**3. Focus on adeno-associated virus (AAV) inverted terminal repeats (ITR)**

The use of vectors derived from the adeno-associated virus (AAV) for gene delivery encounters a growing success for the treatment of a variety of human diseases

*The Diversity of Parvovirus Telomeres DOI: http://dx.doi.org/10.5772/intechopen.102684*


#### **Figure 4.**

*Principal component analysis (PCA) conducted on the five-prime telomere repeats data set. The PCA analysis was conducted using the R software and the Factoshiny package. (a) Contribution graph of each variable. (b) Clusterisation of the 40 parvovirus TR. (c) Correspondence of each parvovirus in the 5 clusters.*

[19]. Nevertheless, the scientific community has recently faced tragic toxicity of AAV vectors administered intravenously at high doses in several clinical trials [20]. AAV vectors are generated by inserting a recombinant genome usually flanked by AAV-2 inverted terminal repeats (ITR) in an AAV capsid. The recent side-effects observed in human trials have raised the question of DNA sensing, in particular, ITR detection and subsequent cellular responses [21]. Considering the importance of providing new knowledge in this field, a special focus on AAV-2 ITR was included in our study.

The homotelomeric AAV2 possesses two identical ITR of 145 nucleotide-long. The first 125 bases contain three palindromic sequences allowing the ITR to form a T-shape structure composed of two small inverted repeat sequences (BB<sup>0</sup> and CC<sup>0</sup> ) and a larger repeated sequence (AA<sup>0</sup> ) (**Figure 5b**). According to our analysis, AAV2 ITR belongs to group H2 (**Figure 5c**). A fourth proximal region called D remains single-stranded if not annealed to the opposite polarity strand or not in an intramolecular manner to the D<sup>0</sup> region in 3<sup>0</sup> . Each ITR can be found in two alternative configurations termed "flip" and "flop" distinguishable by the BB'-CC' orientation (**Figure 5a**), as a direct result of the replication mechanism. There are nomenclature inconsistencies in the literature. Here, ITR regions are named based on Lusby and Berns' publication [22] and ordered as followed ABB<sup>0</sup> CC<sup>0</sup> A0 D from 5<sup>0</sup> to 3<sup>0</sup> .

For historical reasons and the sake of convenience, most of the AAV vectors contain the ITR of AAV serotype 2, the sole viral sequences required for the

*Recent Advances in Canine Medicine*

#### **Figure 5.**

*Inverted terminal repeats of the adeno-associated virus (AAV) serotype 2. (a) Scheme of five prime and three-prime ITRs of the wild-type AAV serotype 2. (b) Two-dimensional drawing of the five-prime ITR in flip configuration. (c) Predictive folding of the five-prime AAV2 ITR using RNAfold. The color code is the same that for Figure 2 (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi). (d) Putative human transcription factor binding sites in AAV2 ITR. ITR regions are named based on Lusby and Berns' publication [22].*

replication and packaging of the recombinant genome in AAV capsids. Additional functions of AAV2 ITR have been described, such as a promoter activity [23], a role in the virus persistence either through genome integration [24] or recombination to form monomeric or concatemeric episomes [25].

Strikingly, the GC content of AAV2 ITR corresponds to the highest score of all studied parvoviral telomeres (69%). No predictive G4 was found using stringent parameters, unlike Satkunanathan *et al.* who described 18 QGRS inside the AAV2 ITR sequences [14]. In addition, no potential triplex structure was found. According to the PCA (**Figure 4**), AAV2 belongs to cluster 1 with several other *Dependoparvoviruses*.

Putative binding sites for human transcription factors (TF) have already been described in AAV2 ITR [26, 27]. We completed this work by analyzing human TF for AAV serotypes 1 to 7 ITRs using the Alggen-Promo tool with 0% sequence dissimilarity (**Table 2**) [28, 29]. Five human TF sites were found in AAV ITR: C/EBPbeta, Pax-5, YY1, AP-2alphaA, and GR-alpha. The C/EBPbeta was found in most of the


#### **Table 2.**

*Putative recognition sites of human transcription factors in inverted terminal repeats of AdenoAssociated virus serotypes. Predictions was realized using Alggen-promo tool with 0% sequence dissimilarity (http://alggen.lsi.upc.e s/cgi-bin/promo\_v3/promo/promoinit.cgi?dirDB=TF\_8.3).*

AAV serotypes and unlike the other found TF, is mainly involved in immune responses. In our study, the GR-alpha was only found in AAV5 ITR contains two predictive TF binding sites, one for C/EBPbeta and other for YY1 (**Figure 5d**). YY1 participates in the initial steps of replication by binding to the p5 promoter region of AAV [30, 31].
