Transcript gC-eGFP - International Consortium Of Gene Therapy

Joseph C. Glorioso, PhD
University of Pittsburgh
School of Medicine
Applications of HSV Vectors
Therapeutic Agents
Gene Tools
1. Replicating Oncolytic Vectors
(GBM, liver, and bladder cancers)
2.
2.
(PNS: chronic pain, neuropathy:
CNS: Huntington’s, epilepsy)
3.
(Cancer Biology,
normal development)
1) High payload capacity and efficiency of transgene expression
2) Persistence in non-dividing cells without integration
3) Proven safety in patient trials
Non-Replicating Vector Engineering and
Therapeutic Gene Delivery
To the Nervous System
• Design of Non-Cytotoxic, Non-Replicating HSV Platform
• Localized or Targeted Delivery Methods
• Persistence as latent-like, non-integrated virus genome
without the possibility of virus reactivation
• Capable of long-term, regulated therapeutic
gene expression using Vector Retargeting, Specific
Promoter-Enhancers and/or Cellular microRNA
Essential: delete one or more to create replication defective vectors
US7
US8
US9
US10
US11
US12
US1
US2
US3
US4
US5
UL55
UL56
LAT
ICP0
ICP34.5
UL50
UL51
UL43
UL44
UL45
UL46
UL47
UL39
UL40
UL41
UL23
UL24
UL20
UL21
UL16
UL13
UL10
UL11
UL2
UL3
UL4
GLYCOPROTEIN
SPIKE
ICP4
UL
US6
ICP4
UL52
UL53
UL54
UL48
UL49
UL42
UL25
UL26
UL27
UL28
UL29
UL30
UL31
UL32
UL33
UL34
UL35
UL36
UL37
UL38
UL22
UL17
UL18
UL19
UL14
UL15
UL12
UL5
UL6
UL7
UL8
UL9
UL1
ICP34,5
ICP0
LAT
HSV
DNA
CAPSID
TEGUMENT ENVELOPE
Accessory: delete one or more to create oncolytic vectors
US
HSV-1 Genome(152 Kb)
84 genes-more than 100 Functions
ab
IE
E
ICP34.5
UL26.5
UL47
UL27
UL28
UL2 UL4
UL29
UL30
oriL
ca
LAT
LAT UL1 UL3 UL5
ICP0
UL26
L1
L2
b‘ a‘ c‘
UL7
UL6
UL9 UL11 UL13
UL8
UL36
UL38
UL31 UL33 UL35
UL48 UL49a UL51 UL53 UL55
LAT
UL18 UL20/UL20.5 UL22 UL24
UL10 UL12 UL14 UL17
UL12.5
UL16
UL32 UL34
ICP27
UL49 UL50 UL52 UL54 UL56
UL15
ICP34.5
ICP0
ICP4
UL19
VP5
UL40
UL21
UL23 UL25
tk
UL42 UL44 UL46
UL37
UL39 UL41 UL43 UL45
vhs
UL43.5
ICP22
US1
US8.5 US11
US1.5
US3 US5 US7 US9 oriS
oriS US2 US4 US6 US8
US12 ICP4
gD
US10 ICP47
Viral entry to cell
Vector Design for
PNS Applications
Viral DNA
transported to
nucleus
Cascade of Viral
Gene expression
VP16
transported to
nucleus
Immediate early
gene expression
Essential
in vitro
ICP4
ICP27
ICP0
ICP22
ICP47
Nonessential
in vitro
Immune evasion
Early gene
expression
Viral DNA replication
Late gene
expression
Virion synthesis
Essential HSV Gene Deletion Coupled with Gene Complementation
in Engineered Cell Lines Permits Vector Designs with Few or
No Expressed Viral Functions
ICP22
ICP4
IE
-+
VP
16
ICP0
-
+
-+
-
E
Mutant
HSV-1
L
Requires Complementation
Of ICP4/27 Using Engineered
Engineered Cells
ICP27
ICP27
No Viral
replication
ICP4
D-JOINT
ICP4
WT HSV INFECTION
DEFECTIVE HSV VECTOR INFECTION
Latent-like
x Lytic
rolling strand
DNA synthesis
a (IE)
b (E)
LAT mRNA
g (L)
2.6 kb stable
intron
x
Reactivation
CMV-EGFP IF
Treatment of Glioblastome
Multiforme with Oncolytic HSV
vectors
ONCOLYTIC VIRAL THERAPY for GBM
Virus attaches to &
Infects tumor cell
Tumor cell is lysed.
New virus particles released
Virus De-envelope;
DNA relication;
Viral protein synthesis begin
New viruses produced
Oncolytic Viral Therapy of GBM
What doesn’t work: Oncolytic HSV vectors rapidly kill GBM tumor cells in
culture and destroy tumors in numerous preclinical models, but has not
proven to be effective in patients.
