Gene Therapy Approaches to Infectious Disease Treatment and Prevention

Alexander Pereboev

GTC

Gene TherapyCenter at UAB

GTC

Gene TherapyCenter at UAB

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 Approaches to Infectious Disease Treatment and Prevention

•Dendritic cells (DC) are the most potent

professional antigen-presenting cells.

Dendritic Cells

CD8

MHCClass I

CD4

MHCClass II

T helperCTL precursor

DC picks up antigen

Skin

In transit to the lymph node, DC processes Ag and matures

Peripheral lymph node

DC presents Ag to T cells

Activated T cells perform effector functions

DC As Antigen Presenting Cells

Nucleus

CD8

mRNA

Protein

ERGoldgi

MHCClass I

Proteasome

CTL precursor

Ad Transduced DC Stimulates CTL

Normal pathway:Endogenous proteins are processed

as MHC Class I peptides

Nucleus

CD4

MHCClass II

Endo/Lysosome

Nucleus

CD4

mRNA

Protein MHCClass II

T helper T helper

Endosome targeting sequence at C-terminus

Ad Transduced DC Stimulates T Helpers

Normal pathway:Exogenous antigens are processed

as MHC Class II peptides

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

•Genes are delivered by vectors, both non-viral and viral.

•Adenovirus (Ad) is the most commonly used vector for gene therapy:

Adenovirus As 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.

Both mouse and human DC are deficient in Ad receptor (CAR) expression.

Means to target Ad to DC are needed.

Untargeted Ad Ad targeted to DC

No transduction Transduced DC

Ad Vectors to Transduce DC

Targeting Adenovirus for Gene Therapy

Molecular adaptorsDirect genetic

incorporation of targeting ligands into

Ad capsid

Ligand fused to CAR

Chemical AB conjugatePeptides scFv

Ad Fiber Protein Structure and Function

Ad fiber knob is a homotrimer responsible for Ad binding to its receptor – CAR. Shaft Knob

Ad fiber protein

Ad fiber knob Cell receptor of interest

Molecular adapter

Molecular Adapter to Target Ad

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.

Ad Fiber Protein Structure and Function

CAR

X-ray studies reveal that three CAR molecules can bind one trimeric fiber knob

CAR

CAR

Ad fiber knob Cell receptor of interest

Trimeric molecular adapter

Trimeric Adaptor Is More Efficient

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

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

D1 D2 H H H HHH

D1 D2 H H H HHH

D1 D2 H H H HHH

CAR Fibritin mouse CD40 ligand

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.

B UB B UB B UB B UB

Ant

i-C

AR

Ant

i-F

ibri

tin

Ant

i-6H

is

Ant

i-C

D40

L

CFm40L – Adapter to Target Mouse DC

CFm40L was able to bind both Ad fiber knob and mouse CD40 in ELISA

CFm40L ELISA

1.6

1.2

0.8

0.4

0.0O

D4

90

Ad5 knob

mCD40

No AG

3.9 7.8 15.6 31.3 62.5 125.0 250.0 500.0

CFm40L (ng/well)

CFm40L Enhances DC Transduction with Ad

CFm40L dramatically augments of both mouse and human DC

Untargeted AdLuciferase reporter

Luciferase assay

48h

CD40-targeted AdLuciferase reporter

Mouse or human DC

1E+7

1E+6

1E+5

1E+4

1E+3

1E+2

1E+1

1E+0

Lu

cife

rase

ac

tiv

ity

(RL

U)

Mouse DC Human DC

1E+7

1E+6

1E+5

1E+4

1E+3

1E+2

1E+1

1E+0

U/T LPS Ad only CFm40L Ad + CFm40L

120

100

80

60

40

20

0

IL-1

2 C

on

cen

trat

ion

(p

g/m

l)

Targeted DC Transduction Activates DC

Untargeted Ad

IL-12 ELISA

48h

CD40-targeted Ad

Murine DC Targeted Ad and CFm40L alone induce IL-12 secretion.

This is an indication of DC activation.

CD4+ response CD8+ response

0

200

400

600

800

1000

1200

pg

/ml

Ad only Ad plus CFm40L

0

40

80

120

160

200

pg

/ml

Ad only Ad plus CFm40L

Targeted Ad Elicits Immune Response in vivo

Untargeted AdModel Ag

CD40-targeted AdModel Ag

or

Targeted Ad stimulates CTL and T helper responsein vivo

CD4+ assay

14 days

Lymphocytes

CD8+ assay

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

Gene Therapy of Infectious Disease. WNV Vaccine

5’Structural Nonstructural

3’

C prM 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

Vaccines remain the front line of defense for West Nile virus encephalitis

Gene Therapy of Infectious Disease. WNV Vaccine

E NS1

E NS1

CMVLITR promoter poly A Adenoviral DNA RITR

Encapsidation signal

E-NS1 fusion cDNA

HYPOTHESIS: An Ad vector encoding WNV envelope and NS1 protein will

be an effective vaccine against the disease.

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 Antibody Gene Delivery

S SS S

SS

SSS

S

SS

S SS S

SS

SSS

S

SS

S

SS S

S SS

S

VH

VL

CH1CH2 CH3

Sig COOHNH2

IgG heavy chainVariable Constant

VH JH CH AAA 3’5’ Sig

IgG heavy chain mRNA

C C C C C C C C C C CSig COOHNH2

IgG light chainVariable Constant

VL JL CL AAA 3’5’ Sig

IgG light chain mRNA

C C C

Structure of Human IgG

Immunoglobulin is the product of two genes

CL

hinge

C C

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

SS

S

SS

S

SS

S

S

VH

VL

Fc

Recombinant Antibodies. scFv

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.

VH VL

NH2

COOH

27-29 kDa

VH VL

NH2

COOH

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

Recombinant Antibodies. Minibodies

VH VL

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~ 80 kDa (dimer)

SS

CH3

SS

SS

SS

VL VH

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)

SS

SS

SS

SS

S S

VH VL VL VH

CH2

CH3

hinge

Protein A binding site

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 6,400 128,000

mAb 9E2 1,024,000 256,000 64,000 512,000 64,000 512,000 1,024,000

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

VH VL

NH2

COOH

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

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Un

dil

ute

d

1/2

1/4

1/8

1/1

6

1/3

2

1/6

4

1/1

28

200 ng/well WNE

No Ag

OD

49

0

scFv 9E2 dilution

LITR

Encapsidation signal

Fc9E2 fusion ORF

HYPOTHESIS: adenovirus encoding Fc9E2 will be an efficient vector to deliver neutralizing Ab gene in vivo

Fc VH VLSig

Stop

CMVpromoter RITRPoly A

Ad DNA

Ad Vector Encoding Fc9E2

0.0

0.1

0.2

0.3

0.4

25

6.0

12

8.0

64

.00

32

.00

16

.00

8.0

00

4.0

00

2.0

00

1.0

00

0.5

00

0.2

50

0.1

25

Fc9E2 concentration (μg/ml)

OD

40

5

200 ng/well WNE

No Ag

SS

SS

SS

SS

S S

VH VL VL VH

CH2

CH3

hinge

WNV-Neutralizing Fc9E2?

Ad encoding Fc9E2 has been generated

The recombinant antibody demonstrated strong binding to WNE

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