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Gene Therapy – hype or hope ?
Farzin Farzaneh
Department of Haematological Medicine
King’s College London
Gene Therapy – Inherited Monogenic disorders
• Successful gene therapy of common  Chain cytokine
receptor defect (SCID)-X1 Disease:
9 children cured and off treatment!
Alain Fischer – Institut Pasteur, Paris.
Adrian Thrasher – Institute of Child Health, London.
Science 2000, Vol. 288: 669-672.
Science 2003, Vol. 302: 415-419.
• Successful gene therapy of ADA deficiency (SCID)
2 children cured and off treatment!
Caludio Bordignon – Hospital San Rafael, Milan
Shimuon Slavin – Hadasa University Hospital, Jerusalem
Science 2002, Vol. 296: 2410-2413.
Cure for two fatal genetic disorders!
Clinical trial for X-SCID
(Alain Fischer – Paris, Adrian Thrasher - London)
- SCID due to deficiency of common
interleukin receptor c chain
- lethal at 4 months if untreated
- survival prognosis - 10 years under
sterile conditions
1998-2000: Successful gene transfer in 10 out of 11 patients
Oct 2002: 1st report describing development of leukaemic syndrome
Jan 2003: 2nd report of identical adverse event
Jan 2005: 3rd report of leukaemia
Mar 2007: 4th report of leukaemia
Retroviral life cycle
Viral DNA is integrated into the host cell genome
Nucleus
Integrated
provirus DNA
Integrated viral
genome is
transcribed into
genomic RNA and
viral mRNA
Unintegrated
provirus DNA
Viral RNA genome
is transcribed into
provirus DNA by
Virus
receptor reverse
transcriptase
ψ
Assembly and
release of viral
particles
Infectious virions
Budding
Shed virus
Protein synthesis, processing and
assembly
Virus attachment to
receptors on the
host cell
Replication defective (helper dependent) retroviral vectors
cDNA inserts
gag pol env
LTR

LTR

cDNA
The genome of a
typical retrovirus
neo
Helper dependent
retroviral vector

gag
pol
cDNA
env
neo

Host cell
DNA
gag pol env
Viral
RNA
Retrovirus
producer Cell
Retroviral Packaging
Cell
cDNA
neo

Infected target cell – no
virus production
Helper dependent
retrovirus
Retroviral insertional mutagenesis
Provirus DNA
Genomic DNA sequence
LTR
puro
LTR
regulatory gene
Pseudorandom provirus
integration into the host cell
genome
Insertional inactivation
LTR
puro
LTR
Insertional activation
LTR
puro
LTR
truncated transdominant products
LTR
puro
LTR
C antisense
LMO2 insertional
mutagenesis:
LMO2
LMO2 From:
Actin -
P5
exon 1
Integration
exon 2
exon 5
LMO2 antisense
P5
N. T Cell ()
P4
N. T Cell ()
MEL-F4N
RPMI - 8402
CHO + MO2
C sense
CHO
3 of 15 SCID-X1 (C)
children developed T-cell
leukaemia after the
retroviral transfer of C
gene to the CD34+
haematopoietic stem
cells
Hacein-Bey-Abina et al (2003). Science
302: 415-419.
2 kb
P4
Integration
Possible factors contributing to development of T cell
leukaemia in the C clinical trial
• Use of retroviral vectors – hence inherent risk of insertional
mutagenesis
• Selective growth advantage of T cells expressing C
• The inherent anti-apoptotic effect of C gene expression
• Genetic modification of haematopoietic stem cells
• Genetic modification of large numbers of cells – hence
increased numbers of cells at risk of mutagenesis
• The immune suppressed status of the host
• Reduced endogenous numbers of competing T-cells
• Potential predisposing cytogenetic abnormalities
Retroviral insertional mutagenesis
A problem turned on its head
- functional analysis of the genome
Functional analysis of the genome
Objective:
• Identification of phenotypic / physiological function
• Determination of rate-limiting, regulatory steps
• Identification of causally associated rather than
consequential changes
Strategy:
- Retroviral cDNA library expression cloning
- RNA interference (siRNA) library based inhibition cloning
- Retroviral insertional mutagenesis
Retroviral cDNA library expression cloning

cDNA

neo
cDNA
neo
gag pol env

cDNA
neo
Infect cells with
cDNA library
gag pol env


cDNA
cDNA
neo
Select phenotype
and expand
neo
gag pol env
gag pol
env

Introduce viral genes
to rescue cDNA vector
cDNA
neo
Confirm cDNA encodes the
selected function
Identify cDNA
Williams & Farzaneh (2004). Cancer Immunol. Immunother. 53: 160-165.
