Gene Therapy Approaches to Infectious Disease Treatment and Prevention Alexander Pereboev

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

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 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

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

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

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 Molecular adaptors Chemical AB conjugate Ligand fused to CAR Peptides scFv

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

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.

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

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. 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

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

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

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

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

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

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’ 3’ Structural Nonstructural 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

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.

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

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

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

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

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)

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)

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

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

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

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

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