Chemical Modifications of the Capsid for Redirecting and Improving the Efficacy of Adeno-Associated Virus Vectors

Adeno-associated virus (AAV) vector-directed gene therapy is one of the most exciting modalities of biotechnology as more applications enter clinical stage. Although AAV vectors generally feature low toxicity, high stability, and long-lasting transgene expression, potential challenging issues of AAV include high vector dose, limited tissue tropism, and the host immune response and inflammation, which are all related to the capsid protein. To overcome these challenges, various strategies have been developed to engineer AAV capsids. Apart from widely employed genetic engineering of capsid protein, powerful and versatile chemical modification strategies are underexploited. This minireview summarizes recent advances and our perspectives for future direction in AAV capsid chemical modification to enhance its therapeutic use for gene therapy

Mechanistic understanding of \u3ci\u3eN\u3c/i\u3e-glycosylation in Ebola virus glycoprotein maturation and function

The Ebola virus (EBOV) trimeric envelope glycoprotein (GP) precursors are cleaved into the receptor-binding GP1 and the fusion-mediating GP2 subunits and incorporated into virions to initiate infection. GP1 and GP2 form heterodimers that have 15 or two N-glycosylation sites (NGSs), respectively. Here we investigated the mechanism of how N-glycosylation contributes to GP expression, maturation, and function. As reported before, we found that, although GP1 NGSs are not critical, the two GP2 NGSs, Asn563 and Asn618, are essential for GP function. Further analysis uncovered that Asn563 and Asn618 regulate GP processing, demannosylation, oligomerization, and conformation. Consequently, these two NGSs are required for GP incorporation into EBOV-like particles and HIV type 1 (HIV-1) pseudovirions and determine viral transduction efficiency. Using CRISPR/Cas9 technology, we knocked out the two classical endoplasmic reticulum chaperones calnexin (CNX) and/or calreticulin (CRT) and found that bothCNXand CRT increase GP expression. Nevertheless, NGSs are not required for the GP interaction with CNX or CRT. Together, we conclude that, although Asn563 and Asn618 are not required for EBOV GP expression, they synergistically regulate its maturation, which determines its functionality

Adiponectin receptor fragmentation in mouse models of type 1 and type 2 diabetes

The protein hormone adiponectin regulates glucose and fatty acid metabolism by binding to two PAQR-family receptors (AdipoR1 and AdipoR2). Both receptors feature a C-terminal segment which is released by proteolysis to form a freely circulating C-terminal fragment (CTF) found in the plasma of normal individuals but not in some undefined diabetes patients. The AdipoR1-CTF344-376 is a competitive inhibitor of tumor necrosis factor α cleavage enzyme (TACE) but it contains a shorter peptide domain (AdipoR1 CTF351-362) that is a strong non-competitive inhibitor of insulin-degrading enzyme (IDE). The link between adiponectin receptor fragmentation and diabetes pathology is unclear but could lead to new therapeutic strategies. We therefore investigated physiological variations in the concentrations of CTF in non-obese diabetic (NOD/ShiLtJ) mice and C57BL/6 mice with diet-induced obesity (DIO) as models of diabetes types 1 and 2, respectively. We tested for changes in adiponectin receptor signaling, immune responses, disease progression, and the abundance of neutralizing autoantibodies. Finally, we administered exogenous AdipoR1-CTF peptides either containing or lacking the IDE-binding domain. We observed the more pronounced CTF shedding in the TACE-active NOD mice, which represents an inflammatory autoimmune phenotype, but fragmentation was also observed to a lesser extent in the DIO model. Autoantibodies to CTF were detected in both models. Neither exogenous CTF peptide affected IgG-CTF plasma levels, body weight or the conversion of NOD mice to diabetes. The pattern of AdipoR1 fragmentation and autoantibody production under physiological conditions of aging, DIO, and autoimmune diabetes therefore provides insight into the association adiponectin biology and diabetes

Arms Race between Enveloped Viruses and the Host ERAD Machinery

Enveloped viruses represent a significant category of pathogens that cause serious diseases in animals. These viruses express envelope glycoproteins that are singularly important during the infection of host cells by mediating fusion between the viral envelope and host cell membranes. Despite low homology at protein levels, three classes of viral fusion proteins have, as of yet, been identified based on structural similarities. Their incorporation into viral particles is dependent upon their proper sub-cellular localization after being expressed and folded properly in the endoplasmic reticulum (ER). However, viral protein expression can cause stress in the ER, and host cells respond to alleviate the ER stress in the form of the unfolded protein response (UPR); the effects of which have been observed to potentiate or inhibit viral infection. One important arm of UPR is to elevate the capacity of the ER-associated protein degradation (ERAD) pathway, which is comprised of host quality control machinery that ensures proper protein folding. In this review, we provide relevant details regarding viral envelope glycoproteins, UPR, ERAD, and their interactions in host cells

A novel class of self-complementary AAV vectors with multiple advantages based on cceAAV lacking mutant ITR

Self-complementary AAV vectors (scAAV) use a mutant inverted terminal repeat (mITR) for efficient packaging of complementary stranded DNA, enabling rapid transgene expression. However, inefficient resolution at the mITR leads to the packaging of monomeric or subgenomic AAV genomes. These noncanonical particles reduce transgene expression and may affect the safety of gene transfer. To address these issues, we have developed a novel class of scAAV vectors called covalently closed-end double-stranded AAV (cceAAV) that eliminate the mITR resolution step during production. Instead of using a mutant ITR, we used a 56-bp recognition sequence of protelomerase (TelN) to covalently join the top and bottom strands, allowing the vector to be generated with just a single ITR. To produce cceAAV vectors, the vector plasmid is initially digested with TelN, purified, and then subjected to a standard triple-plasmid transfection protocol followed by traditional AAV vector purification procedures. Such cceAAV vectors demonstrate yields comparable to scAAV vectors. Notably, we observed enhanced transgene expression as compared to traditional scAAV vectors. The treatment of mice with hemophilia B with cceAAV-FIX resulted in significantly enhanced long-term FIX expression. The cceAAV vectors hold several advantages over scAAV vectors, potentially leading to the development of improved human gene therapy drugs