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) 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
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 3Day 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 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 n Purity n Lack of contamination by adventitious agents, including RCV n Strength n The active concentration for toxicity and efficacy
Characterization of Viral Vector Strength Physical determination –1 OD 260 = 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 n Collision between Virus and Detector n Brownian motion n Concentration gradient n External forces
ul/ well ul/ well ul/ well Observed Positives Calculated Titer 3.5 x x x10 10 gal - Static Titer Determination (vp= 8 x ) 0.142cm0.284cm0.568cm
External Forces displacement 1 x gcentrifuged d = S RCF vt
Observed Positives Calculated Titer 3.5 x x x to 82 vp:iu Observed Positives Calculated Titer 1.5 x x x to 9 vp:iu gal - Titer Determination after 90 min at 1000 RCF (0.398 cm) ul/ well ul/ well ul/ well 0.142cm0.284cm0.568cm
Single or Multiple Detection with Virion Displacement Pw = n (1 - e ) -(PC V t (d + I ) t ) [5] (PC t (d + I ) t ) V = - n (1 - ) Pw n 1 [6]
Observed Positives Calculated NAS Titer 3.3 x x x to 2.4 vp:iu Observed Positives Calculated NAS Titer 6.4 x x x to 1.9 vp:iu gal - Titer Determination after 90 min at 1000 RCF (0.398 cm) ul/ well ul/ well ul/ well 0.142cm0.284cm0.568cm
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 1L1D 2R2D 1L1D Fixed Point
Transduction MOI MOI*>10e16 MOI = 1