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About the AAV structure
AAV genome according to the industry experts is developed of ssDNA (single stranded deoxy-ribonucleic acid). It can be negative or positive sensed having a length of about 4.7 kilobase. There are present ITRs (inverted terminal repeats) at the DNA strand’s both ends including 2 ORFs (Open reading frames), namely: cap and rep. The latter comprises of 4 overlapping genes that encodes Rep proteins needed for AAV lifecycle, while the former comprises of overlapping capsid protein nucleotide sequences, namely: VP1-2-3, interacting with one another to form icosahedral symmetry capsid.
The sequences of AAV structure
ITR (Inverted Terminal Repeat) sequences is said to comprise of 145 bases and had been named so after their symmetry, essential for efficient and effective AAV genome multiplication. These sequences have features which provide them with the ability to create hairpin, thereby contributing to so-called self-priming allowing 2nd DNA Strand’s primase independent synthesis. Integration of both AAV DNA to host cell genome (in humans the 19th chromosome) and to rescue from the same, including for efficient AAV DNA encapsidation together with fully assembled generated deoxyribonuclease resistant AAV particles, were displayed by the ITRs.
When gene therapy is concerned, the ITRs appear to be perhaps the only required sequences, in cis following therapeutic gene: packaging (rep) and structural (cap) proteins, with delivery to be made in trans. Having this particular assumption, several methods had been established for effective and efficient AAV Vector System production, comprising of therapeutic or reporter gene. It had been published that the only elements necessary in cis to ensure effective encapsidation and replication is not just limited to the ITRs. Some research groups have found a sequence that has been designated CARE (cis acting Rep dependent element) within the rep gene’s coding sequence. Encapsidation and replication in cis is found to be augmented by CARE.
Rep proteins and rep gene
Genome’s left side has two promoters known as p19 & p5 from where 2 overlapping mRNAs (messenger ribonucleic acids) of varying length is produced. Each is said to comprise of an intron that either is spliced or not. with such possibilities, there are 4 different mRNAs and 4 consequent different Rep proteins that can be synthesized in overlapping sequence. Their sizes are depicted by their names in kDa (kilodaltons): Rep40, Rep52, Rep68 and Rep78. The ITR created hairpin can be specifically bonded in self priming act & cleave at a designated termination resolution site, particular region in the hairpin. Also they were found to be essential for AAVS1 specific AAV packaging genome integration. ATP is shown to be bound by all the 4 Rep proteins and did display helicase activity. They also upregulated the transcription, right from p40 promoter, however, downregulated both p19 and p5 promoters.
BP Proteins & cap gene
The positive sensed AAV expression system’s right side genome encodes 3 capsid proteins’ overlapping sequences, VP1-2-3 that begin from one particular promoter, which is the designated p40. These proteins come with a molecular weight of 62, 72 & 87 kiloDaltons.
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