Phenotypic selection of cellular function
(e.g. resistance to differentiation, apoptosis, etc.)
Immobilized cells in semi-solid
culture (e.g. pluripotent cells in soft agar)
Induction of differentiation, apoptosis,
or other selectable functions
Isolation of clonal population of cells
with the selected phenotype
Alternative strategies:
- Ligand and antibody mediated selection of cells
with specific surface markers
- Tissue/function specific promoters for drug mediated
selection of cells with the appropriate phenotype
Protein Phosphatase 4: an inducer of apoptosis!
cDNA library transfer selection of apoptosis
resistant cells
Protein Phosphatase 4: an inducer of apoptosis!
cDNA library transfer selection of apoptosis
resistant cells
W7.2 + Dex
W7.2/4n10 + Dex
Protein Phosphatase 4: an inducer of apoptosis!
cDNA library transfer selection of apoptosis
resistant cells
Insert identified:
C-terminal catalytic subunit of
PP4 – induces apoptosis
resistance ( PP4 breakdown)
W7.2 + Dex
W7.2/4n10 + Dex
Protein Phosphatase 4: an inducer of apoptosis!
cDNA library transfer selection of apoptosis
resistant cells
W7.2 + Dex
Insert identified:
C-terminal catalytic subunit of
PP4 – induces apoptosis
resistance ( PP4 breakdown)
W7.2/4n10 + Dex
Number of colonies
120
100
Vector
80
PP4-Cat.
60
40
20
0
γ
UV
Dex
γ
UV
Dex
(1000cGy)
(20J/m2)
(60nM)
(1000cGy)
(20J/m2)
(60nM)
W7.2 cells
PP4 – a new apoptosis regulator
(member of the superfamily of serine/threonine phosphatases)
• Expression of the catalytic subunit of PP4
(C-terminal fragment*):
- steady-state levels of PP4 RNA and protein
- blocks induction of apoptosis by UV, γ-irradiation or
dexamethasone
- target site: TTCTAATAAAAGAAGAAAAAT
- reduces
• Over-expression of full-length PP4 induces apoptosis in
mouse and human cell lines
Mourtada-Maarabouni et al. (2003) Cell Death Differ. 10:1016-24.
Apoptosis control by naturally expressed
regulatory RNA species
Induction of resistance to UV (254nm, 20J/m2), X-rays
(1000cGy), steroids (60nM Dex) and etoposide (1nM)
• Growth Arrest Specific transcript 5 (GAS5):
A non-coding regulatory RNA
• rFAU:
A non-coding antisense transcript identified both by cDNA expression
cloning and expressed by Finkel-Biskis-Reilly sarcoma virus (FBRSV)
PP4:
Mourtada-Marabouni et al. (2003) Cell Death & Diff. 10: 1016-1024.
rFau:
Mourtada-Marabouni et al. (2004) Oncogene 23: 9419-9426.
RACK1: Mourtada-Marabouni et al. (2005) J Leuk. Biol. 78: 503-514.
Functional studies of the genome
(RIM, cDNA, RNAi libraries)
Rate-limiting regulatory gene products:
Direct identification of controlling genes
(i.e. causal rather than consequential changes)
Cancer Gene Therapy
- some of the main strategies
Expression of tumour-suppressor genes
• Expression of p53 induces growth
arrest and increased apoptosis in
response to chemo/radio-therapy.
•
p53 expression also blocks
angiogenesis by ↓ VEGF and by ↑
expression of thrombospondin and
IGF-1 BP.
Anti-sense RNA, ribozyme and RNA interference mediated
inhibition of oncogene expression
Oncogenes examined:
c-erbB2, c-erbB4, K-ras,
H-ras, HPV E6/E7, bcl-2,
Telomerase, c-met, c-myc.
Suicide gene therapy
Enzyme
HSV-tk
Prodrug
GCV/ACV
Active product
GCV/ACV triphosphate
Mechanism
Blocks DNA synthesis
Cytosine deaminase
5-Fluorocytosine
5-Fluorouracil (5-FU)
Blocks DNA/RNA synth.
Nitroreductase
Nitrobenzyloxcarbonyl Anthracyclines
anthracyclines
Carboxylesterase
CPT-11
DNA crosslinking
SN38
Topoisomerase
inhibitor
Cytochrome p450
Cyclophosphamide
Phosphoramide mustard
DNA alkylating agent
Purine nucleoside
phosphorylase
6-mercaptopurine-DR
6-mercaptopurine
Purine antagonist
Conditionally replicating / oncolytic viruses
Normal cell: abortive replication
Productive
replication, cell
lysis
Virus kills
tumour cell,
spreads to
neighbours
Oncolytic virus
Tumour cell
Replication of a conditionally replicating virus. ONYX-015, in a cancer cell from a patient with head and
neck cancer during Phase-II clinical trial. 109 infectious E1B defective Adenovirus particles were
injected over a 5 day period. After 8 days biopsy was performed and analysed by electron microscopy.
