Targeted Adenoviral Gene Therapy

1
Targeted Adenoviral Gene Therapy
Shawn E. Lupold, Ph.D. Post Doctoral Fellow Brady
Urological Institute Johns Hopkins Hospital
2
Project 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

3
Purified 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)

4
CX7C-Phage Library
5
Phage Binding Increases with Selection Rounds
6
Tag-xPSM Anti-Phage ELISA
7
Synthetic Peptides Enhance Enzymatic Activity
8
Selected Phage Bind LNCaP Cells
9
Cell Binding Blocked by Soluble PSMA
500 nM PSMA Specifically Blocks Cell Binding by
37
10
PSMA 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

11
PSMA 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
12
Project 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

13
The pFex Fiber Exchange System
Fiber Shuttle
pFex
14
(No Transcript)
15
Small shuttle vector (5,000 bp)
X
Uni-directional Gene Transfer
Large viral vector (35,000 bp)
16
Fiber Shuttle Vectors

• Current Peptide Cloning Sites
• BspEI HI Loop
• Directional HI Loop
• Future Peptide Cloning Sites
• Carboxy-terminal
• IRES

17
(No Transcript)
18
R5-XC1 Targeted Fiber Knob
Underlined extra sequences added to fiber (13
total) Highlighted Selected peptide
19
R4-C9 Targeted Fiber Knob
Underlined extra sequences added to fiber (13
total) Highlighted Selected peptide
20
RGDTP Targeted Fiber Knob
Underlined extra sequences added to fiber (9
total) Highlighted Control peptide
21
Fiber 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)
23
Xho 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
24
24/24 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
25
pFex 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

26
E1 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
27
Future 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

28
Potential 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