1. Gene Therapy Approaches to Infectious Disease Treatment and Prevention
Alexander Pereboev

2. Gene therapy approaches can be used for immunotherapy applications
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

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

4. DC As Antigen Presenting Cells
Activated T cells perform effector functions
Skin
Peripheral
lymph node
MHC
Class I
MHC
Class II
DC picks up antigen
CD8
CD4
CTL precursor
T helper
In transit to the lymph node,
DC processes Ag and matures
DC presents Ag to T cells

5. 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

6. 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

7. 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

8. 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.

9. 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

10. 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

11. Ad Fiber Protein Structure and Function
Ad fiber knob is a homotrimer responsible for Ad binding to its receptor – CAR.
Shaft Knob

12. 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.

13. Ad Fiber Protein Structure and Function
X-ray studies reveal that three CAR molecules can bind one trimeric fiber knob
CAR
CAR
CAR

14. 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.

15. 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

16. 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. H D1 D2 H D1 D2 H H H H H H
CAR Fibritin mouse
CD40 ligand
B UB B UB B UB B UB
Anti-CAR
Anti-6His
Anti-Fibritin
Anti-CD40L

17. CFm40L – Adapter to Target Mouse DC
1.6
1.2
0.8
0.4
0.0
Ad5 knob
mCD40
No AG
CFm40L ELISA
OD490
CFm40L (ng/well)
CFm40L was able to bind both Ad fiber knob and mouse CD40 in ELISA

18. DC Transduction with Ad
CFm40L Enhances
DC Transduction with Ad
Untargeted Ad
Luciferase reporter
CD40-targeted Ad
Luciferase reporter
1E+7
1E+6
1E+5
1E+4
1E+3
1E+2
1E+1
1E+0
Luciferase activity (RLU)
Mouse DC Human DC
Mouse or human DC
48h
CFm40L dramatically augments of both mouse and human DC
Luciferase assay

19. Targeted DC Transduction Activates DC
Untargeted Ad
CD40-targeted Ad
U/T LPS Ad only CFm40L Ad +
CFm40L
120
100 80 60 40 20
IL-12 Concentration (pg/ml)
Murine DC
Targeted Ad and CFm40L alone
induce IL-12 secretion.
This is an indication of DC activation.
48h
IL-12 ELISA

20. Targeted Ad Elicits Immune Response in vivo
Untargeted Ad
Model Ag
CD40-targeted Ad
Model Ag
CD4+ response CD8+ response
200
1200
1000
160 or
800
120
pg/ml
pg/ml
600 80
400 40
200
14 days
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

21. 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

22. 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 A 2B A 4B
Pr M
Envelope protein shown to induce strong protective humoral response
Nonstructural protein 1 shown to induce strong protective CTL response

23. 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.

24. 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

25. Immunoglobulin is the product of two genes
Structure of Human IgG VL JL CL
AAA 3’ 5’
Sig
IgG light chain mRNA VH JH CH
AAA 3’ 5’
Sig
IgG heavy chain mRNA
Sig
COOH
NH2
IgG light chain
Variable Constant
Sig
COOH
NH2
IgG heavy chain
Variable Constant
C C
C C C
C C
C C C C C C C C C VH
CH1 S S
CH2
CH3 S S S
hinge VL S S 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

26. Recombinant Antibodies. scFv
VH VL
NH2
COOH VL S S S S S S S S VH
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

27. 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
VH VL
NH2
COOH
27-29 kDa

28. 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
VH VL
VL VH S S S
CH3
~ 80 kDa (dimer)

29. 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 S
VH VL
VL VH
CH2
CH3
hinge
Protein A
binding site
~ 110 kDa (dimer)

30. 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 AB
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
mAb 9E2
1,024,000
256,000
512,000

31. WNV-Neutralizing scFv 9E2
VH VL
NH2
COOH
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Undiluted
1/2
1/4
1/8
1/16
1/32
1/64
1/128
200 ng/well WNE
No Ag
OD490
scFv 9E2 dilution
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

32. Ad Vector Encoding Fc9E2 Fc9E2 fusion ORF CMV promoter LITR Poly A
RITR
Sig Fc VH VL
Stop
Encapsidation signal
Ad DNA
HYPOTHESIS: adenovirus encoding Fc9E2 will be an efficient vector to deliver neutralizing Ab gene in vivo

33. WNV-Neutralizing Fc9E2? Ad encoding Fc9E2 has been generated
VH VL
VL VH
CH2
CH3
hinge
0.0
0.1
0.2
0.3
0.4
256.0
128.0
64.00
32.00
16.00
8.000
4.000
2.000
1.000
0.500
0.250
0.125
Fc9E2 concentration (μg/ml)
OD405
200 ng/well WNE
No Ag
Ad encoding Fc9E2 has been generated
The recombinant antibody demonstrated strong binding to WNE

34. 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