Inverted terminal repeats

In recent years, there have been significant improvements in production and purification of rAAV vectors. The major improvements in production have included enhanced output of the number of DNase resistant particles (drp) per cell and the emergence of scaleable systems. The most widely utilized rAAV vector production methods require four genetic elements (Hermonat and Muzyczka, 1984 Tratschinetal., 1984) (1) mammalian tissue culture cells, (2) vector sequences containing a transgene flanked by AAV inverted terminal repeats, (3) AAV helper sequences comprising... 

Fig. 3.2. Schematic of recombinant AAV dual-vector strategies for gene therapy for hemophilia A. (A) depicts the individual expression of the 5 and 3 ends of the factor VIII protein followed by heterodimerization to generate a function protein. (B) shows concatamerized 5 and 3 vectors in the correct head-to-tail orientation. Splicing of vectors to remove the intron and inverted terminal repeats leads to expression of the entire factor VIII protein from a single mature mRNA transcript. ITR, AAV inverted terminal repeat SD, splice donor SA, splice acceptor An, poly A.

AAV virions are small nonenveloped particles (20-25 nm) that carry a linear single-stranded DNA (ssDNA) genome, which is 4.7 kb in size. Two open reading frames (ORFs), rep and cap, have been identified in the viral genome and are flanked by T-shaped inverted terminal repeats (ITRs). The cap ORF encodes for the structural proteins that form the capsid, whereas the regulatoiy proteins are produced from the rep ORF (Figure L4). [For more details see (1).]... 

Fig. 2.1 The single-stranded DNA genome of AAV. The inverted terminal repeats (ITRs) flank the two open reading frames rep and cap. The rep gene encodes four nonstructural proteins -Rep78, Rep68, Rep52, and Rep40. The cap gene encodes three structural proteins - VPl, VP2, and VP3. The location of the promoters, p5, pl9, and p40 are depicted by arrows...

Since McClintock s early work on mobile elements in corn, transposons have been identified in other eukaryotes. For Instance, approximately half of all the spontaneous mutations observed in Drosophila are due to the Insertion of mobile elements. Although most of the mobile elements in Drosophila function as retrotransposons, at least one—the P element—functions as a DNA transposon, moving by a cut-and-paste mechanism similar to that used by bacterial insertion sequences. Current methods for constructing transgenic Drosophila depend on engineered, high-level expression of the P-element transposase and use of the P-element Inverted terminal repeats as targets for transposition. 

The core of an adenoviral particle consists of the DNA genome complexed with four polypeptides (pV, pVII, mu, TP). Figure 10.1-4 schematically shows the structure of an adenovirus genome. The extremities contain inverted terminal repeat (ITR) sequences (100-140bp), covalently linked to terminal proteins (TPs), which function as replication origins [23]. The nearby / sequence at the left end of the... 

Figure 10.1-3. Schematic structure of an adenovirus particle. Major capsid (hexon, penton, fiber) and core (pV, pVII, Mu) proteins are shown. Abbreviation ITR—inverted terminal repeat TP—terminal protein. (Adapted from Ref. 27.)...

Figure 10.1-4. The adenovirus genome. The early (E1-E4) and late (L1-L5) transcription units are identified by the arrows. The inverted terminal repeats (ITRs), packaging sequence ( /), and major late promoter (MLP) are also shown. (Adapted from Ref. 26.)...

Ishino, M., Sawada, Y., Yaegashi, T., Demura, M. et al. (1987) Nucleotide sequence of the adenovirus type 40 inverted terminal repeat close relation to that of adenovirus type 5. Virology, 15, 414-416. 

Cni Z, Geurts AM, Liu G, Kaufman CD, Hackett PB (2002) Structure-function analysis of the inverted terminal repeats of the Sleeping Beauty transposon. J Mol Biol 318 1221-1235. 

Hsiao CD, Hsieh FJ, Tsai HJ (2001) Enhanced expression and stable transmission of transgenes flanked by inverted terminal repeats from adeno-associated virus in zebrafish. Dev Dyn 220 323-336. 

The most essential and unique component in AAV is its inverted terminal repeats (ITRs) at both ends of the viral genome (57). In AAV-2, the ITR is composed of 145 self-complementary nucleotides (Fig. 2). This is an extremely GC (guanosine and cytosine)-rich sequence with a GC content of higher than 80% (the average GC content in human chromosomal DNA is less than 60% (58)). The overall structure ofthe ITR is a T-shaped hairpin with a three-way junction (Fig. 2). The A and A fragments form a duplex stem which terminates with two small palindromes (defined as B, B and C, C arms). Depending on the orientation of these two small palindromes, AAV ITRs can be classified as flip (B arm is closer to the 3 end) or flop (C arm is closer to the 3 end) (29). Biophysical studies have indicated that the ITR is composed of largely... 

Ren J, Qu X, Chaires JB, Trempe JP, Dignam SS, Dignam JD. Spectral and physical characterization of the inverted terminal repeat DNA struettrre fiom adeno-assoeiated virus 2. Nttcleic Acids Res 1999 27 1985-1990. 

Wang XS, Ponnazhagan S, Srivastava A. Rescue and replication of adeno-assoeiated virus type 2 as well as vector DNA sequences from recombinant plasmids containing deletions in the viral inverted terminal repeats selective encapsidation of viral genomes in progeny virions. J Virol 1996 70 1668-1677. 

Wang XS, Qing K, Ponnazhagan S, Srivastava A. Adeno-assoeiated vims type 2 DNA rephcalion in vivo mutation analyses of the D sequence in viral inverted terminal repeats. J Virol 1997 71 3077-3082. 

In other transcriptionally regulated Ad constructs described to date, the El A promoter has been removed and replaced with tumor or tissue-specific promoters (81-84). A list of different promoters that have been used to date is shown in Table 1. In some cases, interference from the nearby inverted terminal repeat (ITR) sequence can result in the loss of promoter strength and fidelity (85). Selectivity can be enhanced with the use of silencer (86) or insulator sequences (87). For example, the AdEla04i viras using the artificial a-fetoprotein (AFP) promoter to express ElA needed six copies of a silencer sequence to repress the promoter activity in normal hepatocytes. This virus showed specificity and potency for hepatoma carcinoma tumors and not in non-AFP- producing cells or in normal hepatocytes (86). 

Figure 1 Map of adenoviral vectors derived from wild-type adenovims. (a) First-generation vectors, (b) Second-generation vectors, (e) Ibird-generation veetors. Abbreviations Ad, adenovirus vector E, early region viral genes ITR, inverted terminal repeat L, late region viral genes WT, wild-type TS, temperature-sensitive D, deletion of an early or late region gene , with or without...

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