Reasons include: (i) poor virus growth related to poor infectivity or
subsequent replication, and is an attribute of the attenuating mutations,
(ii) limited virus spread, (iii) innate immune responses to the vector and
(iv) tumor cell infiltration of normal brain.
Solutions: Tumor targeted vector that retains the full complement of
viral functions including those that block internal cellular defense
mechanisms and whose replication is limited to tumor cells. This vector
can be armed with genes that promote virus spread within the tumor,
prevent tumor cell migration and induce anti-tumor cellular responses.
HSV Vector Targeting
•
Direct Injection: intratumoral
• Transduction: modification of envelope for targeted
receptor recognition
• Transcription: promoter/enhancer selection or cellular
microRNAs that allow oncolytic virus replication in
tumor cells but not in normal tissue.
HSV Receptors and Ligands
• Glycoprotein D: Nectin-1, Nectin-2, other Nectins?, HVEM (TNF
receptor family), 3-O-sulfated HS (3-OS HS)
• gH/gL: integrins (αvβ3), Toll-like receptor 2 (TRL2), other
unknown
• gB (class III fusion protein): heparan sulfate (HS), paired
immunoglobulin-like type 2 receptor alpha (PILRa), non-muscle
myosin heavy chain IIA (NMHC-IIA), type II cell surface
membrane protein (B5), Toll-like receptor 2 (TRL2)
• gC: HS, complement component (C3b)
• gE/gI: IgFc
Cell membrane
gB
Nectin-1
HVEM
gH/gL
gD
Viral envelope
Fusion machinery of HSV. The minimal set of proteins required for HSV membrane
fusion is depicted to scale. gB is structurally conserved across all herpesviruses, while
gH/gL is more variable. gD is the accessory fusion protein required for membrane fusion
in HSV. gD determines cell tropism by binding cellular receptors HVEM or Nectin-1. All
proteins are colored according to their structural regions, as defined previously for HSV
gD, HSV gH/gL, HSV gB, HVEM and Nectin-1.
Eisenberg et al. Viruses 2012, 4, 800-832
Receptor-restricted HSV-1 mutant viruses
A-
C-
100 genomes/cell; 8 hpi; VP16 staining
C-
Retargeted entry is inefficient
HVEM-selective HSV
Nectin-1 ABLATED
Selection of enhanced entry variants
Amplification
Re-selection
J1.1-2 cells transduced with gDbinding impaired Nectin-1
J1.1-2 cells transduced with
HVEM
Rate of Entry into Vero Cells
J/ EGFR
100
No. of plaques
K-NTscEGFR
KOS
50
K-scEGFR
0
0
10
20
30
Time (min)
D285N/A549T
40
50
60
N/T gB mutations create a” metastable form” of gB that increases entry
Pre-fusion
Post-fusion
Proposed model of
pre-fusion gB
549
Front view
285
549
top view
549
549
Atanasiu, JV 2013, Aug 14
Entry assay (anti-ICP4 at 6 hpi)
0.3
3
30
300 (MOI)
KOS
K-gB:N/T
K-gB:668N
K-gH:K/V
K-gB:N/T
-gH:K/V
K-gB:668N
-gH:K/V
gH:K/V Further Enhances Virus Entry into CHO-K1 Cells
gH:K/V alone mutations have limited effect on rate of entry
Rate of Entry Assay (Vero cells)
120
100
Entry (%)
A
80
37oC
KOS
K-gB:NT
K-gH:KV
K-gB:NT-gH:KV
60
40
20
0
0
10
B
20
30
40
50
60
Time (min)
Entry (%)
120
30oC
100
KOS
K-gB:NT
K-gH:KV
K-gB:NT-gH:KV
80
60
40
20
0
0
10
20
30
40
Time (min)
50
60
KV mutations enhance plaque size
KOS
K-gB:668N
K-gB:N/T
K-gH:K/V
K-gB:668N-gH:K/V
K-gB:N/T-gH:K/V
gH:KV mutations but not gB:N/T mutations
promote virus spread to receptor deficient cells
gH:KV mutations allow virus egress from receptor deficient cells
Global and local effects of the gH:KV mutations
A
gH:wt
gH:KV
C
H1
H2
B
gH:wt
gH:KV
N753/K75
3 H3
Flap:R717_D726
A778/V778
Flap:R717_D726
Flap:E707_K716
0
20
40
60
Time (ns)
80
100
EGFR Selectivity
a-ICP4 @
T40
a-ICP4 @
T21
Safety
NT at 5x10^7
KOS at 10^2
Vector biodistribution
1.E+07
Genome Copies/100 ng DNA
Tumor volume (mm3)
Tumor killing
PBS
sc-NT
0
10
20
Days after virus injection
30
1.E+06
1.E+05
1.E+04
1.E+03
1.E+02
1.E+01
1.E+00
KOS
KOS
EGFR retargeted vector
EGFR retargeted vector
EGFR Expression on Human GBM Primary Cells
Tumor Marker Heterogenicity will require oHSV
retargeting to multiple tumor cell markers
T2-weighted brain MRI images of nude mice bearing GBM 30
Pre
d3
d10
d15
(4 d post GBM)
pmlantation
PBS
K-NT
scEGFR
Retargeted HSV treatment of a nude mouse model
Of primary human glioblastoma
K-NTscEGFR
PBS
Animal Survival
100%
75%
50%
25%
0%
0
10
Virus
108 gc
20
30
40
50
60
70
Days Post GBM30 Implantation
80
90
HSV vectors can be engineered to achieve high level of
infectivity and replication in tumor cells.
