Gene%20Therapy%20Approaches%20to%20Infectious%20Disease%20Treatment%20and%20Prevention

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Gene Therapy Approaches to Infectious Disease
Treatment and Prevention
Alexander Pereboev
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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
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Dendritic Cells

• Dendritic cells (DC) are the most potent
  professional antigen-presenting cells.

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DC As Antigen Presenting Cells
Activated T cells perform effector functions
Skin
Peripheral lymph node
MHC Class II
MHC Class I
DC picks up antigen
CD4
CD8
T helper
CTL precursor
In transit to the lymph node, DC processes Ag
and matures
DC presents Ag to T cells
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Ad Transduced DC Stimulates CTL
Nucleus
mRNA
Protein
MHC Class I
Goldgi
ER
Proteasome
CD8
CTL precursor
Normal pathway Endogenous proteins are processed
as MHC Class I peptides
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Ad Transduced DC Stimulates T Helpers
Nucleus
Nucleus
mRNA
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
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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

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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.

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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
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Targeting Adenovirus for Gene Therapy
Direct genetic incorporation of targeting ligands
into Ad capsid
Molecular adaptors
Chemical AB conjugate
Ligand fused to CAR
Peptides
scFv
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Ad Fiber Protein Structure and Function
Ad fiber protein
Ad fiber knob is a homotrimer responsible for Ad
binding to its receptor CAR.
Shaft Knob
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Molecular Adapter to Target Ad
Ad fiber knob
Cell receptor of interest
Molecular adapter
Molecular adapter protein is a bi-specific
molecule able to bind both Ad capsid protein and
a cellular receptor of interest. The cell
become susceptible to Ad transduction.
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Ad Fiber Protein Structure and Function
X-ray studies reveal that three CAR molecules can
bind one trimeric fiber knob
CAR
CAR
CAR
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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.
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Search for DC Marker
CD40 is a regulatory molecule specifically
expressed on DC. 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.
CD40 ligand
CD40
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CFm40L Adapter to Target Mouse DC
New adapter protein consists of the ectodomain of
CAR fused to mouse CD40 ligand via a
trimerization motif fibritin. The fusion
protein has been produced in a stable cell line.
Western blot confirmed the presence of all
three functional parts of the adapter.
D1
D2
D1
D2
D1
D2
H
H
H
H
H
H
CAR Fibritin mouse
CD40 ligand
Anti-6His
Anti-CAR
Anti-Fibritin
Anti-CD40L
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CFm40L Adapter to Target Mouse DC
1.6 1.2 0.8 0.4 0.0
Ad5 knob mCD40 No AG
CFm40L ELISA
OD490
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
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CFm40L Enhances DC Transduction with Ad
Untargeted Ad Luciferase reporter
CD40-targeted Ad Luciferase reporter
Mouse or human DC
48h
CFm40L dramatically augments of both mouse and
human DC
Luciferase assay
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Targeted DC Transduction Activates DC
Untargeted Ad
CD40-targeted Ad
Murine DC
Targeted Ad and CFm40L alone induce IL-12
secretion. This is an indication of DC
activation.
48h
IL-12 ELISA
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Targeted Ad Elicits Immune Response in vivo
Untargeted Ad Model Ag
CD40-targeted Ad Model Ag
CD4 response CD8 response
1200
200
1000
160
or
800
120
pg/ml
pg/ml
600
80
400
40
200
14 days
0
0
Ad only Ad plus CFm40L
Ad only Ad plus CFm40L
Lymphocytes
Targeted Ad stimulates CTL and T helper
response in vivo
CD4 assay
CD8 assay
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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
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Gene Therapy of Infectious Disease. WNV Vaccine
Vaccines remain the front line of defense for
West Nile virus encephalitis
5
3
Structural
Nonstructural
C prM E 1
2A 2B 3 4A 4B 5
Envelope protein shown to induce strong
protective humoral response
Nonstructural protein 1 shown to induce strong
protective CTL response
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Gene Therapy of Infectious Disease. WNV Vaccine
E NS1
CMV LITR promoter
poly A Adenoviral DNA
RITR
E NS1
Encapsidation signal
E-NS1 fusion cDNA
HYPOTHESIS An Ad vector encoding WNV envelope
and NS1 protein will be an effective vaccine
against the disease.
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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
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Structure of Human IgG
C C
C C
C C C C C C C C C
C C C
VH
CH1
S
CH2
CH3
S
S
S
hinge
S
S
S
VL
S
S
S
S
S
S
S
S
S
CL
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
Immunoglobulin is the product of two genes
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Recombinant Antibodies. scFv
VL
S
S
S
S
S
S
VH
S
S
27-29 kDa
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
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.

S
S
S
S
Fc
S
S
S
S
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Recombinant Antibodies. scFv

• scFv is the minimal portion of an antibody
  retaining antigen-binding properties.
• Advantages
• scFv is encoded by single gene
• Small size. Better tissue penetration
• Disadvantages
• Small size. Rapid clearance
• Purification tag needed
• Lack of effector functions

27-29 kDa
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Recombinant Antibodies. Minibodies

• A minibody is an scFv fused to the CH3
• domain plus hinge.
• Advantages
• Minibody is encoded by single gene
• Good tissue penetration
• Longer half-life
• Bivalent. Higher avidity
• Disadvantages
• Purification tag needed
• Lack of effector functions

CH3
80 kDa (dimer)
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Recombinant Antibodies. Fc-scFv Fusions

• An Fc-scFv is an scFv fused to an Fc portion of
  IgG.
• Advantages
• Fc-scFv is encoded by single gene
• Half-life comparable to whole IgG
• Bivalent. Higher avidity
• Protein A binding site present. Convenient
  purification
• Fc provides effecter functions

110 kDa (dimer)
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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) Titers of ABs in neutralization test (reverse values) Titers of ABs in neutralization test (reverse values) Titers of ABs in neutralization test (reverse values) Titers of ABs in neutralization test (reverse values) Titers of ABs in neutralization test (reverse values) Titers of ABs in neutralization test (reverse values)
WNV strains WNV strains WNV strains WNV strains WNV strains WNV strains WNV strains
AB Vlg-27889 Vlg-27924 Hp-94 A-1640 Tur-2914 ?-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
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WNV-Neutralizing scFv 9E2
scFv 9E2 has been generated from cDNA synthesized
from 9E2 hybrydoma mRNA In ELISA scFv 9E2 showed
specific binding to the C-terminal fragment of
WNV E protein Importantly, scFv 9E2 demonstrated
some neutralizing activity against WNV isolates
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Ad Vector Encoding Fc9E2
Fc9E2 fusion ORF
CMV promoter
Poly A
LITR
RITR
Fc
VH
VL
Sig
Stop
Encapsidation signal
Ad DNA
HYPOTHESIS adenovirus encoding Fc9E2 will be an
efficient vector to deliver neutralizing Ab gene
in vivo
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WNV-Neutralizing Fc9E2?
Ad encoding Fc9E2 has been generated The
recombinant antibody demonstrated strong binding
to WNE
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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