Frank McCormick 2001, Nature Reviews 1: 130-141.
ONYX-015 plus Cisplatin/5-FU
Baseline
Cycle 1, Day 22
Yoon LTK, Laquerre S, Kasahara N (2001) Curr
Cancer Drug Targets 1: 85-106.
Cycle 3, Day 22
ONYX-015 plus Cisplatin/5-FU
Baseline
Cycle 1, Day 22
Yoon LTK, Laquerre S, Kasahara N (2001) Curr
Cancer Drug Targets 1: 85-106.
Cycle 3, Day 22
ONYX-015 plus Cisplatin/5-FU
Baseline
Cycle 1, Day 22
Yoon LTK, Laquerre S, Kasahara N (2001) Curr
Cancer Drug Targets 1: 85-106.
Cycle 3, Day 22
Oncolytic virus therapy – problems:
– robust immune response
(and other intratumoural barriers):
rapid clearance of virus
– basis for attenuation / tumor selectivity:
not well understood
Retro- and lenti-virus vectors
High-titre vectors for • functional analysis of the genome
• immune gene therapy of poor prognosis acute
myeloid leukaemia (AML)
Retro- and lenti-virus vectors
High-titre vectors for • functional analysis of the genome
• immune gene therapy of poor prognosis acute
myeloid leukaemia (AML)
Generation of biotinylated retroviral vectors
Biotin
succinimide
ester
Hughes et al (2001) Molecular Therapy 3: 623-630.
Biotinylated retro- and lenti-virus vectors:
Vector concentration
Paramagnetic labelling and
concentration of the vector/s
Attachment of targeting ligands
Biotin / avidin
mediated attachment
of targeting ligands
Casimir et al (2004). J. Gene Medicine 6: 1189-1196.
Chan et al (2005) J. Virol. 79: 13190-13194.
Paramagnetic bead concentration of
retroviral vectors
1x1010
125x concentration
(i.e. reduction in volume)
Paramagnetic particle concentrated
lentiviral vectors
4200 X
1x109
Titre (cfu/ml)
Titre (cfu/ml)
1.00E+10
1x108
1x107
1.00E+09
1.00E+08
7229x
646x
Control titre
1.00E+07
PMP-concentrate
1.00E+06
1.00E+05
1.00E+04
Amphotropic
1x106
1x105
1x
VSV-G
Envelope pseudotype
Efficient transduction of primary CD34+ blasts:
71 + 23 % of all cells express transgene after
a single round of infection at an MOI of 3
Paramagnetically targeted
retrovirus delivery
International Society for Cell & Gene Therapy of Cancer
Packaging cells producing endogenously
biotinylated retrovirus vectors
Signal peptide
Transmembrane
domain
Extracellular domain
BAP
LNGFR
Endogenously
biotinylated
LNGFR
SPH-1
SPH-1
…LGGA KEAC GGGLNDIFEAQKIbEWHE ACPTGL…
Biotin
BAP
LNGFR
External domain
LNGFR
Transmembrane
domain
BirA
Nesbeth et al. 2006, Mol. Ther. 13: 814-822
Envelope / receptor independent vector
concentration & targeting
Vector concentration
(K562 stable colonies)
1x 1011
Amphotropic
vector
Amphotropic producer cell
Titre (cfu/ml)
1x 1010
1x 109
1x 108
1x 107
1x 106
Control
Envelope / receptor independent vector
concentration & targeting
Vector concentration
(K562 stable colonies)
1x 1011
Titre (cfu/ml)
1x 1010
Amphotropic
vector
1x 108
1x 107
Amphotropic producer cell
Amphotropic vector
(surface B7.1)
B7.1 cDNA transduced
packaging cells
1x 109
1x 106
Control
-B7.1
CTLA4
Envelope / receptor independent vector
concentration & targeting
Vector concentration
(K562 stable colonies)
1x 1011
Titre (cfu/ml)
1x 1010
Amphotropic
vector
1x 109
1x 108
1x 107
Amphotropic producer cell
Amphotropic vector
(surface B7.1)
B7.1 or LNGFR cDNA
transduced packaging cells
Amphotropic vector
(surface LNGFR)
1x 106
Control
-B7.1
CTLA4
-LNGFR
Envelope / receptor independent vector
concentration & targeting
Vector concentration
(K562 stable colonies)
1x 1011
Titre (cfu/ml)
1x 1010
Amphotropic
vector
1x 109
1x 108
1x 107
Amphotropic producer cell
Amphotropic vector
(surface B7.1)
1x 106
Control
Amphotropic vector
(surface LNGFR)
SCF cDNA transduced
packaging cells
Producer cell with surface
expressed SCF
Amphotropic vector
(surface SCF)
Relative transduction efficiency
B7.1 or LNGFR cDNA
transduced packaging cells
8
-B7.1
CTLA4
-LNGFR
Targeting to c-kit+/CD34
Bone Marrow Cells
6
4
2
0
ampho
neo
SCF-ampho
Casimir et al (2004). J. Gene Medicine 6: 1189-1196.
