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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! - PowerPoint PPT Presentation
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Farzin FarzanehDepartment of Haematological MedicineKing’s College London
Gene Therapy – hype or hope ?
Gene Therapy – Inherited Monogenic disorders
• Successful gene therapy of common Chain cytokinereceptor 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
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
1998-2000: Successful gene transfer in 10 out of 11 patients
Retroviral life cycle
Integrated provirus DNA
Nucleus
Viral DNA is integrated into the host cell genome
Unintegrated provirus DNA
Integrated viral genome is transcribed into genomic RNA and viral mRNA Virus
receptor
Viral RNA genome is transcribed into provirus DNA by reverse transcriptase
Budding
Shed virus
Assembly and release of viral particles
Infectious virions
ψ
Virus attachment to receptors on the host cell
Protein synthesis, processing and assembly
Replication defective (helper dependent) retro-viral vectors
Retroviral Packaging Cell
cDNA neo
Helper dependent retroviral vector
gag pol env
LTR LTR
The genome of a typical retrovirus
cDNA inserts
gag pol env
Retrovirus producer Cell
Helper dependent retrovirus
cDNA neo
gag pol env
cDNA neo
Viral RNA
Infected target cell – no virus production
Host cell DNA
Retroviral insertional mutagenesis
Provirus DNAGenomic DNA sequence
regulatory gene
LTR LTRpuro
Insertional inactivation
LTR LTRpuro
Insertional activationLTR LTRpuro
Pseudorandom provirus integration into the host cell
genome
LTR LTRpuro
truncated transdominant products
CH
O
CH
O +
MO
2
RP
MI
- 84
02
ME
L-F
4N
N. T
Cel
l (
)
P4
N. T
Cel
l ()
P5
LMO2 -
Actin -
P4Integration
P5Integration
C antisense
C sense
LMO2 antisense
LMO2
2 kb
exon 1 exon 2 exon 5
LMO2 insertional mutagenesis:
3 of 15 SCID-X1 (C) children developed T-cell leukaemia after the retroviral transfer of C gene to the CD34+ haematopoietic stem cells
From:
Hacein-Bey-Abina et al (2003). Science 302: 415-419.
• 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
Possible factors contributing to development of T cell leukaemia in the C clinical trial
Retroviral insertional mutagenesis
A problem turned on its head
- 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
Functional analysis of the genome
Strategy:- Retroviral cDNA library expression cloning
- RNA interference (siRNA) library based inhibition cloning
- Retroviral insertional mutagenesis
Retroviral cDNA library expression cloning
cDNA neo
gag pol env
cDNA neo
gag pol env
Infect cells with cDNA library
Select phenotypeand expand
cDNA neo
Introduce viral genesto rescue cDNA vector
gag pol env
Confirm cDNA encodes the selected function
cDNA neo
gag pol env
cDNA neo
Identify cDNA
cDNA neo
Williams & Farzaneh (2004). Cancer Immunol. Immunother. 53: 160-165.
Immobilized cells in semi-solidculture (e.g. pluripotent cells in soft agar)
Induction of differentiation, apoptosis, or other selectable functions
Isolation of clonal population of cellswith the selected phenotype
Phenotypic selection of cellular function (e.g. resistance to differentiation, apoptosis, etc.)
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!
W7.2 + Dex W7.2/4n10 + Dex
cDNA library transfer - selection of apoptosis resistant cells
Protein Phosphatase 4: an inducer of apoptosis!
W7.2 + Dex W7.2/4n10 + Dex
cDNA library transfer - selection of apoptosis resistant cells
Insert identified: C-terminal catalytic subunit of PP4 – induces apoptosis resistance ( PP4 breakdown)
Protein Phosphatase 4: an inducer of apoptosis!
W7.2 + Dex W7.2/4n10 + Dex
PP4-Cat.
