# Presentation: 228346

## 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

- ITR

## Adenoviruses as Vectors

- Package up-to 105%
- Manipulate Circular Form

## Adenovirus for Gene Therapy

- <date/time>

**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

**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

- 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 RCV
- Strength
- 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

**Collision between Virus and Detector**
- Brownian motion
- Concentration gradient
- External forces

## gal - Static Titer Determination

**Observed Positives**      **1.7****	****       ****	****2.1****	****            2.0**
**Calculated Titer****	**** 3.5****x10****11             ****2.1x10****11****	****              ****9.8x10****10**

- gal - Static Titer Determination
- (vp= 8 x 1012)

## External Forces

## gal - Titer Determination after

**Calculated Titer****	**** 3.5****x10****11             ****2.1x10****11****	****              ****9.8x10****10****	****22 to 82 vp:iu**
**Observed Positives**      **7.3****	****       ****	****13.1****	****            17.6**
**Calculated Titer****	**** 1.5****x10****12             ****1.3x10****12****	****              ****8.8x10****11****	****5 to 9 vp:iu**

- gal - Titer Determination after
- 90 min at 1000 RCF (0.398 cm)

## Single or Multiple Detection with Virion Displacement

- Pw = n (1 - e

- )

- [5]

- (PC

- t

- (d + I      )

- t

- )

- V = -n (1 -      )

- 1

- [6]

## gal - Titer Determination after

**Calculated NAS Titer****	**** 3.3****x10****12             ****4.2x10****12****	****              ****6.4x10****12****	****1.3 to 2.4 vp:iu**
**Observed Positives**      	**7.3****	****       ****	****13.1****	****            17.6**
**Calculated NAS Titer****	**** 6.4****x10****12             ****5.4x10****12****	****              ****4.3x10****12****	****1.3 to 1.9 vp:iu**

- gal - Titer Determination after
- 90 min at 1000 RCF (0.398 cm)

## 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

- 2R1D

- 1L1D

- 1L1D

- 2R2D

- 1L1D

- Fixed Point

## Transduction

**MOI*>10e16**

**MOI = 1**