AAV Helper-Free System*

Viral-based System for Gene Delivery

As we move from gene discovery to understanding gene function, the need to study gene expression in a native host is becoming increasingly important. The problem is that many of these hosts are difficult or impossible to transfect which means these studies are limited to hosts which easily accept DNA using traditional transfection methods. To solve this problem, viral-based gene delivery systems, like the AAV Helper-Free System, have been created for high-efficiency gene delivery to a broader range of hosts.1

Efficient Gene Delivery, Stable Expression

Recombinant adeno-associated virus (AAV) is a proven research and therapeutic tool1. This system is used to introduce genes into cells for gene expression or gene therapy studies. Using this system, genes can be delivered into a wide range of hosts including many different human and non-human cell lines or tissues. Since gene delivery is made possible in mammalian systems, expressed proteins have all of the necessary posttranslational modifications and folding. Recombinant AAV are capable of transducing a broad range of cell types and transduction is not dependent on active host cell division. High titers, > 108 vp/ml, are easily obtained in the supernatant and ³ 1011 -1012 vp/ml with further concentration. The gene of interest is integrated into the host genome so expression is long term and stable.

Helper-Free, Safe System

AAV is naturally replication-deficient and normally requires coinfection with a unrelated helper virus, like adenovirus, to generate AAV virions. This novel system uses a vector containing the necessary genes from adenovirus (pHelper vector) to induce the lytic phase of AAV producing recombinant, replication-defective AAV virions ready to deliver a gene of interest to target cells. Eliminating the need for helper virus means:

Additionally, in traditional viral delivery systems, regeneration of wild-type virus by recombination is a major concern. In the AAV Helper-Free system, the AAV-2 ITR-containing plasmid (pAAV-LacZ) and the rep/cap-gene containing plasmid (pAAV-RC), do not share any regions of homology, preventing the production of wild-type AAV-2 through recombination.

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.

Easy, Three-Step Procedure for Gene Expression

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 2). 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.
 

pCMV-MCS Vector

The pCMV-MCS is a 4.5 kb cloning vector. The gene of interest is cloned into the multiple cloning site. After cloning, the expression cassette is removed from this vector using the flanking NotI sites for subsequent cloning into the pAAV-LacZ vector.

pAAV-MCS

The pAAV-MCS vector contains the CMV promoter and other elements for high-level gene expression in mammalian cells when a gene of interest is cloned into the multiple cloning site (MCS). This vector contains AAV-2 inverted terminal repeats (ITRs), which direct viral replication and packaging.

pAAV-IRES-hrGFP vector

The vector pAAV-IRES-hrGFP contains a dicistronic expression cassette in which the Renilla reniformis green fluorescent protein (hrGFP) gene is expressed as a second open reading frame downstream of the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES). The hrGFP gene serves as a useful marker for titering the virus and ascertaining the infection efficiency for the desired target cell type. Another unique feature of this vector is the FLAG sequence (3x) at the c-terminal of MCS. The foreign gene could be cloned in frame with the tag and the protein could be further purified or detected by anti-FLAG antibody.
 

pAAV-LacZ Vector

The 7.3 kb pAAV-LacZ is the replication-deficient AAV vector. This vector serves two purposes; first, as a cloning vector; second, as a control vector. As a cloning vector, this vector is cut with NotI, the LacZ cassette is lost and the expression cassette, harboring the gene of interest, from the pCMV-MCS vector will be cloned into the complementing NotI sites of this vector. The recombinant vector will be cotransfected into a viral packaging cell line for production of recombinant, replication-deficient AAV virions. This vector is also a control using the LacZ reporter enzyme.

pAAV-hrGFP

The pAAV-hrGFP is a nice alternative to the pAAV-LacZ positive control vector. This vector allows the you to determine the titer (i.e. the number of infectious virus particles per unit volume) of virus preparations, and confirm the transfection efficiency of their packaging cell line in vivo using the humanized Renilla GFP (hrGFP) reporter gene. This reporter gene allows for easy detection of expression in vivo using fluorescence microscopy, fluorescence-activated cell sorting (FACS) analysis.
 

pAAV-RC Vector

This 7.3 kb vector harbors the cap and rep genes that make the capsid and DNA replication proteins required to make infectious virions. This vector is cotransfected into the packaging cell line for production of recombinant, replication-deficient AAV virions.

pHelper Vector

This 11.6 kb vector carries the adenovirus genes (E2A, E4 and VA RNAs) required for inducing the lytic phase of AAV. This vector is cotransfected into the packaging cell line to produce recombinant, replication-deficient AAV virions.

Sequence Analysis

pAAV-LacZ
 

pAAV-IRES-hrGFP

pAAV-MCS


pAAV-RC


pCMV-MCS

pHelper

 

pAAV-hrGFP



Ordering Information

AAV Helper-Free System
  VECTORS CONTENTS USE CATALOG #
Additional
Cloning Vector
pAAV-IRES-
hrGFP
10 µg
  • Express both the recombinant protein and humanized Renilla GFP (hrGFP) reporter from same mRNA transcript
  • Cloning vector containing MCS and ITR sequences for direct cloning in this vector
  • Expression cassette from pCMV-MCS vector can be transferred to this ITR-containing vector
240075
Additional Positive Control Vector pAAV-hrGFP 20 µg
  • Control vector harboring humanized Renilla GFP (hrGFP) reporter gene
  • Expression cassette from pCMV-MCS vector can be transferred to this ITR-containing vector
240074
Complete System pCMV-MCS 10 µg
  • Cloning vector and source of expression cassette
240071
pAAV-MCS 10 µg
  • Cloning vector containing MCS and ITR sequences for direct cloning in this vector
pAAV-LacZ 10 µg
  • Expression cassette with gene of interest cloned into replication-defiicent AAV vector
  • Original vector with LacZ is a control vector
pAAV-RC 20 µg
  • Carries the rep and cap AAV genes required for generating recombinant, replication-deficient AAV virions
pHelper 20 µg
  • Carries the adenovirus genes required to induce lytic phase of AAV to generate recombinant, replication-deficient AAV virions
 

 

  1. Matsushita, T., et al. (1998) Gene Thereapy 5: 938-945

  *Legal Use of the AAV Helper-Free System is covered by U.S. Patent Nos. 5,622,856, 5,945,335, 6,001,650, 6,004,797 and 6,027,931 and is limited to use solely for research purposes. Any other use of the AAV Helper-Free System requires a license from Avigen, Inc.