Vector100
0
20
60
40
80
120
Dex (60nM)
γ(1000cGy)
Dex (60nM)
γ(1000cGy)
UV(20J/m2)
UV(20J/m2)
Nu
mb
er
of
co
lon
ies
W7.2 cells
cDNA library transfer - selection of apoptosis resistant cells
Insert identified: C-terminal catalytic subunit of PP4 – induces apoptosis resistance ( PP4 breakdown)
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.
• 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)
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)
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)
Direct identification of controlling genes
(i.e. causal rather than consequential changes)
Rate-limiting regulatory gene products:
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 Prodrug Active product MechanismHSV-tk GCV/ACV GCV/ACV triphosphate Blocks DNA synthesis
Cytosine deaminase 5-Fluorocytosine 5-Fluorouracil (5-FU) Blocks DNA/RNA synth.
Nitroreductase Nitrobenzyloxcarbonyl Anthracyclines DNA crosslinking anthracyclines
Carboxylesterase CPT-11 SN38 Topoisomerase inhibitor
Cytochrome p450 Cyclophosphamide Phosphoramide mustard DNA alkylating agent
Purine nucleoside 6-mercaptopurine-DR 6-mercaptopurine Purine antagonistphosphorylase
Conditionally replicating / oncolytic viruses
Frank McCormick 2001, Nature Reviews 1: 130-141.
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.
Productive replication, cell
lysis
Virus kills tumour cell, spreads to neighbours
Oncolytic virus
Normal cell: abortive replication
Tumour cell
ONYX-015 plus Cisplatin/5-FU
Cycle 1, Day 22 Cycle 3, Day 22Baseline
Yoon LTK, Laquerre S, Kasahara N (2001) Curr Cancer Drug Targets 1: 85-106.
ONYX-015 plus Cisplatin/5-FU
Cycle 1, Day 22 Cycle 3, Day 22Baseline
Yoon LTK, Laquerre S, Kasahara N (2001) Curr Cancer Drug Targets 1: 85-106.
ONYX-015 plus Cisplatin/5-FU
Cycle 1, Day 22 Cycle 3, Day 22Baseline
Yoon LTK, Laquerre S, Kasahara N (2001) Curr Cancer Drug Targets 1: 85-106.
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
Attachment of targeting ligands
Paramagnetic labelling and concentration of the vector/s
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.
1x109
1x1010
1x108
1x107
1x106
1x105
Control
Biotin
SE conc
DMSO c
onc
Biotin
SEDM
SO
1x
4200 X
125x concentration(i.e. reduction in volume)
Tit
re (
cfu
/ml)
Paramagnetic bead concentration of retroviral vectors
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
…LGGA KEAC GGGLNDIFEAQKIbEWHE ACPTGL…
SPH-1SPH-1
Signal peptide
BAP LNGFRExtracellular domain
Transmembrane domain
Endogenously biotinylated LNGFR
Packaging cells producing endogenously biotinylated retrovirus vectors
BirALNGFRTransmembrane domain
LNGFRExternal domain
BAP
Biotin
Nesbeth et al. 2006, Mol. Ther. 13: 814-822
Envelope / receptor independent vector concentration & targeting
Amphotropic producer cell
Amphotropic vector
Vector concentration (K562 stable colonies)
Tit
re (
cfu
/ml)
1x 1010
1x 1011
1x 106
1x 107
1x 108
1x 109
Control
Envelope / receptor independent vector concentration & targeting
Amphotropic producer cell
B7.1 cDNA transduced packaging cells
Amphotropic vector
Amphotropic vector (surface B7.1)
Vector concentration (K562 stable colonies)
Tit
re (
cfu
/ml)
1x 1010
1x 1011
-B7.11x 106
1x 107
1x 108
1x 109
CTLA4Control
Envelope / receptor independent vector concentration & targeting
Amphotropic producer cell
B7.1 or LNGFR cDNA transduced packaging cells
Amphotropic vector
Amphotropic vector (surface LNGFR)
Amphotropic vector (surface B7.1)
Vector concentration (K562 stable colonies)
Tit
re (
cfu
/ml)
1x 1010
1x 1011
-B7.11x 106
1x 107
1x 108
1x 109
CTLA4 -LNGFRControl
Envelope / receptor independent vector concentration & targeting
Amphotropic producer cell
SCF cDNA transduced packaging cells
Casimir et al (2004). J. Gene Medicine 6: 1189-1196.
