if p53 is left undegraded?
• p53 will
• Cause activation of p21, which will prevent
  phosphorylation of RB and the release of E2F
• Activate the mitochondrial apoptotic pathway

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Adenovirus-Ad proteins function
E2 region encodes TP and Ad DNA Pol Functions
Ori
ITR
ITR
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Adenovirus-Ad proteins function
E2 region encodes TP and Ad DNA Pol Functions
ITR
ITR
TP
Pol
NF-1
Oct-1
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Adenovirus-Ad proteins function

• E3 region encodes 11.6Kda (Adenovirus Death
  Protein, ADP) and Receptor Internalization and
  Degradation complex (RID) (10.4 and 14.5 Kda)
• Functions
• ADP efficient lysis of Ad-infected cells
• RID internalization of Fas and TRAIL receptors

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Adenovirus-Ad proteins function

• E4 region encodes E4orf4 and E4orf6
• Functions
• E4orf4 p53-independent apoptosis at later stages
  after infection
• E4orf6 in a complex with E1B55Kd binds to p53
  for its ubiquitination and proteosomal degradation

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Adenovirus-Ad proteins function
L region encodes hexon, penton and fiber proteins
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Adenovirus-Ad proteins function
L region encodes hexon, penton and fiber proteins
Shaft
Knob
Hexon
Penton base
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Adenovirus-Summary

• Ad encodes various proteins that
• Control cell cycle
• Prevent cell death early after infection
• Promote cell death late after infection
• Act as building units of the capsid

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Adenovirus-rAd
L
E1
E3
WtAd
ITR
ITR
E2
E4
DE1
rAd
ITR
ITR
Requires helper cell line (293, 911 or PerC6)
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Adenovirus-rAd applications
Protection against cell death Normal cells
Induction of cell death Cancer treatment
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Adenovirus-Heart cells

• rAds can be used as efficient vehicles for the
  delivery of protecting proteins

Sod/Cat
DE1
rAd (DE1/DE3)
ITR
ITR
(Abou El Hassan et al., BJC, 2003 CGT, 2003)
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Adenovirus-Heart cells
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Adenovirus-Heart cells
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Adenovirus-Heart cells
Dox AdCat/Sod
Control
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Adenovirus-conclusion

• The adenovirus-mediated Sod or Cat overexpression
  did not protect against doxorubicin-induced
  cardiotoxicity in vitro

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Adenovirus- NSCLC

• rAds can be used as efficient vehicles for the
  delivery of pro-apoptotic proteins

TRAIL
DE1
rAd (DE1/DE3)
ITR
ITR
(Abou El Hassan et al., BJC, 2004)
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Adenovirus- NSCLC

• TRAIL is one of the TNF family

• TRAIL is constitutevily expressed in many tissues
  and cells

Target cell
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Adenovirus- NSCLC

• Induce apoptosis in many tumor cell lines both in
  vitro and in vivo.

• Has no/low effect on normal cells e.g.
  fibroblasts and mouse hepatocytes

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Adenovirus- NSCLC
Death recepors
Decoy recepors
1 2 3 4
Out
In
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Adenovirus- NSCLC

• H460 cells infected with different MOIs (50-200
  pfu/cell), harvest after 1,2 and 3 day PI/Western

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Adenovirus- NSCLC
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Adenovirus- NSCLC

• H460 cells treated with different concentrations
  of doxorubicin, taxol or cisplatin 24 h PI with
  AdTRAIL

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Adenovirus- NSCLC
H460
3D
Control
Conc. (mM)
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Adenovirus- NSCLC
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Adenovirus- NSCLC
NHF
3D
Control
Conc. (mM)
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Adenovirus- conclusion

• AdTRAIL produced efficient cell kill of the
  resistant NSCLC cell line NCI-H460
• AdTRAIL sensitized H460 cells to chemotherapy
• AdTRAIL was much more specific to tumor cells
  compared to conventional chemotherapeutic agents

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Adenovirus-conditionally replicating
L
E1
E3
WtAd
ITR
ITR
E2
E4
D24E1A/D55E1B
CRAds
ITR
ITR
Replicate only in RB- or P53-mutated cancer cells
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CRAd
Normal cell Wt RB/WtE1A
E1A
E2F
RB
Normal cell Wt RB/mutant E1A
E2F
RB
E2F
Cancer cell Mutant RB/mutant E1A
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CRAd
(Heise and Kirn, JCI, 2000)
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CRAd applications

• CRAds proved effective as anti-cancer agents in
  vitro
• Their combined treatment with chemotherapeutic
  agents showed promising results
• (Suzuki et al., CCR, 2001)

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CRAd applications

• How does the CRAd kill the infected cancer cell?
• Is it apoptosis or necrosis??

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CRAd applications
zVAD
-
Blank
CRAd?24RGD
FasL CHX
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CRAd applications
AnnexinV
PI
Phase
2D
- zVAD
CRAd(E3) MOI 25
- zVAD
FasLCHX (16 h)
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Gene therapy-conclusion

• CRAds induce their oncolytic effect via other
  routes than the basic apoptotic machinery
• (Abou El Hassan et al., JVI, 2004)

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Introduction of Cancer Gene Therapy to the
Egyptian NCI
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Why Ad-GT ?

• Offers a new cancer (genetic-based disorders!!)
  treatment modality in Egypt
• CRAds offers an efficient tool, which overcomes
  all the cell death blocking proteins, which are
  the main cause of cancer resistance to
  conventional chemotherapy

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Why Ad-GT ?

• CRAds, are very specific to tumors rather than to
  normal cells
• Can be used to deliver Apoptotic proteins
• e.g. TRAIL

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Why TRAIL ?

• One of the most specific anticancer agents
  discovered so far
• Has a potent anti-cancer effect against a wide
  range of tumors both in vitro and in vivo

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Why AdTRAIL ?

• Production of TRAIL in a context of a CRAd offers
  a continuous source of production of TRAIL at the
  site of the tumor

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Then?

• We play our expected role as pioneers of cancer
  treatment in the middle east ???

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How Ad-GT to the NCI ?
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How Ad-GT to the NCI ?
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How Ad-GT to the NCI ?
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How Ad-GT to the NCI ?
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How Ad-GT to the NCI ?
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How Ad-GT to the NCI ?