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Overview of Adenoviral Vectors and Titer
Determination
Historical Overview
• 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)
ITRITR
Adenoviruses as Vectors
Package up-to 105%
Manipulate Circular Form
Adenovirus for Gene Therapy
- Replication deficientReplication deficient
- 8kb foreign DNA8kb foreign DNA
- High titer productionHigh titer production
- Infect variety of tissuesInfect variety of tissues
- High expression in non-replicating High expression in non-replicating
tissuestissues
• 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
Adenovirus for Gene Therapy
EG
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?
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.
weeks
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
Purity Lack of contamination by adventitious
agents, including RCVStrength
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
-9 -10 -11 -12
50 ul/ well
-9 -10 -11 -12
100 ul/ well
-9 -10 -11 -12
200 ul/ well
Observed Positives 1.7 2.1 2.0
Calculated Titer 3.5x1011 2.1x1011 9.8x1010
gal - Static Titer Determination(vp= 8 x 1012)
0.142cm 0.284cm 0.568cm
External Forces
displacement
1 x g centrifuged
d = S RCF v t
Observed Positives 2.6 2.8 2.7
Calculated Titer 3.5x1011 2.1x1011 9.8x1010 22 to 82 vp:iu
Observed Positives 7.3 13.1 17.6
Calculated Titer 1.5x1012 1.3x1012 8.8x1011 5 to 9 vp:iu
gal - Titer Determination after90 min at 1000 RCF (0.398 cm)
-9 -10 -11 -12
50 ul/ well
-9 -10 -11 -12
100 ul/ well
-9 -10 -11 -12
200 ul/ well
0.142cm 0.284cm 0.568cm
Single or Multiple Detection with Virion Displacement
Pw = n (1 - e ) -(PC Vt (d + I )t )[5]
(PC t (d + I )t )V = -n (1 - )
Pwn
1[6]
Observed Positives 2.6 2.8 2.7
Calculated NAS Titer 3.3x1012 4.2x1012 6.4x1012 1.3 to 2.4 vp:iu
Observed Positives 7.3 13.1 17.6
Calculated NAS Titer 6.4x1012 5.4x1012 4.3x1012 1.3 to 1.9 vp:iu
gal - Titer Determination after90 min at 1000 RCF (0.398 cm)
-9 -10 -11 -12
50 ul/ well
-9 -10 -11 -12
100 ul/ well
-9 -10 -11 -12
200 ul/ well
0.142cm 0.284cm 0.568cm
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
• Crucial for managing the manufacturing processes
• Crucial for maintaining consistent QC• Crucial for dose escalation studies• Crucial for a true product
Standard as an address not an absolute
2R1D 2R1D1L1D1L1D
2R2D
1L1D
Fixed Point
Transduction
MOI
MOI*>10e16MOI = 1
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