Vector DNA Metabolism

For rAAV vectors to transduce a cell, the ss DNA genome must be converted into a double-stranded form (33), as shown in Fig. 3. Several reports, based on transduction of cells in vitro, indicated that this single- strand to double-strand (DS) conversion step might be rate limiting in the absence of helper virus coinfection (165-167). The adenovirus E4 ORF6 protein has been shown to increase the level of transgene expression and to increase second-strand DNA synthesis (168). More recently, a cellular protein that bound to the single-stranded D-sequence of the AAV ITR was identified as the well-known FK506-binding protein (FKBP-52). This protein has been implicated in controlling the conversion of ss to ds DNA in vector-infected cells and phosphorylation of FKBP-52 influences its ability to bind the D sequence (169,170). When phos-phorylated at tyrosine residues (by the epidermal growth factor receptor protein tyrosine kinase), FKBP-52 binds to the ss D sequence region of the ITR that is present in infecting vector genomes, and second-strand DNA synthesis is impaired. The efficiency of transduction in a number of cell types in vitro and in vivo correlates with the phosphorylation state of FKBP-52. In HeLa cells, overexpression of a cellular phosphatase (TC-PTP) that can use the FKBP-52 protein as a substrate, led to dephosphorylation of the FKBP-52, an increase in AAV second-strand DNA synthesis, and an increase in transgene expression. Transgenic mice expressing either the wt or a cata-lytically mutant form of the phosphatase were created. Hema-topoietic stem cells from transgenic mice expressing the wt TC-PTP phosphatase were transduced by an AAV2 vector, but those from mice expressing the phosphatase-negative mutant were not. These results suggest that the block to second-strand DNA synthesis is due to binding of FKBP-52 to the D-se-quence of infecting vector genomic DNA and that this binding is regulated by phosphorylation.

A second mechanism for conversion of ss DNA to ds DNA has been proposed. In this model, annealing of negative and positive sense ss DNA genomes (both of which are efficiently packaged in AAV particles) occurs in cells to form ds DNA in the absence of second-strand DNA synthesis. There is some experimental evidence for this self-annealing model both in vitro and in vivo (171), although it is not consistent with observations that AAV infection displays single-hit kinetics (59). However, it is possible that double-stranded DNA may be formed via either pathway in cells transduced with rAAV vectors.

Further support for the idea that conversion of single-stranded vector genomes into transcriptionally active, double-stranded forms is crucial for transduction and is a rate-limiting step comes from a study demonstrating that vector genomes smaller than half the size of the AAV genome are packaged in multiple ways (172). Particles contain either a single vector genome, two copies of the small vector genome, or a cova-lently linked double-stranded hairpin molecule equivalent to a replicative-intermediate formed during vector genome replication. In a separate study, vectors containing these small self-complementary genomes were shown to be relatively insensitive to the enhancing effects of adenovirus on transduction, to be resistant to the effects of DNA synthesis inhibitors, and displayed altered kinetics of transgene expression in vivo (173). When these vectors were administered to mice, an increase in transduction from the usual 5% to more than 50% of hepatocytes was observed (174,175). These results strengthen the model that second-strand DNA synthesis is a rate-limiting step for transduction, at least in hepatocytes.

Once the vector genome is converted into a double-stranded DNA molecule, a number of fates have been reported. One group of studies convincingly demonstrates that the viral genome circularizes, and that these circular monomers recombine at the AAV ITRs to form larger circular con-catemers of head-to-head, head-to-tail, and tail-to-tail arrangements both in vitro and in vivo (79,80,176,177). These genomic concatemeric molecules, containing a ''double-D'' ITR structure (an ITR bracketed on each side by the D sequence and presumably formed by an ITR-ITR recombination event) persist extra chromosomally as episomes and are responsible for the long-term persistent transgene expression seen with rAAV vectors. Recently, it was reported that severe combined immunodeficiency (SCID) mice that are deficient in DNA-dependent protein kinase activity (DNA-PK) lack the ability to convert rAAV genomes into circular concatemers (178). Rather the concatemers formed in these mice appear to be linear molecules, suggesting that DNA-PK activity is involved in the formation of circular episomes. In both cases (normal and SCID mice) transgene expression persisted for 1 year at similar levels. A second group ofreports (171,179,180) suggest that vector genome concatamerization occurs by recombination of linear monomeric genomes. It is possible that both mechanisms of concatamerization are operative and one pathway is more likely to occur than another in a tissue-specific manner (e.g., muscle vs. liver). Despite the differences reported on the substrate for concatemer formation, it is clear that long-term transgene expression is mainly mediated by episomal concatemers of viral genomes rather than integrated molecules (83,84).

Evidence that DNA repair and recombination are directly involved in circularization or concatamerization of AAV vector genomes is supported by recent insights into possible biochemical mechanisms of their formation (178,180). In fibro-blasts from a patient with ataxia telangiectasia (ATM), there is greatly enhanced formation of AAV vector circular forms and enhanced integration of the head-to-tail concatemers as proviral genomes (181). The ATM gene is a PI-3 kinase that regulates the p53-dependent cell-cycle checkpoint and apop-totic pathways, and in these ATM cells the DNA double-strand break (DSB) repair systems that normally can be activated by UV irradiation appear to be already activated maximally.

Consequently, AAV vectors in these cells yield a high level of transduction and this is not activated further by UV irradiation (181), in contrast to the observations in normal cells (165-167). Additional evidence that DSB repair pathways are involved in regulating AAV transduction comes from observations that the proteins Ku86 and Rad52, which are known to recognize DNA hairpin structures and DNA termini and to promote repair of DSB, could associate with the AAV DNA ITR (182).

How To Deal With Rosacea and Eczema

How To Deal With Rosacea and Eczema

Rosacea and Eczema are two skin conditions that are fairly commonly found throughout the world. Each of them is characterized by different features, and can be both discomfiting as well as result in undesirable appearance features. In a nutshell, theyre problems that many would want to deal with.

Get My Free Ebook

Post a comment