Paramagnetically labelled / concentrated lentivirus
1 m particles with attached vector
Nesbeth et al. 2006, Mol. Ther. 13: 814-822
Immune gene therapy of cancer
A genetically modified
autologous cell vaccine for
Acute Myeloid Leukaemia (AML)
Human cancer antigens recognized by T lymphocytes
Cancer-testis antigens:
MAGE-3, BAGE, GAGE, NY-ESO-1
Melanocyte differentiation antigens:
Melan-A/MART-1, Tyrosinase, gp100
Point mutations:
β-catenin, MUM-1, CDK-4, p53, ras
Overexpressed ‘self’ antigens:
Her-2/neu. P53, MUC-1
Viral antigens:
HPV, HBV, HCV, EBV
• Tumour cells can be immunogenic
• There are tumour associated and tumour specific antigens
• Cancer is not the product of immune incompetence
- ELISPOT and MHC/antigen tetramers show increased
presence of tumour targeted T cells
• Tumour editing of the immune system AND immune editing
of the tumour
- a clinical tumour has already escaped immune surveillance
Professional antigen presenting cells:
Schwartz 1992
Professional antigen presenting cells:
Schwartz 1992
Acute Myeloid Leukaemia (AML):
• AML blasts express both HLA class-I, and class-II
• Express AML associated antigens (WT1, PRAME, GP250, etc)
• Common lineage with APCs – efficient antigen presentation
• Express many surface markers present on DC – but not B7.1 (CD80) !
Leukaemogenecity of 32DP210bcr/abl cells modified
to express B7.1, IL-2 or both
% Survival
100
■
●
■
■
●
■
■
■
●
■ ■
● ●
■
■
●
■
■
●
●
●
■ ■
■
32D/B7.1/IL-2
●
32D/B7.1
80
●
32D/IL-2
● ●
■
60
40
■ ■
20
■
0
0
20
■
■
40
60
Days post-challenge
2x107 leukaemic cells iv
80
32D/M3P (vector)
Rejection of established myeloid leukemia (32Dp210) in mice,
by genetically modified leukemia cells expressing B7.1 and IL-2
■
●
100
■
●
■
■
●
■
■
■
● ●
■
●
■
■■
■
●
●
●
■
% Survival
80
● ●
60
●
Cell vaccine
■
●
■
32D/B7.1/IL-2
●
32D/B7.1
32D/IL-2
■
■
40
■
20
■
0
Leukemia initiation
0
20
40
■
■
■
60
Time (days)
(105 32Dp210 cells iv)
(106
Vaccination
irradiated cells)
80
100
32D/M3P (Vector)
Important questions for the clinical
application of immune gene therapy:
Can B7.1/IL-2 expressing AML cells induce T cell
proliferation?
If so, are the stimulated T cells functionally competent
(Cytokine release, cytolytic activity)?
Are AML cells susceptible to T cell mediated lysis?
Can post-chemotherapy ,“remission” T cells, stimulate
cytolytic activity?
Is there any specificity in the cytolytic activity of the
stimulated T cells against the leukaemic cells?
In vitro stimulation of T cells with autologous
primary AML blasts (MLR)
PW – at presentation
AJ – remission, post BMT
CY – remission (no BMT)
IL-2
IL-2
IL2
IL-2/B7
B7/IL-2
B7/IL-2
B7
B7
uninfected
Uninfected
0
2
4
6
8
10
12
14
B7
uninfected
0
10
Stimulation Index
20
30
40
50
0
200
400
IL-2
IL-2/B7
L-2/B7
1000
1200
HM –remission, post BMT
MB – remission (no BMT)
IL-2
800
Stimulation Index
Stimulation Index
MB – at presentation
600
IL-2
B7/IL-2
B7
B7
B7
GFP
GFP
Uninfected
uninfected
uninfected
0
50
100
150
Stimulation Index
200
250
300
0
5
10
15
20
Stimulation Index
25
30
0
100
200
300
Stimulation Index
400
500
IFN-gamma ELISPOT:
1 week stimulation with the indicated autologous AMLs,
assayed on the same unmodified AMLs
CM
Increased numbers
of functionally
competent T cells
generated by the in
vitro culture of T
cells with B7.1/IL-2
expressing AML
cells.
