Title: Targeted Adenoviral Gene Therapy
1Targeted Adenoviral Gene Therapy
Shawn E. Lupold, Ph.D. Post Doctoral Fellow Brady
Urological Institute Johns Hopkins Hospital
2Project Goals
- Part I Identify Prostate Targeting Peptides
- Phage Displayed Peptide Libraries
- Peptide Characterization
- Affinity Maturation
- Part II Incorporate Peptides into Viral Capsid
- Vector Shuttle System
- Adenoviral Fiber Knob Cloning Vectors
- Evaluate Viral Production and Targeting
3Purified PSMA Protein
- Baculovirus expressed and secreted fusion
protein - NAALADase active (Km 16.1 nM, Vmax 2.1 mmol/mg
min) - Two affinity tags
- S-Tag S-protein
- Cellulose Binding Domain (CBD)
4CX7C-Phage Library
5Phage Binding Increases with Selection Rounds
6Tag-xPSM Anti-Phage ELISA
7Synthetic Peptides Enhance Enzymatic Activity
8Selected Phage Bind LNCaP Cells
9Cell Binding Blocked by Soluble PSMA
500 nM PSMA Specifically Blocks Cell Binding by
37
10PSMA Binding Peptides
R5-XC1 CQKHHNYLC R4-C9 CTLVPHTRC
- Bind to both purified and cellular PSMA
- Bind as free peptides or while incorporated
into phage - Estimated Affinity Micromolar
11PSMA Peptide Future Course
- Adenoviral Targeting via Fiber Knob
- Proof of principal GFP tagged virus
- Stealth Liposome Targeting
- Proof of principal reporter assays
- Affinity Maturation
- Phage Display
- Viral Display
XXX-CQKHHNYLC-XXX
12Project Goals
- Part I Identify Prostate Targeting Peptides
- Phage Displayed Peptide Libraries
- Peptide Characterization
- Affinity Maturation
- Part II Incorporate Peptides into Viral Capsid
- Vector Shuttle System
- Adenoviral Fiber Knob Cloning Vectors
- Evaluate Viral Production and Targeting
13The pFex Fiber Exchange System
Fiber Shuttle
pFex
14(No Transcript)
15Small shuttle vector (5,000 bp)
X
Uni-directional Gene Transfer
Large viral vector (35,000 bp)
16Fiber Shuttle Vectors
- Current Peptide Cloning Sites
- BspEI HI Loop
- Directional HI Loop
- Future Peptide Cloning Sites
- Carboxy-terminal
- IRES
17(No Transcript)
18R5-XC1 Targeted Fiber Knob
Underlined extra sequences added to fiber (13
total) Highlighted Selected peptide
19R4-C9 Targeted Fiber Knob
Underlined extra sequences added to fiber (13
total) Highlighted Selected peptide
20RGDTP Targeted Fiber Knob
Underlined extra sequences added to fiber (9
total) Highlighted Control peptide
21Fiber exchange followed by E1 exchange
Ad5 Left
KanR
pShuttle-Fib
3) Kanamycin Selection
Ad5 Right
KanR
1) Co-Transform RP-Fib and pFex into Cre
expressing E. coli and select with Ampicillin and
Sucrose
AmpR
Fiber
2) Digest pShuttle with Pme I and co-transform
with pFex-Fib into Rec A expressing E. coli.
Ad5 Left
pFex-Fib
pFEX-Fib
AmpR
Ad5 Right
pShuttle
Ad5 Right
Pme I
KanR
Ad5 Left
22(No Transcript)
23Xho I Digestion reveals multiple potential
recombination products however, there are also
several contaminant bands, including the 6 Kb
Rp-Fib shuttle band.
Rp-Fib-3
Rp-Fib-1
4
5
9
10
11
12
10
3
1
2
8
9
5 Kb
4 Kb
Recombination specific band (3633 bp) visible
among background bands
3 Kb
2 Kb
2424/24 DH5a Transformants contain the desired
recombination products
pFex-Fib-3-10
pFex-Fib-3-11
pFex-Fib-3-12
pFex-Fib-3-1
pFex-Fib-3-2
pFex-Fib-3-3
pFex-Fib-3-4
pFex-Fib-3-5
pFex-Fib-3-6
pFex-Fib-3-7
pFex-Fib-3-8
pFex-Fib-3-9
pFex
3633 bp
pFex-Fib-1-10
pFex-Fib-1-11
pFex-Fib-1-12
pFex-Fib-1-1
pFex-Fib-1-2
pFex-Fib-1-3
pFex-Fib-1-4
pFex-Fib-1-5
pFex-Fib-1-6
pFex-Fib-1-7
pFex-Fib-1-8
pFex-Fib-1-9
pFex
3633 bp
25pFex Efficiency
- Small scale experiments achieve a calculated
diversity of 2000 with - 101 molar ratio of shuttle to pFex
- 1-5 ul DNA per transfection
- 40 ul electrocompetent cells
- Currently optimizing the system with the goal of
creating viral display libraries
26E1 exchange followed by Fiber exchange
Fiber
pShuttle
KanR
Ad5 Right Hand
KanR
pShuttle-Fib
Pme I
Ad5 Left Hand
1) Pme I digest pShuttle and Co-transform with
pFex into RecA expressing E. coli
3) Co-transform pShuttle-Fex and RPuc-Fib into
Cre expressing E. coli and select with Kanamycin
and Sucrose
Fiber
SacB
SacB
KanR
KanR
pShuttle-FEX
pFEX
2) Kanamycin Selection
AmpR
27Future Course for pFex Project
- Viral Production
- Compare AdTrack-Fib1 with Ad5-AdTrack
- CAR ablated production in 911-S11 cells
- Viral Retargeting
- Positive Control RGD4C CAR (/-) to T24 cells
- PSMA Targeting R5-XC1 CAR () to 293 (/-) PSMA
and PC-3 (/-) PSMA - R4-C9 CAR () to 293 (/-) PSMA
and PC-3 (/-) PSMA
28Potential Future Course
- Affinity maturation of R5-XC1 in virus
- XXX-CQKHHNYLC-XXX library ordered
- Diversity dependant on transfection efficiency
- Selection for specificity on 293 (/-) PSMA
- Small Peptide Library screening
- Direct peptide library XXXXXXX
- Diversity dependant on transfection efficiency
- Fiber serotype switching
- Artificial Fiber