Transcript Overview of Adenoviral Vectors and Titer Determination

Overview of Adenoviral Vectors
and Titer Determination
Historical Overview
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Identified in early 50’s
Etiologic agent of the Common Cold et al?
Linear dsDNA encapsidated in protein shell
Over 100 in the Adenoviral group
wt Adeno used as vaccine in military recruits
Virus Structure
• Icoshedral
– 20 surfaces
– 12 vertices
• 13% DNA
• 87% Protein
• NO LIPID
Gene Structure
and Organization
• 2 origins of replication -ITR
• Transcription Units
– 5 “early” (E1A, E1B, E2, E3, E4)
– 2 “delayed early” (IVa2 and IX)
– 1 major late -> (L1-L5)
ITR
ITR
Adenoviruses as Vectors
Package up-to 105%
Manipulate Circular Form
Adenovirus for Gene Therapy
- Replication deficient
- 8kb foreign DNA
- High titer production
- Infect variety of tissues
- High expression in non-replicating
tissues
Adenovirus for Gene Therapy
EG
• Evolution of Adenovectors
– 1st generation: E1- and E3 +/– 2nd generation: E1-, E2- or E4-, E3 +/– Generation X: E1A+, E1B-, E3 +/– Generation X.1: E1A and/or E1B conditional
– Generation X.2: helper dependent
Gene of
Interest
X
2nd Vector generations?
Day 3
Day 21
O’Neal, W.K. et al. Toxicological comparison of E2a-deleted and first-generation adenoviral vectors expressing a1-antitrypsin after systemic
delivery. Human Gene Therapy, July 1998
Generation X.2?
weeks
Morral, N, et al. High doses of helper-dependent adenoviral vector yield supraphysiological levels of a1-antitrypsin with negligible
toxicity. Human Gene Therapy, Dec. 1998.
Conclusions
• Adenoviruses can be converted into efficient gene
transfer vehicles
• Adenoviral vectors are not inherently dangerous
• Not all adenoviral vectors have equivalent toxicity
profiles
• The dose of vector delivered is related to the toxicity
observed
• Standardization of dose specification is necessary
Characterization of Viral
Vectors
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Purity
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Lack of contamination by adventitious
agents, including RCV
Strength
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The active concentration for toxicity and
efficacy
Characterization of Viral
Vector Strength
• Physical determination
– 1 OD260= 1.1 x 10e12 vp
• Biological determination
– physical characteristics of the method
• distance and time
• likelihood of vector and cell meeting
– functional characteristics of the system
• receptors
• detection
Typical Titer Set Up
Culture Dish
Virus Dilution
Target Cells

Collision between Virus and Detector
 Brownian motion
 Concentration gradient
 External forces
bgal - Static Titer Determination
(vp= 8 x 1012)
-9 -10 -11 -12
50 ul/ well
-9 -10 -11 -12
100 ul/ well 200 ul/ well
0.142cm
Observed Positives
Calculated Titer
1.7
3.5x1011
-9 -10 -11 -12
0.284cm
2.1
2.1x1011
0.568cm
2.0
9.8x1010
External Forces
displacement
1xg
d = S vRCF
t
centrifuged
bgal - Titer Determination after
90 min at 1000 RCF (0.398 cm)
-9 -10 -11 -12
50 ul/ well
-9 -10 -11 -12
100 ul/ well 200 ul/ well
0.142cm
Observed Positives
Calculated Titer
Observed Positives
Calculated Titer
2.6
3.5x1011
7.3
1.5x1012
-9 -10 -11 -12
0.284cm
2.8
0.568cm
2.7
2.1x1011
9.8x1010
13.1
17.6
1.3x1012
8.8x1011
22 to 82 vp:iu
5 to 9 vp:iu
Single or Multiple Detection with Virion
Displacement
Pw = n (1 - e
V = -ln (1 -
Pw
n
-(PC tV (d + I
)
t) )
) [5]
1
(PC t (d + I
t) )
[6]
bgal - Titer Determination after
90 min at 1000 RCF (0.398 cm)
-9 -10 -11 -12
50 ul/ well
-9 -10 -11 -12
100 ul/ well 200 ul/ well
0.142cm
Observed Positives
2.6
Calculated NAS Titer
3.3x1012
Observed Positives
7.3
Calculated NAS Titer
6.4x1012
-9 -10 -11 -12
0.284cm
2.8
0.568cm
2.7
4.2x1012
6.4x1012
13.1
17.6
5.4x1012
4.3x1012
1.3 to 2.4 vp:iu
1.3 to 1.9 vp:iu
What:
quantity, quality
• From bench to bedside
– Original Titer
• V.P. vs I.U., PFU, FFU, etc
– Clinical Titer
–
Nyberg-Hoffmann, C. and Aguilar-Cordova, E. Instability of adenoviral vectors during transport and its implication for
clinical studies. Nature Medicine, August 1999
Need to Standardize
• Definition of how a product will behave
– Benchmarks for comparing the toxicology and efficacy of
the products
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Crucial
Crucial
Crucial
Crucial
for
for
for
for
managing the manufacturing processes
maintaining consistent QC
dose escalation studies
a true product
Standard as an address not an
absolute
Fixed
Point
1L1D
2R2D
2R1D
1L1D
2R1D
1L1D
Transduction
MOI*>10e16
MOI = 1
MOI