IL-2.B7 AML
Unmodified AML
unstimulated
0
50
100
150
200
250
300
number of IFN-gamma secreting cells per 2 x 10^5 cells
AJ
IL-2.B7 AML
Unmodified AML
unstimulated
0
50
100
150
200
250
300
number IFN-gamma secreting cells / 2 x 10^5 cells
Stimulation of cytotoxic activity against unmodified AML blasts
Effectors:
% Lysis
40
30
20
10
0
Donor T cells
Stimulators:
The indicated AML cells
41
18
16
8
4
Target cells:The same, but unmodified,
AML cells
% Lysis
40
30
20
10
0
16
0
1.7
14
SB
9
% Lysis
40
30
20
10
0
AJ
10
0
2
12
6
WB
• AML cells expressing B7.1 & IL-2 can stimulate in vitro CTL activity in donor T cells.
• AML cells are susceptible to CTL mediated lysis.
Unstimulated
% LYSIS
18
16
14
12
10
8
6
4
2
0
30
20
10
Unmodified AML
0
LV.B7.1 AML
Unstimulated
LV.IL-2/B7.1 AML
Unmodified
AML
IL-2.B7 AML
LV.IL-2 AML
25
100:1
50:1
25:1
12:1
6:1
Effector to Target Ratio
% LYSIS
% Lysis
Autologous CTL activity
20
15
11
9
5
Remission PBLs can be stimulated by
B7.1/IL-2 expressing autologous AML
cells to generate cytotoxic activity
- AML cells are not resistant to T cell
mediated lysis
18
10
0
Unstimulated
Unmodified
AML
IL-2.B7 AML
20
% LYSIS
- Remission T cells are not defective
in cytolytic activity
CM
15
11
10
5
0
0
0
Unstimulated
Unmodified
AML
E:T ratio = 50:1
IL-2.B7 AML
PREVIOUS STIMULATION
Specificity of the in vitro stimulated T cells
B7.IL-2 AML
Secondary targets
No target
Unmodified
AML
AML blasts
Remission Bone
Marrow
AML blasts
Unstimulated
0
CD14+
10
20
30
Autologous
Stimulators
unstimulated
(media only)
40
Stimulation Index
(proliferation in a secondary assay)
unmodified
AML cells
IL-2/B7.1 AML
• Greater specificity of the B7.1/IL-2 stimulated T cells against
AML blasts, than against remission bone marrow cells.
Two obstacles to cancer immune therapy:
- tumour editing of the immune system
- immune editing of the tumour
Tumour editing of the immune system:
Chronic immune stimulation (cancer or infection) induces loss
of functional competence, anergy, clonal exhaustion, depletion,
and induction of Tregs.
Tumour editing of the immune system:
Chronic immune stimulation (cancer or infection) induces loss
of functional competence, anergy, clonal exhaustion, depletion,
and induction of Tregs.
Klenerman et al (2002)
Nature Reviews:
Immunology 2: 263-272.
Tumour editing of the immune system:
Chronic immune stimulation (cancer or infection) induces loss
of functional competence, anergy, clonal exhaustion, depletion,
and induction of Tregs.
Klenerman et al (2002)
Nature Reviews:
Immunology 2: 263-272.
Implications for therapeutic vaccination strategies
- the most potent antigens may not provide the best
vaccination targets !
Immune editing of the tumour:
A clinical tumour has undergone selection for
resistance to immune Surveillance hence the need for : - reduced tumour mass
- reconstituted immune
system – if possible !
Chan et al (2006). Cancer Immunol. Immunother 55: 1017-1024.
Poor prognosis AML
Standard Treatment
Day 100+
Donor Leuckocyte Infusion (DLI)
Chemotherapy
if no evidence of GvHD
Allo-HSCT RIC
(Fludarabin, Busulphan,
Campath 1H)
DLI (cells/kg)
CR or PR
5x105
Day 0
Day 28
Day 56
Day 100
106
5x106
107
5x107
108
Poor prognosis AML
Standard Treatment
Day 100+
Donor Leuckocyte Infusion (DLI)
Chemotherapy
if no evidence of GvHD
Allo-HSCT RIC
(Fludarabin, Busulphan,
Campath 1H)
DLI (cells/kg)
CR or PR
Minimal
disease burden
5x105
Day 0
Day 28
Day 56
Reconstituted
immune system
(donor chimerism)
Day 100
106
5x106
107
5x107
108
Poor prognosis AML
B7.1/IL-2 immune gene therapy
Day 100+
Donor Leuckocyte Infusion (DLI)
if no evidence of GvHD
Allo-HSCT RIC
(Fludarabin, Busulphan,
Campath 1H)
Chemotherapy
DLI (cells/kg)
5x105
CR or PR
Day 0
Day 28
Day 56
Reconstituted
immune system
(donor chimerism)
Vaccination and DLI will stop if:
1. GVHD > grade 2
2. Progressive cytopenia
3. Grade-2 toxicity
4. Unexplained side effects
5x106
107
5x107
108
Day 100
105
Minimal
disease burden
106
106
107
108
108
108
Vaccination
B7.1/IL-2 modified ‘autologous’ AML cells
Gene Therapy – Hype or hope?
