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.

Gene therapy is widely used to treat incurable disorders and has become a routine procedure in clinical practice. Since viruses can exhibit specific tropisms, effectively penetrate the cell, and are easy to use, most gene therapy approaches are based on viral delivery of genetic material. However, viral vectors have some disadvantages, such as immune response and cytotoxicity induced by a disturbance of cell metabolism, including miRNA pathways that are an important part of transcription regulation. Therefore, any viral-based gene therapy approach involves the evaluation of side effects and safety. It is possible for such effects to be caused either by the viral vectors themselves or by the delivered genetic material. Many gene therapy techniques use non-coding RNA delivery as an effective agent for gene expression regulation, with the risk of cellular miRNA pathways being affected due to the nature of the non-coding RNAs. This review describes the effect of viral vector entry and non-coding RNA delivery by these vectors on miRNA signaling pathways.