AAV- based vector improvements unrelated to capsid protein modification

Abstract

Recombinant adeno-associated virus (rAAV) is the leading platform for delivering genetic constructs in vivo. To date, three AAV-based gene therapeutic agents have been approved by the FDA and are used in clinical practice. Despite the distinct advantages of gene therapy development, it is clear that AAV vectors need to be improved. Enhancements in viral vectors are mainly associated with capsid protein modifications. However, there are other structures that significantly affect the AAV life cycle and transduction. The Rep proteins, in combination with inverted terminal repeats (ITRs), determine viral genome replication, encapsidation, etc. Moreover, transgene cassette expression in recombinant variants is directly related to AAV production and transduction efficiency. This review discusses the ways to improve AAV vectors by modifying ITRs, a transgene cassette, and the Rep proteins.

Keywords: Rep proteins; a transgene cassette; adeno-associated virus; gene therapy; inverted terminal repeats.

FIGURE 1

Directed structure modification of ITRs. (A) Detailed scheme of the ITR structure in FLIP orientation. A red letter indicates a nucleotide changing in different orientations. This scheme was created based on a figure from (110). (B) Some variants of ITR modification. In order from left to right: 1—11/14 bp terminal deletion does not affect AAV viability, since it is rapidly repaired during following second-strand DNA synthesis; 2—mutagenesis in the RBE site reduces binding with Rep proteins. Dinucleotide substitutions in the core 10 bp sequence decreased binding affinity over 10-fold and single-nucleotide substitution up to fivefold (43); 3—mutagenesis in the trs site decreases Rep nicking. The greatest effect of 3–10-fold Rep nicking reduction has been discovered due to mutagenesis in the core 7 bp site representing two thymines. Mutagenesis in the trs flanking regions has similarly been observed to reduce Rep nicking, but only by 20–50% (44); 4—the deletion of the BB’ and CC’ regions reduces viral productivity by 75%, meanwhile, provides an increased level of transgene expression in vitro and in vivo, reaching up to a 6.6-fold increase in some cases, compared to wild-type ITRs (47); 5—substitution of the entire D region of the 5′ ITR by a non-AAV substitute sequence containing transcription factor binding sites (TF BS) led to increased transduction efficiency, possibly due to increased transgene expression (110). Pink color indicates the sequence containing TF BS 6—CpG depleted ITRs are stable in bacterial passaging, thus facilitating AAV production (54). Red markers used for a schematic depiction of guanine and cytosine substitutions to adenine and thimine. In both pictures, the colors indicate the main structures of ITRs. Black—AA’, BB’, CC’ regions; green—RBE site with the core sequence in the black box; blue—RBE’ site; yellow—trs site with two thymines in the black box between which a single-strand break occurs; purple—D-region containing one nucleotide from trs site underlined on (A). Created with BioRender.com.