Rapid, widespread transduction of the murine myocardium using self-complementary Adeno-associated virus
© Andino et al; licensee BioMed Central Ltd. 2007
Received: 01 October 2007
Accepted: 10 December 2007
Published: 10 December 2007
Adeno-associated virus (AAV) has shown great promise as a gene transfer vector. However, the incubation time needed to attain significant levels of gene expression is often too long for some clinical applications. Self-complementary AAV (scAAV) enters the cell as double stranded DNA, eliminating the step of second-strand synthesis, proven to be the rate-limiting step for gene expression of single-stranded AAV (ssAAV). The aim of this study was to compare the efficiency of these two types of AAV vectors in the murine myocardium. Four day old CD-1 mice were injected with either of the two AAV constructs, both expressing GFP and packaged into the AAV1 capsid. The animals were held for 4, 6, 11 or 21 days, after which they were euthanized and their hearts were excised. Serial sections of the myocardial tissue were used for real-time PCR quantification of AAV genome copies and for confocal microscopy. Although we observed similar numbers of AAV genomes at each of the different time points present in both the scAAV and the ssAAV infected hearts, microscopic analysis showed expression of GFP as early as 4 days in animals injected with the scAAV, while little or no expression was observed with the ssAAV constructs until day 11. AAV transduction of murine myocardium is therefore significantly enhanced using scAAV constructs.
Results and discussion
Adeno-associated virus has become an important tool for gene transfer because of its lack of pathogenicity and its ability to express passenger genes for long periods of time. Although potentially safe as a gene therapy vector, this virus exhibits an extended lag period before transgene expression actually occurs. The reason for this delay in expression is the binding of a cellular protein, FKBP52, to the D-sequence within the inverted terminal repeats (ITRs). Phosphorylated FKBP52 inhibits viral second strand DNA synthesis, needed for transgene expression, consequently leading to delayed transgene expression [1–3]. As an avenue for bypassing this phenomenon, McCarty et al. have introduced the use of a double-stranded form of AAV.
The typical single-stranded AAV (ssAAV) genome is flanked by two, 145 bp ITRs. The 3' ITR serves as the replication origin for the viral genome as well as a packaging signal. During replication, AAV genome dimers are formed as replication intermediates. These dimers are subsequently cleaved by AAV Rep proteins at the junction of the ITR and the D sequence. Wang et al. discovered that if one of the ITRs has a deleted D-sequence and terminal resolution site (trs), cleavage by Rep cannot occur and consequently, the dimers are not resolved into monomers. Therefore, the double-stranded genomes are then packaged as large hairpin DNA molecules.
Although the biology of these constructs has been studied, testing of these constructs for gene delivery to animals is in its early stages. In this report, we demonstrate the efficacy of self complementary AAV (scAAV) in the murine myocardium, a traditionally challenging organ to transduce. Although different viruses have been employed for myocardial gene transfer, each has its own limitations. For example, adenovirus has been shown to be toxic and has short-term expression, and lentiviral vectors stimulate inflammatory responses. Although adeno-associated virus has a delay in onset and small packaging capacity, it has been shown to direct gene expression for long periods of time in the heart without any toxicity [9–11].
We compared the expression profile of both the ssAAV and scAAV constructs in murine myocardia using sub-xiphoid injections in 4 day old CD-1 mice (Charles Rivers). The hearts were assessed using direct immunofluorescence of the tissue 4, 6, 11, and 21 days post-injection.
Once the constructs were packaged, four day old CD-1 mice (Charles Rivers) were anesthetized on ice until movement was barely visible, and injections into the cardiac chamber were carried out as described by Zhang et al. Each animal was injected with 1.85 × 1011 total particles of AAV1 containing either the ssAAV or the scAAV expression cassettes. Three animals were injected per group. Animals were returned to their dams for 4, 6, 11, or 21 days and then euthanized. Their hearts were extracted and then cut into thirds representing the apical region, the middle region and the base region of the heart. The tissue was then fixed for 12 hours with 4% paraformaldehyde, rinsed in PBS and then allowed to equilibrate in 20% sucrose for 12–24 hours. The next day, hearts were frozen in OCT compound (Tissue-Tek). Five micron sections were cut in a cryostat and used for direct visualization of GFP. Slides were then mounted using Vectashield mounting media containing DAPI (Vector Labs). Two sections, each containing 75 μm of tissue, were reserved for quantitative real-time PCR. Because of its high content of adenine dinucleotides, cardiac tissue exhibits a high level of autofluorescence and this background can obscure the detection of GFP even when secondary antibodies are employed. Therefore, we chose to measure the direct GFP signal generated using confocal microscopy of the fixed tissues. This technique allowed us to almost entirely eliminate background fluorescence seen with conventional fluorescent microscopes and filters enabling us to accurately represent the transduced cells. A Leica confocal microscope was used to obtain fluorescent images. Montages consisting of 2–4 fields of view per section were created using Adobe Illustrator CS2 and Adobe Photoshop CS2.
We have described a novel self-complementary AAV vector that contains the chicken β-actin promoter driving the expression of GFP. Despite similar number of vector genomes observed in hearts infected with either scAAV or ssAAV, the scAAV transduced animals showed robust and widespread GFP expression as early as 4 days post-injection, while even at 11 days, expression using ssAAV was minimal. GFP expression could be seen all throughout the heart from the base to the apex. The amount of GFP expression increased over the time course of the experiment in both of the scAAV and ssAAV transduced animals. Nonetheless, the amount of GFP expression seen in the scAAV injected animals was much higher than the ssAAV injected animals at each time point.
Although this construct will not be suitable for packaging large transgenes, this type of vector will be very useful for the delivery of small transgenes and small molecules such as ribozymes and shRNA molecules. Additionally, Wu et al. have demonstrated that the packaging capacity of these self-complementary vectors can be as large as 3.3 kb which is more than what was originally expected. If efficient myocardial transduction can be demonstrated in adult animals, these double-stranded AAV vectors might ultimately prove useful in patients who need rapid expression of therapeutic genes.
The authors' would like to thank the Powell Gene Therapy Center for the assistance in quantitating the AAV vector genomes in the murine cardiac tissue samples. We acknowledge NIH grants EY13729, EY11123, NS36302, EY08571, and grants from the Macular Vision Research Foundation, Foundation Fighting Blindness, Juvenile Diabetes Research Foundation and Research to Prevent Blindness, Inc. for partial support of this work. Lourdes M. Andino was the recipient of an American Heart Association predoctoral fellowship.
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