Producer cell with surface expressed SCF
Amphotropic vector (surface SCF)
B7.1 or LNGFR cDNA transduced packaging cells
Amphotropic vector
Amphotropic vector (surface LNGFR)
Amphotropic vector (surface B7.1)
Vector concentration (K562 stable colonies)
Tit
re (
cfu
/ml)
1x 1010
1x 1011
-B7.11x 106
1x 107
1x 108
1x 109
CTLA4 -LNGFRControl
ampho0
2
4
6
8
neo
Re
lati
ve
tra
ns
du
cti
on
eff
icie
nc
y
SCF-ampho
Targeting to c-kit+/CD34
Bone Marrow Cells
Paramagnetically labelled / concentrated lentivirus
1 m particles with attached vector
Nesbeth et al. 2006, Mol. Ther. 13: 814-822
A genetically modified autologous cell vaccine for
Acute Myeloid Leukaemia (AML)
Immune gene therapy of cancer
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) !
■
●
■■■ ■ ■ ■ ■ ■ ■●●●
●
● ● ●
●
● ●
■ ■
■ ■
■■
■
■
100
80
60
40
20
0
0 20 40 60 80
Days post-challenge2x107 leukaemic cells iv
% S
urv
ival
32D/M3P (vector)
32D/B7.1
32D/IL-2
32D/B7.1/IL-2
Leukaemogenecity of 32DP210bcr/abl cells modified to express B7.1, IL-2 or both
■●
●
■
■
Rejection of established myeloid leukemia (32Dp210) in mice, by genetically modified leukemia cells expressing B7.1 and IL-2
●
100
80
60
40
20
0
% S
urv
ival
■ ■●●
● ●
■ ■
■ ■
■ ■
■■■
●
Time (days)Leukemia initiation
(105 32Dp210 cells iv)
100 20 40 60 80 0
Vaccination (106 irradiated cells)
■
32D/M3P (Vector)
■
●
■
32D/B7.1
32D/IL-2
32D/B7.1/IL-2
Cell vaccine
●
● ●●● ● ●■ ■■■ ■ ■ ■ ■ ■■
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?
Important questions for the clinical application of immune gene therapy:
In vitro stimulation of T cells with autologous primary AML blasts (MLR)
MB – at presentation
0 50 100 150 200 250 300
uninfected
GFP
B7
IL-2/B7
IL-2
PW – at presentation
0 2 4 6 8 10 12 14
uninfected
B7
IL-2/B7
IL-2
Stimulation Index
MB – remission (no BMT)
CY – remission (no BMT)
0 10 20 30 40 50
Uninfected
B7
B7/IL-2
IL2
Stimulation Index
0 5 10 15 20 25 30
uninfected
GFP
B7
IL-2
L-2/B7
Stimulation Index
AJ – remission, post BMT
uninfected
0 200 400 600 800 1000 1200
B7
B7/IL-2
IL-2
Stimulation Index
0 100 200 300 400 500
Uninfected
B7
B7/IL-2
IL-2
HM –remission, post BMT
Stimulation IndexStimulation Index
CM
0 50 100 150 200 250 300
unstimulated
Unmodified AML
IL-2.B7 AML
number of IFN-gamma secreting cells per 2 x 10^5 cells
AJ
0 50 100 150 200 250 300
unstimulated
Unmodified AML
IL-2.B7 AML
number IFN-gamma secreting cells / 2 x 10^5 cells
IFN-gamma ELISPOT: 1 week stimulation with the indicated autologous AMLs, assayed on the same unmodified AMLs
Increased numbers of functionally competent T cells generated by the in vitro culture of T cells with B7.1/IL-2 expressing AML cells.
