Recombinant adeno-associated viral (rAAV) vectors have proven to be efficient vehicles for gene transfer in animal models. The attractive features of this vector system are long-term gene expression with little or no associated toxicities following administration to a variety of tissues. Previous and ongoing clinical trials in humans demonstrate a very good over-all safety profile. However, one of the caveats of this work that has been carried out by several laboratories is the inability to normalize vector doses administered by different investigators to animals and humans. Most of the work to date has involved AAV serotype2 vectors, but vector systems based on other AAV serotypes are being rapidly developed…
Tag: <span>infectious titer</span>
Through the tremendous efforts of the Adenovirus Reference Material Working Group (ARMWG), an adenovirus reference material (ARM) is now available from the American Type Culture Collection (ATCC). The history and progress of the ARM production and characterization has been presented at many meetings and published in numerous journal articles. Although general statements have been made regarding how the ARM should be used, there is no formal directive or specific set of instructions detailing its application in the field. The goals of this paper are (1) to briefly review the objectives for development and implementation of the ARM, (2) to describe a critical assumption necessary to meet those objectives, (3) to outline specific approaches for using the ARM, and (4) to highlight the need for a working group to address the issues raised in the process…
As development proceeds for adenoviral vectors in gene transfer clinical trials, it becomes increasingly important that these products demonstrate a good safety profile, and thereby build confidence in those who must make decisions about risk/benefit ratios, dose escalation, and efficacy. Currently, safety and efficacy are based predominantly upon the analysis of data generated by non-standardized methods, resulting in inconsistent values being reported for virus titer and particle counts…
The Adenovirus Reference Material (ARM) was developed under the guidance of the Adenovirus Reference Material Working Group (ARMWG) and the U.S. Food and Drug Administration (FDA), and was made possible through the donation of services and supplies by a large number of laboratories and institutions from the United States, Canada, France, The Netherlands, Germany, and the United Kingdom. The purpose of the ARM is to provide a reference material for use in validating assays and internal standards for adenoviral particle concentration and infectious titer. The NIH Recombinant DNA Advisory Committee recommended the development of such a reference-testing agent in their report issued January 2002. The ARM consists of purified wild type 5 Adenovirus as described by ATCC’s catalog number VR-5…
A Short-Term Field Use and Shipping Stability Study of a Wild Type Ad5 Adenoviral Reference Material
Adenoviral vectors for gene delivery are being tested in the clinic for a number of indications and therapeutic uses. In order to facilitate the comparison of studies from different laboratories, the Adenovirus Reference Material Working Group (ARMWG) has developed a reference testing reagent, which will be referred to as the Wild Type Ad5 Adenoviral Reference Material (ARM). This ARM will allow laboratories to standardize in-house controls employed in assays for the determination of particle concentration and infectious titer of their own adenoviral preparations. As part of this project, short-term field use and shipping studies were performed on the ARM. The virus was found to be stable under simulated shipping conditions, for one thaw after shipping, and at 4 °C for up to four hours after thawing. However, there was evidence of aggregation in some vials with repeated freeze-thaw cycles. Therefore, we recommend that each vial be treated as a single-use aliquot, and that it be used within four hours of thawing…
Recombinant adeno-associated viral (rAAV) vectors are known to be efficient vehicles for gene transfer in animal models. The attractive feature of this vector system consists primarily of long-term gene expression with little or no associated toxicities following administration to a variety of tissues. Previous and ongoing clinical trials in humans demonstrate a very good overall safety profile, but problems persist due to the lack of any systematic method for normalizing doses administered to animals and humans. To date, most of the work involves AAV serotype 2 vectors, but vector systems based on other AAV serotypes continue to develop rapidly. Administered doses are usually based on titer, but the defective nature of AAV makes determining vector infectious units difficult. Titering methods based on vector genomes (using hybridization, real-time PCR, or spectrophotometry) are more reliable, but give no information as to the infectivity of the vector. Determining infectious titer is critical, as the ratio of infectious virions to vector genome-containing virions helps to determine the dose, potency, and strength of the vector preparation…