Gene Therapy Approaches to Infectious Disease Treatment and

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Transcript Gene Therapy Approaches to Infectious Disease Treatment and

Gene Therapy Approaches to Infectious
Disease Treatment and Prevention
Alexander Pereboev
GTC
Gene
Therapy
Center
at UAB
Gene Therapy Approaches to Infectious
Disease Treatment and Prevention
Gene therapy is the delivery of a gene or genetic
information into cells for the purpose of achieving a
therapeutic effect
Immunotherapy is a treatment that stimulates or modifies
the body's immune response: vaccination , antibody gene
delivery
Gene therapy approaches can be used for immunotherapy
applications
Dendritic Cells
•Dendritic cells (DC) are the most potent
professional antigen-presenting cells.
DC As Antigen Presenting Cells
Activated T cells perform
effector functions
Skin
DC picks up antigen
Peripheral
lymph node
MHC
Class I
MHC
Class II
CD8
CD4
CTL precursor
T helper
In transit to the lymph node,
DC processes Ag and matures
DC presents Ag to T cells
Ad Transduced DC Stimulates CTL
mRNA
Nucleus
Protein
Proteasome
ER
Goldgi
MHC
Class I
CD8
CTL precursor
Normal pathway:
Endogenous proteins are processed
as MHC Class I peptides
Ad Transduced DC Stimulates T Helpers
Nucleus
mRNA
Nucleus
Endo/Lysosome
MHC
Class II
Protein
MHC
Class II
CD4
CD4
T helper
T helper
Normal pathway:
Exogenous antigens are processed
as MHC Class II peptides
Endosome
targeting
sequence at
C-terminus
DC Based Immunotherapy
DC can be isolated from a patient and
loaded with antigen by:
-
Pulsing with peptides/proteins/tumor
cell lyzates;
-
Transfection with DNA/RNA;
-
Viral (including Ad) gene transfer.
Loaded DC are reintroduced back to the
patient
Adenovirus As Vector for Gene Therapy
•Genes are delivered by vectors, both non-viral and
viral.
•Adenovirus (Ad) is the most commonly used vector
for gene therapy:
- Ad5 has an outstanding efficacy of gene transfer in vivo;
- Ad infects both proliferating and differentiated cells;
- Ad grows to high titer;
- Large (up to 7.5 kb) foreign DNA fragments can be
incorporated into the Ad genome.
Ad Vectors to Transduce DC
Untargeted Ad
Ad targeted to DC
Both mouse and human DC are deficient
in Ad receptor (CAR) expression.
Means to target Ad to DC are needed.
No transduction
Transduced DC
Targeting Adenovirus for Gene Therapy
Direct genetic
incorporation of
targeting ligands into
Ad capsid
Peptides
Molecular adaptors
scFv
Chemical AB
conjugate
Ligand fused to
CAR
Ad Fiber Protein Structure and Function
Ad fiber protein
Shaft
Knob
Ad fiber knob is a homotrimer
responsible for Ad binding to its
receptor – CAR.
Molecular Adapter to Target Ad
Ad fiber knob
Cell receptor of interest
Molecular adapter
Molecular adapter protein is a bispecific molecule able to bind both
Ad capsid protein and a cellular
receptor of interest.
The cell become susceptible to Ad
transduction.
Ad Fiber Protein Structure and Function
CAR
CAR
CAR
X-ray studies reveal that three CAR
molecules can bind one trimeric
fiber knob
Trimeric Adaptor Is More Efficient
Ad fiber knob
Cell receptor of interest
Trimeric molecular adapter
Trimeric molecular adapter has
been shown experimentally to have
higher affinity to Ad.
It can potentially bind more cellular
receptor molecules.
Search for DC Marker
CD40 is a regulatory molecule specifically
expressed on DC.
CD40 ligand
CD40
Interaction of CD40 with its natural ligand
– CD40 ligand – causes DC maturation.
DC maturation during antigen processing is
essential to proper antigen presentation.
CD40 ligand is a homotrimer.
CFm40L – Adapter to Target Mouse DC
D1 D2 H H H H H H
New adapter protein consists of the
ectodomain of CAR fused to mouse CD40
ligand via a trimerization motif – fibritin.
