Generating DNA Expression Cassettes Encoding Multimeric Artificial MicroRNA Precursors.

RNA interference (RNAi) is a promising tool for the treatment of chronic viral infection, such as that caused by the hepatitis B virus (HBV). RNAi activators, including expressed primary microRNA (pri-miRNA) mimics, can effectively silence viral gene expression and thereby inhibit viral replication. Here we describe a protocol for the design, generation and functional assessment of cassettes encoding effective single and multimeric pri-miRNA mimics.

AAV-Mediated Expression of Broadly Neutralizing and Vaccine-like Antibodies Targeting the HIV-1 Envelope V2 Region.

HIV-1 infection continues to be a global health challenge and a vaccine is urgently needed. Broadly neutralizing antibodies (bNAbs) are considered essential as they inhibit multiple HIV-1 strains, but they are difficult to elicit by conventional immunization. In contrast, non-neutralizing antibodies that correlated with reduced risk of infection in the RV144 HIV vaccine trial are relatively easy to induce, but responses are not durable.

Design of Effective Primary MicroRNA Mimics With Different Basal Stem Conformations.

Primary microRNA (pri-miRNA) mimics are important mediators of effective gene silencing and are well suited for sustained therapeutic applications. Pri-miRNA mimics are processed in the endogenous miRNA biogenesis pathway, where elements of the secondary RNA structure are crucial for efficient miRNA production. Cleavage of the pri-miRNA to a precursor miRNA (pre-miRNA) by Drosha-DGCR8 typically occurs adjacent to a basal stem of ~11 bp.