Stimulation of cytotoxic activity against unmodified AML blasts
8 4
16
41
18
010203040
0 210 12
6
010203040
Unstim
ulate
d A
ML
B7 AM
L
IL-2
.B7
AML
IL-2
AM
L
SB
WB
0 1.7
16 149
010203040
Unstim
ulate
dAM
L
B7 AM
L
IL-2
.B7
AML
IL-2
AM
L
AJ
Effectors: Donor T cells
Stimulators: The indicated AML cells
Target cells: The same, but unmodified, AML cells
• 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.
% Lysis
% Lysis
% Lysis
Unstim
ulate
d A
ML
B7 AM
L
IL-2
.B7
AML
IL-2
AM
L
Autologous CTL activity
Remission PBLs can be stimulated by B7.1/IL-2 expressing autologous AML cells to generate cytotoxic activity
- Remission T cells are not defective in cytolytic activity
- AML cells are not resistant to T cell mediated lysis
0
10
20
30
Unstimulated Unmodified AML
IL-2.B7 AML
% L
YS
IS
E:T ratio = 50:1
0 0
11
0
5
10
15
20
Unstimulated Unmodified AML
IL-2.B7 AML
PREVIOUS STIMULATION
% L
YS
IS
CM
9
1811
0
5
10
15
20
25
Unstimulated Unmodified AML
IL-2.B7 AML
% L
YS
IS 0
2468
1012141618
100:1 50:1 25:1 12:1 6:1
Effector to Target Ratio
% L
ysis
Unstimulated
Unmodified AML
LV.B7.1 AML
LV.IL-2/B7.1 AML
LV.IL-2 AML
Specificity of the in vitro stimulated T cells
• Greater specificity of the B7.1/IL-2 stimulated T cells against AML blasts, than against remission bone marrow cells.
0 10 20 30 40
Unstimulated
Unmodified AML
B7.IL-2 AML
Stimulation Index (proliferation in a secondary assay)
Remission BoneMarrow
AML blasts
Autologous Stimulatorsunstimulated (media only)
unmodifiedAML cells
IL-2/B7.1 AML
No target CD14+ AML blasts
Secondary targets
- tumour editing of the immune system
- immune editing of the tumour
Two obstacles to cancer immune therapy:
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.
Standard Treatment
5x105 106 5x106 107 5x107 108
Chemotherapy
CR or PR
Allo-HSCT RIC (Fludarabin, Busulphan, Campath 1H)
Day 28 Day 56 Day 100Day 0
DLI (cells/kg)
Day 100+
Donor Leuckocyte Infusion (DLI)
if no evidence of GvHD
Poor prognosis AML
Standard Treatment
5x105 106 5x106 107 5x107 108
Chemotherapy
CR or PR
Allo-HSCT RIC (Fludarabin, Busulphan, Campath 1H)
Day 28 Day 56 Day 100Day 0
DLI (cells/kg)
Day 100+
Donor Leuckocyte Infusion (DLI)
if no evidence of GvHD
Minimal disease burden
Reconstituted immune system
(donor chimerism)
Poor prognosis AML
B7.1/IL-2 immune gene therapy
5x105 106 5x106 107 5x107 108
Chemotherapy
CR or PR
Allo-HSCT RIC (Fludarabin, Busulphan, Campath 1H)
Day 28 Day 56 Day 100Day 0
DLI (cells/kg)
Day 100+
Donor Leuckocyte Infusion (DLI)
if no evidence of GvHD
Minimal disease burden
Reconstituted immune system
(donor chimerism)
105 106 107 108 108 108
VaccinationB7.1/IL-2 modified ‘autologous’ AML cells
Poor prognosis AML
Vaccination and DLI will stop if:
1. GVHD > grade 2
2. Progressive cytopenia
3. Grade-2 toxicity
4. Unexplained side effects
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
King’s College London:
HaematologyLucas Chan David Darling
Steve Devereux Andrea Buggins
Joop Gäken Joanna Galea-Lauri Barbara Guinn Nicola Hardwick
Joti Hannoe Al HoWendy Ingram Aytug Kizilors
Nicholas Lea Daren NesbethJames WellsGhulam Mufti
Head & Neck Oncology
Mahvash Tavassoli
Mayo Clinic: Stephen Russell
University College London:
Mary CollinsAdrian Thrasher
UCLANoriyuki Kasahara
Sharon WilliamsNigel Slater
University of Cambridge:
Imperial College London: Colin CasimirMyrtle GordonNagy Habib
• 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
Mitigating factors in considering the use of replicating MLV vectors for suicide gene therapy of
cancer
PBMCs alonePBMCs with
Unmodified AMLPBMCs with B7.1/IL-2
expressing AML
• Ex-vivo modification of cells followed by lethal irradiation before re-administration (e.g. cancer vaccines).