D1 D2 H H H H H H
D1 D2 H H H H H H
CAR
Fibritin
B UB
B UB
B UB
mouse
CD40 ligand
B UB
The fusion protein has been produced in a
stable cell line.
Anti-CD40L
Anti-Fibritin
Anti-6His
Anti-CAR
Western blot confirmed the presence of all
three functional parts of the adapter.
CFm40L – Adapter to Target Mouse DC
1.6
Ad5 knob
mCD40
1.2
No AG
OD490
CFm40L ELISA
0.8
0.4
0.0
3.9
7.8
15.6 31.3 62.5 125.0 250.0 500.0
CFm40L (ng/well)
CFm40L was able to bind both Ad fiber
knob and mouse CD40 in ELISA
CFm40L Enhances
DC Transduction with Ad
Untargeted Ad
CD40-targeted Ad
Luciferase reporter Luciferase reporter
Luciferase activity (RLU)
Mouse DC
Human DC
1E+7
1E+7
1E+6
1E+6
1E+5
1E+5
1E+4
1E+4
1E+3
1E+3
1E+2
1E+2
1E+1
1E+1
1E+0
1E+0
Mouse or human DC
48h
Luciferase assay
CFm40L dramatically augments of both
mouse and human DC
Targeted DC Transduction Activates DC
CD40-targeted Ad
IL-12 Concentration (pg/ml)
Untargeted Ad
Murine DC
48h
IL-12 ELISA
120
100
80
60
40
20
0
U/T
LPS
Ad only CFm40L
Ad +
CFm40L
Targeted Ad and CFm40L alone
induce IL-12 secretion.
This is an indication of DC activation.
Targeted Ad Elicits Immune Response in vivo
Untargeted Ad
Model Ag
CD40-targeted Ad
Model Ag
200
CD4+ response
1200
CD8+ response
1000
160
or
800
pg/ml
pg/ml
120
600
80
400
40
200
14 days
0
Lymphocytes
CD4+ assay
CD8+ assay
Ad only
Ad plus
CFm40L
0
Ad only
Ad plus
CFm40L
Targeted Ad stimulates CTL and T helper response
in vivo
Gene Therapy of Infectious Disease.
WNV Vaccine
Gene therapy is the delivery of a gene or genetic
information into cells for the purpose of achieving a
therapeutic effect
Immunotherapy is a treatment that stimulates or modifies
the body's immune response: vaccination , antibody gene
delivery
Gene therapy approaches can be used for immunotherapy
applications
Gene Therapy of Infectious Disease.
WNV Vaccine
Vaccines remain the front line of defense for West Nile virus encephalitis
5’
C
Structural
prM
3’
Nonstructural
E
1
2A 2B
3
4A 4B
5
Pr M
Envelope protein shown to induce
strong protective humoral response
Nonstructural protein 1 shown to
induce strong protective CTL response
Gene Therapy of Infectious Disease.
WNV Vaccine
E
LITR
NS1
CMV
promoter
poly A
E
Encapsidation
signal
Adenoviral DNA
RITR
NS1
E-NS1 fusion
cDNA
HYPOTHESIS: An Ad vector encoding
WNV envelope and NS1 protein will be
an effective vaccine against the disease.
Gene Therapy of Infectious Disease.
WNV Antibody Gene Delivery
Gene therapy is the delivery of a gene or genetic
information into cells for the purpose of achieving a
therapeutic effect
Immunotherapy is a treatment that stimulates or modifies
the body's immune response: vaccination, antibody gene
delivery
Gene therapy approaches can be used for immunotherapy
applications
Structure of Human IgG
IgG light chain mRNA
5’
Sig
NH2 Sig
VL
JL
CL
IgG heavy chain mRNA
AAA 3’
5’
IgG light chain
Variable
Constant
C C
C C C COOH
Sig
VH
JH
CH
AAA 3’
IgG heavy chain
Variable
Constant
NH2 Sig
C
C
C C C C C C C C C
VH
CH1
S
CH2
S
S
S
S
S
VL
S
S
CH3
hinge
S
S
S S
S
S
S
S
S S
S
S
S
S
CL
S
S
S
S
S
S
S
S
S
S
Immunoglobulin is the product of two genes
COOH
Recombinant Antibodies. scFv
NH
VL
S
S
S
VH
VL
27-29 kDa
S
S
S
S
S
VH
S
COOH
S
S
S S
S
S
S S
S
S
S
S
S
S
S
S
S
S
S
2
S
S
S
S
Fc
Single chain antibody (scFv) is a construct where
two variable fragments are connected with a flexible
linker.
scFv is the minimal portion of an antibody retaining
antigen-binding properties.