Monogenic inherited disorders:
Over 30 children with incurable SCID
(common  Chain and ADA) cured and
currently off treatment
Malignant disease:
A lot of hype, a great deal of hope and still
a long way to go
Imperial College London:
Colin Casimir
Myrtle Gordon
Nagy Habib
University College London:
Mary Collins
Adrian Thrasher
Mayo Clinic:
Stephen Russell
UCLA
Noriyuki Kasahara
University of Cambridge:
Sharon Williams
Nigel Slater
King’s College London:
Haematology
Lucas Chan
David Darling
Steve Devereux
Andrea Buggins
Joop Gäken
Joanna Galea-Lauri
Barbara Guinn
Nicola Hardwick
Joti Hannoe
Al Ho
Wendy Ingram
Aytug Kizilors
Nicholas Lea
Daren Nesbeth
James WellsGhulam Mufti
Head & Neck Oncology
Mahvash Tavassoli
Mitigating factors in considering the use of
replicating MLV vectors for suicide gene therapy of
cancer
• HSC transduction unlikely with intra-tumoural injection
• Inability to infect HSCs in vivo without growth factors
• No selective growth advantage for the infected cells
• Suicide gene-mediated elimination of infected cells
• Risk versus benefit ratio in poor prognosis malignancies
PBMCs alone
PBMCs with
Unmodified AML
PBMCs with B7.1/IL-2
expressing AML
Current strategies for dealing with the problem of
insertional mutagenesis
Forego stable expression:
• Ex-vivo modification of cells followed by lethal irradiation before re-administration
(e.g. cancer vaccines).
• Use of non-integrating vectors (e.g. adenovirus).
Develop better vectors:
• Use of vectors with preferred genomic sites of integration e.g. adeno-associated
virus (AAV) – need to increase payload.
• Use of episomally maintained vectors based on EBV and EBNA/Ori containing
plasmids (i.e. extra-chromosomal maintenance).
• Development of vectors with targeted chromosomal site/s of integration.
• Incorporation of single or multiple suicide genes into vectors.
siRNA or ncRNA library production and analysis
siRNA/ncRNA Library under the
control of inducible promoter
Transfection into retrovirus
packaging cell line
Library of cells producing the
siRNA/ncRNA retrivirus library
(no expression)
Retrovirus packaging cell
( no expression )
Target cells
Inducible expression of
siRNA/ncRNA
Target cells infected with the
retroviral siRNA/ncRNA library
(no expression of siRNA/ncRNA)
Retroviral
siRNA/ncRNA library
Induced expression of
siRNA/ncRNA
Phenotypic selection
Identification of siRNA or ncRNA
and their targets
RCR vector mediates
highly efficient gene transmission
(NIH3T3 cells, MOI=0.0005)
Day 2:
3.3 %
Day 4:
Day 7:
22.7 %
93.8 %
Logg CR et al. (2001) Hum Gene Ther, 12: 921-932.
RCR vectors for suicide gene therapy
Yeast cytosine deaminase (CD) as a suicide gene
The ACE-CD Vector:
CMV
R U5
gag
pol
env
U3
R U5

IRES
CD
5-fluorocytosine
5-fluorouracil
(non-toxic)
(toxic)
• CD converts the non-toxic prodrug 5-FC to the toxic metabolite 5-FU
• Better bystander effect than HSV-tk/GCV
Logg CR et al. (2001) Hum Gene Ther, 12: 921-932.
Multiple cycles of 5-FC can further improve survival and suggests
persistence of RCR-CD in metastatic intracranial glioma cells
median
survival:
>100 days
Logg CR et al. (2001) Hum Gene Ther, 12: 921-932.
Poor prognosis AML
Current Standard Treatment:
Reduced Intensity Conditioning (RIC) combined with mini-HSCT
Day 100+
Donor Leuckocyte Infusion (DLI)
Chemotherapy
if no evidence of GvHD
Allo-HSCT RIC
(Fludarabin, Busulphan,
Campath 1H)
DLI (cells/kg)
CR or PR
5x105
Day 0
Day 28
Day 56
Day 100
106
5x106
107
5x107
108
Analysis of the transcriptome, proteome, etc.
