Molecular Biology

Neutralizing antibodies (Nabs) are a major challenge in clinical trials of adeno-associated virus (AAV) vector gene therapy, because Nabs are able to inhibit AAV transduction in patients. We have successfully isolated several novel Nab-escaped AAV chimeric capsids in mice by administrating a mixture of AAV shuffled library and patient serum. These AAV chimeric capsid mutants enhanced Nab evasion from patient serum with a high muscle transduction efficacy. In this protocol, we describe the procedures for selection of the Nab-escaped AAV chimeric capsid, including isolation and characterization of Nab-escaping AAV mutants in mice muscle.

Interferon regulatory transcription factor 3 (IRF3) is a transcription factor that upon activation by virus infection promotes the synthesis of antiviral genes, such as the interferons (Hiscott, 2007). In addition to inducing genes, IRF3 triggers antiviral apoptosis by RIG-I-like receptor-induced IRF3 mediated pathway of apoptosis (RIPA), which is independent of its transcriptional activity. RIPA protects against lethal virus infection in cells and mice (Chattopadhyay et al., 2016). In the absence of RIPA, caused by genetic ablation, chemical mutagenesis or inhibition of the pattern recognition receptor (PRR) retinoic acid-inducible gene I (RIG-I), Sendai virus (SeV) infection does not trigger cellular apoptosis and become persistently infected (Peters et al., 2008; Chattopadhyay et al., 2013). IRF3-expressing wild type (WT) cells (U4C) undergo SeV-induced apoptosis; however, the P2.1 cells, which are deficient in IRF3 expression are not capable of triggering viral apoptosis (Figure 1). Ectopic expression of human IRF3 restores the apoptotic activity in P2.1 cells (P2.1/IRF3, Figure 1). SeV is used as a model for studying pathogenic human viruses, which are difficult to work with or require BSL3 facility. We have previously reported that both human and mouse cells can establish SeV persistence in the absence of IRF3’s apoptotic activity (Chattopadhyay et al., 2013). Here, we outline a detailed procedure for the development of a persistently SeV-infected human cell line (Figure 2), which continuously expresses viral protein and produces low levels of infectious viral particles.


Figure 1. SeV-induced apoptosis is IRF3-dependent. HT1080-derived cell lines (U4C, P2.1 and P2.1/IRF3) were infected with Sendai virus and three days post infection culture fields were photographed, scale bar represents 50 µm.

Ribosome-inactivating proteins (RIPs) are enzymes that irreversibly inactivate ribosomes as a consequence of their N-glycosylase (EC 3.2.2.22) activity. The enzyme cleaves the N-glycosidic bond between the adenine No. 4324 from the 28S rRNA and its ribose in rat ribosomes (or the equivalent adenine in sensitive ribosomes from other organisms). This adenine is located in the α-sarcin-ricin loop (SRL) that is crucial for anchoring the elongation factor (EF) G and EF2 on the ribosome during mRNA-tRNA translocation in prokaryotes and eukaryotes, respectively. RIPs have been isolated mainly from plants and examples of these proteins are ricin or Pokeweed Antiviral Protein (PAP). These proteins, either alone or as a part of immunotoxins, are useful tools for cancer therapy. The following protocol describes a method to detect the RNA fragment released when the RIP-treated apurinic RNA from rabbit reticulocyte lysate is incubated in the presence of acid aniline by electrophoresis on polyacrylamide gels. The fragment released (Endo’s fragment) is diagnostic of the action of RIPs.