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Overview of Adenoviral Vectors and Titer Determination

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.

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Overview of Adenoviral Vectors and Titer Determination

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  1. Overview of Adenoviral Vectors and Titer Determination

  2. 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

  3. Virus Structure • Icoshedral • 20 surfaces • 12 vertices • 13% DNA • 87% Protein • NO LIPID

  4. 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

  5. Adenoviruses as Vectors Package up-to 105% Manipulate Circular Form

  6. Adenovirus for Gene Therapy • Replication deficient • 8kb foreign DNA • High titer production • Infect variety of tissues • High expression in non-replicating tissues

  7. Gene of Interest Adenovirus for Gene Therapy • 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 EG X

  8. 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

  9. 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.

  10. 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

  11. Characterization of Viral Vectors • Purity • Lack of contamination by adventitious agents, including RCV • Strength • The active concentration for toxicity and efficacy

  12. 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

  13. Culture Dish Virus Dilution Target Cells Typical Titer Set Up • Collision between Virus and Detector • Brownian motion • Concentration gradient • External forces

  14. -9 -10 -11 -12 100 ul/ well -9 -10 -11 -12 200 ul/ well -9 -10 -11 -12 50 ul/ well 0.142cm 0.284cm 0.568cm bgal - Static Titer Determination (vp= 8 x 1012) Observed Positives1.7 2.1 2.0 Calculated Titer 3.5x1011 2.1x1011 9.8x1010

  15. displacement 1 x g centrifuged d = S RCF v t External Forces

  16. -9 -10 -11 -12 100 ul/ well -9 -10 -11 -12 200 ul/ well -9 -10 -11 -12 50 ul/ well 0.142cm 0.284cm 0.568cm bgal - Titer Determination after 90 min at 1000 RCF (0.398 cm) Observed Positives2.6 2.8 2.7 Calculated Titer 3.5x1011 2.1x1011 9.8x1010 22 to 82 vp:iu Observed Positives7.3 13.1 17.6 Calculated Titer 1.5x1012 1.3x1012 8.8x1011 5 to 9 vp:iu

  17. (d + I ) -(PC V ) t t Pw n Single or Multiple Detection with Virion Displacement ) Pw = n (1 - e [5] 1 [6] V = -ln (1 - ) (d + I ) ) (PC t t

  18. -9 -10 -11 -12 100 ul/ well -9 -10 -11 -12 200 ul/ well -9 -10 -11 -12 50 ul/ well 0.142cm 0.284cm 0.568cm bgal - Titer Determination after 90 min at 1000 RCF (0.398 cm) Observed Positives2.6 2.8 2.7 Calculated NAS Titer 3.3x1012 4.2x1012 6.4x1012 1.3 to 2.4 vp:iu Observed Positives7.3 13.1 17.6 Calculated NAS Titer 6.4x1012 5.4x1012 4.3x1012 1.3 to 1.9 vp:iu

  19. 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

  20. 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

  21. Standard as an address not an absolute Fixed Point 1L1D 2R2D 2R1D 2R1D 1L1D 1L1D

  22. MOI Transduction MOI*>10e16 MOI = 1

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