• How can we improve the safety of these new
generation of OV?
Control of virus growth based on tumor-specific changes in
metabolism particularly those essential to the tumor
phenotype (e.g. miRNAs).
Differential miRNA copy number per cell
between Normal Brain and GBM Tumor
lines
hsa-mir
Normal
Brain
CNS
Tumor
Lines
Fold
Change
21
124
809
8012
14361
1
17.7 
8012 
128
137
122
1495
0
66
122 
22.6 
A. Gaur et al., Cancer Res 2007
ICP27
BAC
ICP4
ICP4
JOINT
Neurons
Glioma cells
HSV replication
mir-124
mir-128
mir-137
mir-21
mir-17
mir-10
Replication of ICP4-T124 virus is blocked by mir-124
ICP27
BAC
D ICP4
D JOINT
107
ICP4-T124
106
104
103
102
ICP4-wt
ICP4-T124
101
Gli68
Gli82
pCDH-eGFP
pCDH-mir124
no pCDH
pCDH-eGFP
pCDH-mir124
100
no pCDH
pfu/ml
105
MOI=5
oHSV titration on U2OS cells of
supernatants collected at 5 dpi.
Toxicity testing in immunodeficient mice
BAC
KG
ICP27
gC-eGFP
BAC
ICP4-miR124t
ICP4
D JOINT
ICP27
D ICP4
gC-eGFP
D JOINT
BOX
1
2
Virus
KG
ICP4-miR124t
gc injected
4.8 x 109
4.8 x 109
Volume Injected
4μl
4μl
Mice
4
4
ICP4-T124
• 4μl containing 4.8 x 109 gc of either KG or ICP4-miR124 were administered in
3 weeks/old BALB/c mice by intracranial stereotactic injection into the right
temporal lobe (cortex) for a total of 4 mice per virus.
• three of the four KG-injected animals had died by 5dpi.
Toxicity testing in immunodeficient mice
KG
19
Weight (grams)
21
19
Weight (grams)
ICP4-miR124t
21
17
15
17
15
13
13
11
11
9
9
0
5
10
15
20
25
Days Post Virus Injection
N
R
L
B
30
35
0
5
10
15
20
25
30
Days Post Virus Injection
N
R
L
35
B
KG virus was extremely pathogenic while infection with the miR
controlled vector proved to be benign.
ICP4t124 virus is safe even at 4.8 x 109 genome
copies
Safety Study
qPCR of viral genomes present in the brain
ICP4T124
80
60
40
Control
20
0
0
7
14
21
28
Days Post Injection
35
% of Injected genome copies
% of mice
100
200%
150%
100%
25%
20%
15%
10%
5%
0%
KG-NT
KG-NT4T124
5
14
21
33
Days Post Injection
miR-124 HSV treatment of a nude mouse model
Of primary human glioblastoma
Percent survival
100
PBS
9C3 - Ctrl
10A16 - T124
80
60
40
20
0
0
15
30
45
60
75
90
Days Post GBM30 Injection
Virus
Inje ction
gD-scFvEGFR
BAC gB N/T
ICP27
D ICP4
gC-eGFP
D JOINT
ICP4-T124
Summary and Future Directions
1. HSV can be used for treatment of nervous system diseases that include
targeted delivery to sensory neurons and cancer.
2. Pain applications will be further enhanced by drug-activated specific
silencing methods and shRNA control of channel activation and expression.
3. Cancer therapies will be enhanced by (i) further development of methods for
systemic vector delivery and (ii) by arming vectors with genes that enhance
vector spread, suppress tumor cell migration and metastasis and activate
specific immune activities.
U Pittsburgh
Senior Scientists
,
U Michigan
U Ferrara
U Fukuoka
U Seoul
Fellows and Students
Cell lines,
Viruses and
Antibodies
U Milan