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