• Use of non-integrating vectors (e.g. adenovirus).
Current strategies for dealing with the problem of insertional mutagenesis
• 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.
Forego stable expression:
Develop better vectors:
siRNA or ncRNA library production and analysis
Retrovirus packaging cell ( no expression )
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)
Retroviral siRNA/ncRNA library
Target cells
Target cells infected with the retroviral siRNA/ncRNA library
(no expression of siRNA/ncRNA)
Induced expression ofsiRNA/ncRNA
Phenotypic selection
Inducible expression of siRNA/ncRNA
Identification of siRNA or ncRNA and their targets
RCR vector mediates highly efficient gene transmission
(NIH3T3 cells, MOI=0.0005)
3.3 %
Day 2:
22.7 %
Day 4:
93.8 %
Day 7:
Logg CR et al. (2001) Hum Gene Ther, 12: 921-932.
RCR vectors for suicide gene therapy
• CD converts the non-toxic prodrug 5-FC to the toxic metabolite 5-FU• Better bystander effect than HSV-tk/GCV
Yeast cytosine deaminase (CD) as a suicide gene
gag pol envU5RCMV U5RU3
IRES CD
5-fluorocytosine (non-toxic)
5-fluorouracil (toxic)
The ACE-CD Vector:
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.
Current Standard Treatment:
Reduced Intensity Conditioning (RIC) combined with mini-HSCT
5x105 106 5x106 107 5x107 108
Chemotherapy
CR or PR
Allo-HSCT RIC (Fludarabin, Busulphan, Campath 1H)
Day 28 Day 56 Day 100Day 0
DLI (cells/kg)
Day 100+
Donor Leuckocyte Infusion (DLI)
if no evidence of GvHD
Poor prognosis AML
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 ?
• At the molecular/biochemical level– e.g. kinases, proteases, etc.
~ 30,000 genes(~ 100,000 protein coding RNA)
~ 1/3 known biochemical role
What function ?
• At the molecular/biochemical level– e.g. kinases, proteases, etc.
• 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.)
~ 30,000 genes(~ 100,000 protein coding RNA)
~ 1/2 identified physiological role
~ 1/3 known biochemical role
What function ?
• At the molecular/biochemical level– e.g. kinases, proteases, etc.
• 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, trans-differentiation, etc.
~ 30,000 genes(~ 100,000 protein coding RNA)
~ 1/2 identified physiological role
Few have rate-limiting regulatory functions ?
~ 1/3 known biochemical role
• Objective: • Identification of phenotypic / physiological function
• Determination of rate-limiting, regulatory steps
• Identification of causally associated rather than consequential changes
Functional analysis of the genome
- 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
• the availability of genomic sequences
• increased retroviral titres
Substantially enhanced by:
Functional analysis of the genome(RIM, cDNA, siRNA and ncRNA libraries)
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!
Determination of physiological role & identification of rate-limiting regulatory gene products:
U937 K564
NB4 MAR(Primary AML)
Lentiviral (VSV-G) infection of established and primary myeloid leukaemia cells
MOI3.0
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.
PBMCs + unmodified AML
PBMCs + IL2 expressing AML
PBMCs + B7.1/IL2 expressing AML
FACS analysis of NK cells
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!
W7.2 + Dex W7.2/4n10 + Dex
PP4-Cat.
