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
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
pAAV-MCS
pCMV-MCS
pHelper
pAAV-hrGFP
*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.