• Comparison of transcripts or proteins expressed
in cell or tissue A with B
• Advantage:
-
Rapid screening of large
number of changes
• Disadvantage:
-
No discrimination between
cause and consequence
What function ?
~ 30,000 genes
(~ 100,000 protein coding RNA)
What function ?
~ 30,000 genes
(~ 100,000 protein coding RNA)
• At the molecular/biochemical level
– e.g. kinases, proteases, etc.
~ 1/3 known biochemical
role
What function ?
~ 30,000 genes
(~ 100,000 protein coding RNA)
• At the molecular/biochemical level
– e.g. kinases, proteases, etc.
~ 1/3 known biochemical
role
• At the cellular level
– Specific (e.g. phosphorylation of cell cycle
proteins, response to growth hormones)
– General (e.g. involvement or regulation of
DNA repair, protein synthesis, etc.)
~ 1/2 identified
physiological role
What function ?
~ 30,000 genes
(~ 100,000 protein coding RNA)
• At the molecular/biochemical level
– e.g. kinases, proteases, etc.
~ 1/3 known biochemical
role
• At the cellular level
– Specific (e.g. phosphorylation of cell cycle
proteins, response to growth hormones)
– General (e.g. involvement or regulation of
DNA repair, protein synthesis, etc.)
• At the phenotypic/physiological level
– e.g. rate limiting regulatory factors controlling
cell survival, apoptosis, differentiation, transdifferentiation, etc.
~ 1/2 identified
physiological role
Few have ratelimiting regulatory
functions ?
Functional analysis of the genome
• Objective:
• Identification of phenotypic / physiological function
• Determination of rate-limiting, regulatory steps
• Identification of causally associated rather than consequential changes
- Retroviral insertional mutagenesis
disruption cloning
- Retroviral cDNA library expression cloning
- RNA interference (siRNA) library based
repression cloning
- Non-coding RNA (ncRNA) library based
regulation cloning
Substantially enhanced by:
• the availability of genomic
sequences
• increased retroviral titres
Functional analysis of the genome
(RIM, cDNA, siRNA and ncRNA libraries)
Determination of physiological role & identification
of rate-limiting regulatory gene products:
Advantage:
- Direct identification of controlling genes
(i.e. identification of causal rather than
consequential changes)
Disadvantage:
- Requires selectable phenotype
(e.g. resistance to apoptosis, differentiation, etc.)
- limited by inefficient library transfers (…..no longer!)
- Requires adequate knowledge of the genome (now
available!)
- Requires robust validation!
Lentiviral (VSV-G) infection of established and
primary myeloid leukaemia cells
U937
NB4
K564
MAR
(Primary AML)
MOI
3.0
0.3
Efficient transduction of primary
AML blasts
Efficiency of primary AML transduction:
MOI ~ 1 (43 ng p24) > 40%
MOI ~ 5 (200ng p24) > 95 %
Chan et al (2005) J. Virol. 79 (20): 13190-13194.
Human myeloid leukaemia cells infected with SIN lentiviral
vectors encoding B7.1, IL-2 or both
Chan et al (2005) Mol. Therapy 11: 120-131.
FACS analysis of NK cells
PBMCs + unmodified
AML
PBMCs + IL2 expressing
AML
PBMCs + B7.1/IL2
expressing AML
CD56dim: Account for >90% of NK cells in peripheral blood.
Express perforin and KIRs.
Subpopulation express CD16 and responsible for ADCC.
Publications suggesting CD16neg population responsible for
cytotoxicity against tumour cells.
CD56bright: Produce cytokines e.g. IFN-, TNF-, IL-10.
Protein Phosphatase 4: an inducer of apoptosis!
cDNA library transfer selection of apoptosis
resistant cells
Insert identified:
C-terminal catalytic subunit of
PP4 – induces apoptosis
resistance ( PP4 breakdown)
Number of colonies
120
100
Vector
80
PP4-Cat.
60
40
20
0
γ
UV
Dex
γ
UV
Dex
(1000cGy)
(20J/m2)
(60nM)
(1000cGy)
(20J/m2)
(60nM)
W7.2 cells
% reduction in endogenous PP4
W7.2 + Dex
W7.2/4n10 + Dex
70
60
50
40
30
20
10
0
100
200
No. of colonies
after Dex treatment
Protein Phosphatase 4: an inducer of apoptosis!
cDNA library transfer selection of apoptosis
resistant cells
Insert identified:
C-terminal catalytic subunit of
PP4 – induces apoptosis
resistance ( PP4 breakdown)
100
Vector
80
PP4-Cat.