Vector100
0
20
60
40
80
120
Dex (60nM)
γ(1000cGy)
Dex (60nM)
γ(1000cGy)
UV(20J/m2)
UV(20J/m2)
Nu
mb
er
of
co
lon
ies
W7.2 cells
70
60
50
40
30
20
10
0 100 200
No. of coloniesafter Dex treatment
% r
ed
uc
tio
n i
n e
nd
og
en
ou
s P
P4
cDNA library transfer - selection of apoptosis resistant cells
Insert identified: C-terminal catalytic subunit of PP4 – induces apoptosis resistance ( PP4 breakdown)
Protein Phosphatase 4: an inducer of apoptosis!
W7.2 + Dex W7.2/4n10 + Dex
PP4-Cat.
Vector100
0
20
60
40
80
120
Dex (60nM)
γ(1000cGy)
Dex (60nM)
γ(1000cGy)
UV(20J/m2)
UV(20J/m2)
Nu
mb
er
of
co
lon
ies
W7.2 cells
70
60
50
40
30
20
10
0 100 200
No. of coloniesafter Dex treatment
% r
ed
uc
tio
n i
n e
nd
og
en
ou
s P
P4
Vector0
50
100
150
200
PP4-Cat.
CEM-C7 cells
Nu
mb
er
of
co
lon
ies
cDNA library transfer - selection of apoptosis resistant cells
Insert identified: C-terminal catalytic subunit of PP4 – induces apoptosis resistance ( PP4 breakdown)
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)
1A II III IV V VI VII VIII
AAA
Provirus
RARα:Mutants resistant to RA only.
HL-60 cells HL-60 cells + Retinoic acid PAGER D cells + Retinoic acid
Insertional mutagenesis in myeloid cells (HL-60 differentiation)
1A II III IV V VI VII VIII
AAA
Provirus
RARα:Mutants resistant to RA only.
MPSV retroviral insertional mutagenesis of HL-60 cells: mutants resistant to RA, DMSO and Vit. D3
AT
G
11 4111
AT
G
c-myb:Mutants resistant to RA, DMSO, Vit. D3
HL-60 cells HL-60 cells + Retinoic acid PAGER D cells + Retinoic acid
0
500
1000
1500
2000
2500
IFN-γ TNF-α IL-2 IL-10 1L-6 IL-4
pg
cyto
kin
e /
10^
5 c
ell
s
Unstimulated
Unmodified AML
Lv.IL-2.B7 AML
The profile of cytokine secretion by the in vitro stimulated T cells
(Cytokine Bead Array – CBA)
• The in vitro stimulated T cells have a predominantly Th1 phenotype
n=3
IFN
IL-4
TNF-
IL-2
IL-6IL-10
IFN
IL-4
TNF-
IL-2
IL-6IL-10
IFN
IL-4
TNF-
IL-2
IL-6IL-10
VUD and sibling RIC transplants
VUD n = 56Sibling n = 31
0 500 1000 1500 20000
50
100
days from transplant
Per
cen
t S
urv
iva
l
0 500 1000 1500 2000
100
0
50
days from transplant
%Overall Survival
Relapse
Ho et al (2004) Blood 104: 1616-1623.
Endogenously biotinylated retro- and lenti-virus vectors:
Vector concentration
Attachment of targeting ligands
Paramagnetic labelling and concentration of the vector/s
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.
– 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 GCV-P; 5-fluorocytosine 5-flurouracil)
– availability of anti-retroviral drugs
No viraemia and clearance of the infected cells in mice and monkeys
Replicating MLV retrovirus as a cancer therapeutic
HSV-tk CD
(non-toxic) (non-toxic) (toxic) (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 DNAGenomic DNA sequence
regulatory gene
LTR LTRSelectable marker
Insertional inactivation / activation(gain or loss of function)
LTR LTR
cDNA expression cloning (gain or loss of function)
LTR LTR
cDNA library
Pseudorandom provirus integration into the host cell
genome
Oncolytic virus therapy – problems:
– robust immune response:rapid clearance of virus
– Inadequate targeting/specificity: “even a brick can kill tumour cells”