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

Multiple Reaction Monitoring Profiling (MRM-Profiling) of Lipids To Distinguish Strain-Level Differences in Microbial Resistance in Escherichia coli

The worldwide increase in antimicrobial resistance is due to antibiotic overuse in agriculture and overprescription in medicine. For appropriate and timely patient support, faster diagnosis of antimicrobial resistance is required. Current methods for bacterial identification rely on genomics and proteomics and use comparisons with databases of known strains, but the diagnostic value of metabolites and lipids has not been explored significantly. Standard mass spectrometry/chromatography methods involve multiple dilutions during sample preparation and separation. To increase the amount of chemical information acquired and the speed of analysis of lipids, multiple reaction monitoring profiling (MRM-Profiling) has been applied. The MRM-Profiling workflow includes a discovery stage and a screening stage. The discovery stage employs precursor (PREC) ion and neutral loss (NL) scans to screen representative pooled samples for functional groups associated with particular lipid classes. The information from the first stage is organized in precursor/product ion pairs, or MRMs, and the screening stage rapidly interrogates individual samples for these MRMs. In this study, we performed MRM-Profiling of lipid extracts from four different strains of Escherichia coli cultured with amoxicillin or amoxicillin/clavulanate, a β-lactam and β-lactamase inhibitor, respectively. t tests, analysis of variance and receiver operating characteristic (ROC) curves were used to determine the significance of each MRM. Principal component analysis was applied to distinguish different strains cultured under conditions that allowed or disallowed development of bacterial resistance. The results demonstrate that MRM-Profiling distinguishes the lipid profiles of resistant and nonresistant E. coli strains

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

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

Chemical modification of AAV9 capsid with N-ethyl maleimide alters vector tissue tropism

Abstract Although more adeno-associated virus AAV-based drugs enter the clinic, vector tissue tropism remains an unresolved challenge that limits its full potential despite that the tissue tropism of naturally occurring AAV serotypes can be altered by genetic engineering capsid vie DNA shuffling, or molecular evolution. To further expand the tropism and thus potential applications of AAV vectors, we utilized an alternative approach that employs chemical modifications to covalently link small molecules to reactive exposed Lysine residues of AAV capsids. We demonstrated that AAV9 capsid modified with N-ethyl Maleimide (NEM) increased its tropism more towards murine bone marrow (osteoblast lineage) while decreased transduction of liver tissue compared to the unmodified capsid. In the bone marrow, AAV9-NEM transduced Cd31, Cd34, and Cd90 expressing cells at a higher percentage than unmodified AAV9. Moreover, AAV9-NEM localized strongly in vivo to cells lining the calcified trabecular bone and transduced primary murine osteoblasts in culture, while WT AAV9 transduced undifferentiated bone marrow stromal cells as well as osteoblasts. Our approach could provide a promising platform for expanding clinical AAV development to treat bone pathologies such as cancer and osteoporosis. Thus, chemical engineering the AAV capsid holds great potential for development of future generations of AAV vectors

Fast and high-throughput LC-MS characterization, and peptide mapping of engineered AAV capsids using LC-MS/MS

Adeno-associated virus (AAV) has emerged as a leading platform for gene therapy. With the skyrocketing rate of AAV research and the prevalence of many new engineered capsids being investigated in preclinical and clinical trials, capsid characterization plays a vital role in serotype confirmation and quality control. Further, peptide mapping the capsid proteins might inevitably be a future requirement by regulatory agencies since it is a critical step in good manufacturing practice (GMP) for biotherapeutic characterization. To overcome many challenges that traditional methods like SDS-PAGE and western blots carry, liquid chromatography and mass spectrometry (LC-MS) allows high resolution and sensitivity with great accuracy in characterizing the AAV capsid proteins. Our optimized LC-MS method provides quick sample preparation, a fast and high-throughput 4-min run, and high sensitivity, which allows for very efficient characterization of wild-type and engineered capsids. This study also reports the usage of LC-MS/MS peptide mapping of AAV capsid proteins to determine the most accessible lysine residues targeted by chemical modifications. Our detailed protocols are anticipated to promote the development and discovery of AAV variants with high accuracy and efficiency

Cryptic resolution sites in the vector plasmid lead to the heterogeneities in the rAAV vectors

Recombinant adeno-associated virus (rAAV) vectors carry a cassette of interest retaining only the inverted terminal repeats (ITRs) from the wild-type virus. Conventional rAAV production primarily uses a vector plasmid as well as helper genes essential for AAV replication and packaging. Nevertheless, plasmid backbone related contaminants have been a major source of vector heterogeneity. The mechanism driving the contamination phenomenon has yet to be elucidated. Here we identified cryptic resolution sites in the plasmid backbone as a key source for producing snapback genomes, which leads to the increase of vector genome heterogeneity in encapsidated virions. By using a single ITR plasmid as a model molecule and mapping subgenomic particles, we found that there exist a few typical DNA break hotspots in the vector DNA plasmid backbone, for example, on the ampicillin DNA element, called aberrant rescue sites. DNA around these specific breakage sites may assume some typical secondary structures. Similar to normal AAV vectors, plasmid DNA with a single ITR was able to rescue and replicate efficiently. These subgenomic DNA species significantly compete for trans factors required for rAAV rescue, replication, and packaging. The replication of single ITR contaminants during AAV production is independent of size. Packaging of these species is greatly affected by its size. A single ITR and a cryptic resolution site in the plasmid work synergistically, likely causing a source of plasmid backbone contamination