60
40
20
0
γ
UV
Dex
γ
UV
Dex
(1000cGy)
(20J/m2)
(60nM)
(1000cGy)
(20J/m2)
(60nM)
W7.2 cells
W7.2/4n10 + Dex
200
70
60
Number of colonies
Number of colonies
120
% reduction in endogenous PP4
W7.2 + Dex
50
40
30
20
10
150
100
50
0
0
100
200
No. of colonies
after Dex treatment
Vector
PP4-Cat.
CEM-C7 cells
Insertional mutagenesis in myeloid cells
(HL-60 differentiation)
HL-60 cells
HL-60 cells + Retinoic acid
PAGER D cells + Retinoic acid
Insertional mutagenesis in myeloid cells
(HL-60 differentiation)
HL-60 cells
HL-60 cells + Retinoic acid
RARα:
Mutants resistant to
RA only.
PAGER D cells + Retinoic acid
AAA
1A
II
Provirus
III IV V VI VII
VIII
Insertional mutagenesis in myeloid cells
(HL-60 differentiation)
HL-60 cells
HL-60 cells + Retinoic acid
RARα:
Mutants resistant to
RA only.
AAA
II
Provirus
III IV V VI VII
VIII
ATG
ATG
1A
c-myb:
Mutants resistant
to RA, DMSO, Vit.
D3
PAGER D cells + Retinoic acid
11 4111
MPSV retroviral insertional mutagenesis of HL-60 cells:
mutants resistant to RA, DMSO and Vit. D3
The profile of cytokine secretion by the in vitro
stimulated T cells
(Cytokine Bead Array – CBA)
Unstimulated
2500
pg cytokine / 10^5 cells
IL-2
IL-4
IL-6
IL-10
TNF-
IFN
Unmodified AML
Lv.IL-2.B7 AML
2000
1500
IL-2
IL-4
IL-6
IL-10
TNF-
IFN
1000
500
0
IFN-γ
TNF-α
IL-2
IL-10
1L-6
IL-4
n=3
• The in vitro stimulated T cells have a predominantly
Th1 phenotype
IL-2
IL-4
IL-6
IL-10
TNF-
IFN
VUD and sibling RIC transplants
Overall Survival
Percent Survival
100
50
0
0
500
1000
1500
2000
days from transplant
%
100
VUD n = 56
Sibling n = 31
Relapse
50
0
0
500
1000
1500
2000
days from transplant
Ho et al (2004) Blood 104: 1616-1623.
Endogenously biotinylated retro- and lenti-virus vectors:
Vector concentration
Paramagnetic labelling and
concentration of the vector/s
Attachment of targeting ligands
Biotin / avidin
mediated attachment
of targeting ligands
Casimir et al (2004). J. Gene Medicine 6: 1189-1196.
Chan et al (2005) J. Virol. 79: 13190-13194.
Replicating MLV retrovirus as a
cancer therapeutic
– simple virus, well understood
– poor immunogenicity
– infects proliferating cells only
– transcriptional control of replication – use of tissue/ tumour
specific promoters
– stable integration, not directly cytolytic – therefore possibility
of sustained presence
– can provide prodrug-activated cell death by suicide genes
(e.g. GCV
HSV-tk
(non-toxic)
GCV-P;
(toxic)
5-fluorocytosine
(non-toxic)
– availability of anti-retroviral drugs
CD
5-flurouracil)
(toxic)
Microarray & Proteomics
• Comparison of transcripts or proteins expressed in cell
A with cell B
• Comparison of the same cell under different biological
conditions
• Advantage:
-
Rapid screening of large
numbers of transcripts/proteins
• Disadvantage:
-
No discrimination between
cause and consequence
Descriptive analysis of genome
Subtractive cloning strategies (PCR Select):
Identification of regulatory iron binding proteins:
IREG1 (iron transporter)
McKie et al (2000). Molecular Cell 5: 299-309.
Dcyt.B (Ferric reductase)
McKie et al (2001). Science 291: 1755-1759.
Microarray analysis:
Identification of host factors responsible for resistance to HIV
infection
A number of candidates identified!
Retroviral insertional mutagenesis
Provirus DNA
Genomic DNA sequence
LTR
cDNA
library
Selectable
marker
regulatory gene
Pseudorandom provirus
integration into the host cell
genome
Insertional inactivation / activation
(gain or loss of function)
LTR
LTR
cDNA expression cloning (gain or
loss of function)
LTR
LTR
LTR
Oncolytic virus therapy – problems:
– robust immune response:
rapid clearance of virus
– Inadequate targeting/specificity:
“even a brick can kill tumour cells”