Viral-Based System For High-Efficiency Gene Delivery

Viral-Based System for High-Efficiency Gene Delivery

• Safe, Helper-Free System—No Adenovirus Required
• High-Efficiency Gene Delivery—Nearly 100% Transduction Efficiency
• Broad Host Range Including Both Dividing and Nondividing Cells

Stratagene’s AAV Helper-Free System^Σ is a powerful gene delivery system for stable, long-term gene expression. The AAV Helper-Free System improves upon recombinant adeno-associated virus-2 (AAV-2) technology by eliminating the need for helper virus. It allows safe, high-efficiency gene delivery to a broad range of hosts, overcoming the limitations of traditional transfection methods.

Powerful Gene Delivery and Expression System

Stratagene’s new AAV Helper-Free System offers the high-efficiency gene transfer of viral-based systems with increased safety over other viral-based systems. These important characteristics have lead to increased interest in the use of AAV vectors for many applications including gene therapy and drug discovery. In fact, the Stratagene vectors described here have been used successfully in human phase I clinical trials¹ and use of these vectors to generate cell-based assays for high-throughput screening has increased due to efficient stable cell line production over a broad range of cell types.

Unparalleled Safety

AAV is naturally replication-deficient and normally requires co-infection with an unrelated helper virus, like adenovirus, to replicate. Recently, helper-free AAV-2 production systems have been developed. Stratagene’s AAV Helper Free System is composed of the minimum number of adenoviral gene products required for helper-free transient production of virus based on the system developed at Avigen, Inc. by Peter Colosi and colleagues.² Eliminating the need for helper virus coupled with the fact that the AAV virus has never been associated with any known human disease gives this system a high biosafety profile. (Figure 1)

Easy, Three-Step Procedure

Expressing genes of interest with the AAV Helper-Free System is easy and consists of three steps: 1) transfection of a packaging cell line, 2) collection of high-titer recombinant virus from the cell lysates, and 3) transduction of target cells for expression studies.

When preparing the recombinant AAV-2 vector to express your gene of interest, you have two options, 1) a single-cloning step or 2) a double-cloning step. The single-cloning step involves cloning your expression cassette directly into your choice of two ITR-containing vectors with a multiple cloning site (Figure 1). However, insertion of certain sequences directly into an ITR-containing vector may lead to rearrangements due to homologous recombination between the ITRs in E. coli. In such cases, it is best to first insert the gene of interest downstream of the CMV promoter in the shuttle vector pCMV-MCS and then easily transfer the expression cassette into your choice of the four ITR-containing vectors. Use of the pCMV-MCS shuttle is also recommended if subsequent modifications (e.g. mutagenesis, promoter replacement, etc.) are to be performed on the gene of interest prior to production of virus. Upon transduction of target cells, high-level gene expression may be achieved as soon as 3 days post-infection (Figure 2).

Novel Helper-Free Titer Protocol

To determine the titer (i.e. the number of infectious virus particles per unit volume) of virus preparations, a reporter virus is produced in parallel in this system. Stratagene has developed a novel 3-day titering method that eliminates the need for co-infection with adenovirus. Traditionally, co-infection with adenovirus shortened the time required to generate titers from 10 to 3 days. Stratagene’s new helper-free method involves applying chemical reagents known to both enhance transduction efficiency and increase expression from the CMV promoter. Stratagene’s novel chemical induction method* eliminates the need for co-infection with adenovirus and titers are obtained quickly in 2 to 3 days.
Now you can deliver your gene to a broader range of hosts with extremely high efficiency for long-term, stable gene expression using a viral-based system with unmatched safety.

REFERENCES
1. Kay, M. A., et al. (2000) Nature Genetics 24: 257-261
2. Matsushita, T., et al. (1998) Gene Therapy 5: 938-945

* Patent pending
^{Ψ Σ}tradmarks and patents

Contributing Scientists
Stratagene: Vivian Zhang, Yanchun Miao, Peter Vaillancourt