Recombinant Antibodies. scFv
scFv is the minimal portion of an antibody retaining
antigen-binding properties.
NH
Advantages:
2
VH
VL
COOH
•scFv is encoded by single gene
•Small size. Better tissue penetration
Disadvantages:
27-29 kDa
•Small size. Rapid clearance
•Purification tag needed
•Lack of effector functions
Recombinant Antibodies. Minibodies
A minibody is an scFv fused to the CH3
domain plus hinge.
Advantages:
•Minibody is encoded by single gene
VL
S S
VL
S S
VH
VH
•Good tissue penetration
•Longer half-life
S
S
S
•Bivalent. Higher avidity
S
CH3
Disadvantages:
•Purification tag needed
~ 80 kDa (dimer)
•Lack of effector functions
Recombinant Antibodies. Fc-scFv Fusions
An Fc-scFv is an scFv fused to an Fc portion
of IgG.
VH
VL
VL
Advantages:
VH
S
S
S
hinge
S
•Fc-scFv is encoded by single gene
S
S
S
S
S
S
CH2
CH3
•Half-life comparable to whole IgG
Protein A
binding site
•Bivalent. Higher avidity
•Protein A binding site present. Convenient
purification
•Fc provides effecter functions
~ 110 kDa (dimer)
WNV-Neutralizing mAb 9E2
Hybridoma secreting antibody 9E2 has been generated. 9E2 demonstrated
specific binding to C-terminal portion of WNV envelope protein.
mAb 9E2 shows strong neutralizing activity against a variety of WNV strains
Titers of ABs in neutralization test (reverse values)
WNV strains
Vlg-27889
Vlg-27924
Hp-94
A-1640
Tur-2914
А-72
Eg-101
Immune
serum
128,000
64,000
400
32,000
100
6,400
128,000
mAb 9E2
1,024,000
256,000
64,000
512,000
64,000
512,000
1,024,000
AB
WNV-Neutralizing scFv 9E2
NH
2
3.5
VH
200 ng/well WNE
VL
3.0
No Ag
Importantly, scFv 9E2 demonstrated
some neutralizing activity against
WNV isolates
1.0
scFv 9E2 dilution
1/128
1/64
1/32
1/16
0.0
1/8
0.5
1/4
In ELISA scFv 9E2 showed specific
binding to the C-terminal fragment of
WNV E protein
1.5
1/2
scFv 9E2 has been generated from
cDNA synthesized from 9E2
hybrydoma mRNA
2.0
Undiluted
COOH
OD490
2.5
Ad Vector Encoding Fc9E2
LITR
Fc9E2 fusion ORF
CMV
promoter
Poly A
Sig
Fc
VH
RITR
VL
Stop
Encapsidation
signal
Ad DNA
HYPOTHESIS: adenovirus encoding
Fc9E2 will be an efficient vector to
deliver neutralizing Ab gene in vivo
WNV-Neutralizing Fc9E2?
VH
VL
VL
VH
200 ng/well WNE
0.4
No Ag
S
S
S
CH2
OD405
S
S
hinge
S
0.3
0.2
S
S
S
S
0.1
Fc9E2 concentration (μg/ml)
Ad encoding Fc9E2 has been
generated
The recombinant antibody
demonstrated strong binding to WNE
0.125
0.250
0.500
1.000
2.000
4.000
8.000
16.00
32.00
64.00
128.0
0.0
256.0
CH3
Conclusions
•Ad vectors can be efficiently targeted to DC using molecular
adaptors
•DC-targeted Ad gene therapy vectors encoding viral antigens
may elicit protective immunity
•Gene engineering techniques allow generation of functionally
active recombinant antibodies
•Ad gene therapy vectors may be efficient tool to rapidly induce
protective humoral immunity by delivering neutralizing antibody
genes