Browsing by Author "Agbandje-McKenna, Mavis"

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Adeno-Associated Virus (AAV) Capsid Stability and Liposome Remodeling During Endo/Lysosomal pH Trafficking
(2020-06) Lins-Austin, Bridget; Patel, Saajan; Mietzsch, Mario; Brooke, Dewey; Bennett, Antonette; Venkatakrishnan, Balasubramanian; Van Vliet, Kim; Smith, Adam N.; Long, Joanna R.; McKenna, Robert; Potter, Mark; Byrne, Barry; Boye, Sanford L.; Bothner, Brian; Heilbronn, Regine; Agbandje-McKenna, Mavis
Adeno-associated viruses (AAVs) are small, non-pathogenic ssDNA viruses being used as therapeutic gene delivery vectors for the treatment of a variety of monogenic diseases. An obstacle to successful gene delivery is inefficient capsid trafficking through the endo/lysosomal pathway. This study aimed to characterize the AAV capsid stability and dynamics associated with this process for a select number of AAV serotypes, AAV1, AAV2, AAV5, and AAV8, at pHs representative of the early and late endosome, and the lysosome (6.0, 5.5, and 4.0, respectively). All AAV serotypes displayed thermal melt temperatures that varied with pH. The stability of AAV1, AAV2, and AAV8 increased in response to acidic conditions and then decreased at pH 4.0. In contrast, AAV5 demonstrated a consistent decrease in thermostability in response to acidification. Negative-stain EM visualization of liposomes in the presence of capsids at pH 5.5 or when heat shocked showed induced remodeling consistent with the externalization of the PLA2 domain of VP1u. These observations provide clues to the AAV capsid dynamics that facilitate successful infection. Finally, transduction assays revealed a pH and temperature dependence with low acidity and temperatures > 4 °C as detrimental factors.
Analysis of phospholipase activity in adeno-associated virus particles by liquid-chromatography/mass-spectrometry
(2013-03) Brooke, Dewey; Bothner, Brian; Agbandje-McKenna, Mavis
Adeno-associated virus (AAV) belongs to the Parvovridae, a family of small, non-enveloped isosahedral viruses. The viral capsid has T=1 symmetry and is composed of 60 subunits, made up from three proteins (VP1, VP2, VP3) in a ratio of 1:1:10. The minor proteins are the same as VP3 in their C-termini region, but they have additional domains on their N-termini that play essential roles in cellular entry and trafficking. Structural studies of AAV have shown that the N-termini of VP1 and VP2 are initially internalized in the capsid and become externalized, most likely during endocytosis. Based on sequence and structural similarity, VP1 contains a phospholipase A2 domain (PLA2) which, when mutated, dramatically reduces infectivity. Currently, little is known about the mechanism of VP1 externalization or the role of the lipase in escape of the virus particle from the endosome. Also, due to low sequence similarity, there is even concern over whether this is a true PLA2 type domain. To address these questions, we have developed a liquid-chromatography mass-spectrometry based assay for lipase activity. To date, we have tested factors such as receptor binding, heat, and pH on the externalization of the PLA2 and are addressing the question of substrate specificity.
Structural Dynamics and Activity of B19V VP1u during the pHs of Cell Entry and Endosomal Trafficking
(MDPI AG, 2022-08) Lakshmanan, Renuk V.; Hull, Joshua A.; Berry, Luke; Burg, Matthew; Bothner, Brian; McKenna, Robert; Agbandje-McKenna, Mavis
Parvovirus B19 (B19V) is a human pathogen that is the causative agent of fifth disease in children. It is also known to cause hydrops in fetuses, anemia in AIDS patients, and transient aplastic crisis in patients with sickle cell disease. The unique N-terminus of Viral Protein 1 (VP1u) of parvoviruses, including B19V, exhibits phospholipase A2 (PLA2) activity, which is required for endosomal escape. Presented is the structural dynamics of B19V VP1u under conditions that mimic the pHs of cell entry and endosomal trafficking to the nucleus. Using circular dichroism spectroscopy, the receptor-binding domain of B19V VP1u is shown to exhibit an α-helical fold, whereas the PLA2 domain exhibits a probable molten globule state, both of which are pH invariant. Differential scanning calorimetry performed at endosomal pHs shows that the melting temperature (Tm) of VP1u PLA2 domain is tuned to body temperature (37 °C) at pH 7.4. In addition, PLA2 assays performed at temperatures ranging from 25–45 °C show both a temperature and pH-dependent change in activity. We hypothesize that VP1u PLA2 domain differences in Tm at differing pHs have enabled the virus to “switch on/off” the phospholipase activity during capsid trafficking. Furthermore, we propose the environment of the early endosome as the optimal condition for endosomal escape leading to B19V infection.
Thermal stability as a determinant of AAV serotype identity
(2017-09) Bennett, Antonette; Patel, Saajan; Mietzsch, Mario; Jose, Ariana; Lins-Austin, Bridget; Yu, Jennifer C.; Bothner, Brian; McKenna, Robert; Agbandje-McKenna, Mavis
Currently, there are over 150 ongoing clinical trials utilizing adeno-associated viruses (AAVs) to target various genetic diseases, including hemophilia (AAV2 and AAV8), congenital heart failure (AAV1 and AAV6), cystic fibrosis (AAV2), rheumatoid arthritis (AAV2), and Batten disease (AAVrh.10). Prior to patient administration, AAV vectors must have their serotype, concentration, purity, and stability confirmed. Here, we report the application of differential scanning fluorimetry (DSF) as a good manufacturing practice (GMP) capable of determining the melting temperature (Tm) for AAV serotype identification. This is a simple, rapid, cost effective, and robust method utilizing small amounts of purified AAV capsids (âˆ¼25 Î¼L of âˆ¼1011 particles). AAV1-9 and AAVrh.10 exhibit specific Tms in buffer formulations commonly used in clinical trials. Notably, AAV2 and AAV3, which are the least stable, have varied Tms, whereas AAV5, the most stable, has a narrow Tm range in the different buffers, respectively. Vector stability was dictated by VP3 only, specifically, the ratio of basic/acidic amino acids, and was independent of VP1 and VP2 content or the genome packaged. Furthermore, stability of recombinant AAVs differing by a single basic or acidic amino acid residue are distinguishable. Hence, AAV DSF profiles can serve as a robust method for serotype identification of clinical vectors.