Methods Matter: Standard Production Platforms for Recombinant AAV Produce Chemically and Functionally Distinct Vectors.

Different approaches are used in the production of recombinant adeno-associated virus (rAAV). The two leading approaches are transiently transfected human HEK293 cells and live baculovirus infection of Spodoptera frugiperda (Sf9) insect cells. Unexplained differences in vector performance have been seen clinically and preclinically. Thus, we performed a controlled comparative production analysis varying only the host cell species but maintaining all other parameters. We characterized differences with multiple analytical approaches: proteomic profiling by mass spectrometry, isoelectric focusing, cryo-EM (transmission electron cryomicroscopy), denaturation assays, genomic and epigenomic sequencing of packaged genomes, human cytokine profiling, and functional transduction assessments in vitro and in vivo, including in humanized liver mice. Using these approaches, we have made two major discoveries: (1) rAAV capsids have post-translational modifications (PTMs), including glycosylation, acetylation, phosphorylation, and methylation, and these differ between platforms; and (2) rAAV genomes are methylated during production, and these are also differentially deposited between platforms. Our data show that host cell protein impurities differ between platforms and can have their own PTMs, including potentially immunogenic N-linked glycans. Human-produced rAAVs are more potent than baculovirus-Sf9 vectors in various cell types in vitro (p < 0.05-0.0001), in various mouse tissues in vivo (p < 0.03-0.0001), and in human liver in vivo (p < 0.005). These differences may have clinical implications for rAAV receptor binding, trafficking, expression kinetics, expression durability, vector immunogenicity, as well as cost considerations.

BKCa channel regulates calcium oscillations induced by alpha-2-macroglobulin in human myometrial smooth muscle cells.

The large-conductance, voltage-gated, calcium (Ca(2+))-activated potassium channel (BKCa) plays an important role in regulating Ca(2+)signaling and is implicated in the maintenance of uterine quiescence during pregnancy. We used immunopurification and mass spectrometry to identify proteins that interact with BKCain myometrium samples from term pregnant (≥37 wk gestation) women. From this screen, we identified alpha-2-macroglobulin (α2M). We then used immunoprecipitation followed by immunoblot and the proximity ligation assay to confirm the interaction between BKCaand both α2M and its receptor, low-density lipoprotein receptor-related protein 1 (LRP1), in cultured primary human myometrial smooth muscle cells (hMSMCs). Single-channel electrophysiological recordings in the cell-attached configuration demonstrated that activated α2M (α2M*) increased the open probability of BKCain an oscillatory pattern in hMSMCs. Furthermore, α2M* caused intracellular levels of Ca(2+)to oscillate in oxytocin-primed hMSMCs. The initiation of oscillations required an interaction between α2M* and LRP1. By using Ca(2+)-free medium and inhibitors of various Ca(2+)signaling pathways, we demonstrated that the oscillations required entry of extracellular Ca(2+)through store-operated Ca(2+)channels. Finally, we found that the specific BKCablocker paxilline inhibited the oscillations, whereas the channel opener NS11021 increased the rate of these oscillations. These data demonstrate that α2M* and LRP1 modulate the BKCachannel in human myometrium and that BKCaand its immunomodulatory interacting partners regulate Ca(2+)dynamics in hMSMCs during pregnancy.

N-terminal isoforms of the large-conductance Ca²âº-activated K⁺ channel are differentially modulated by the auxiliary ß1-subunit.

The large-conductance Ca(2+)-activated K(+) (BK(Ca)) channel is essential for maintaining the membrane in a hyperpolarized state, thereby regulating neuronal excitability, smooth muscle contraction, and secretion. The BK(Ca) α-subunit has three predicted initiation codons that generate proteins with N-terminal ends starting with the amino acid sequences MANG, MSSN, or MDAL. Because the N-terminal region and first transmembrane domain of the α-subunit are required for modulation by auxiliary ß1-subunits, we examined whether ß1 differentially modulates the N-terminal BK(Ca) α-subunit isoforms. In the absence of ß1, all isoforms had similar single-channel conductances and voltage-dependent activation. However, whereas ß1 did not modulate the voltage-activation curve of MSSN, ß1 induced a significant leftward shift of the voltage activation curves of both the MDAL and MANG isoforms. These shifts, of which the MDAL was larger, occurred at both 10 µM and 100 µM Ca(2+). The ß1-subunit increased the open dwell times of all three isoforms and decreased the closed dwell times of MANG and MDAL but increased the closed dwell times of MSSN. The distinct modulation of voltage activation by the ß1-subunit may be due to the differential effect of ß1 on burst duration and interburst intervals observed among these isoforms. Additionally, we observed that the related ß2-subunit induced comparable leftward shifts in the voltage-activation curves of all three isoforms, indicating that the differential modulation of these isoforms was specific to ß1. These findings suggest that the relative expression of the N-terminal isoforms can fine-tune BK(Ca) channel activity in cells, highlighting a novel mechanism of BK(Ca) channel regulation.

Effects of progesterone treatment on expression of genes involved in uterine quiescence.

An important action of progesterone during pregnancy is to maintain the uterus in a quiescent state and thereby prevent preterm labor. The causes of preterm labor are not well understood, so progesterone action on the myometrium can provide clues about the processes that keep the uterus from contracting prematurely. Accordingly, we have carried out Affymetrix GeneChip analysis of progesterone effects on gene expression in immortalized human myometrial cells cultured from a patient near the end of pregnancy. Progesterone appears to inhibit uterine excitability by a number of mechanisms, including increased expression of calcium and voltage-operated K(+) channels, which dampens the electrical activity of the myometrial cell, downregulation of agents, and receptors involved in myometrial contraction, reduction in cell signal components that lead to increased intracellular Ca(2+) concentrations in response to contractile stimuli, and downregulation of proteins involved in the cross-linking of actin and myosin filaments to produce uterine contractions.

Nardilysin convertase regulates the function of the maxi-K channel isoform mK44 in human myometrium.

In smooth muscle, large-conductance Ca(2+)- and voltage-activated K(+) channels from the gene KCNMA (maxi-K channels) generate isoforms with disparate responses to contractile stimuli. We previously showed that the human myometrium expresses high levels of the splice variant of the maxi-K channel containing a 44-amino acid insertion (mK44). The studies presented here demonstrate that nardilysin convertase, a Zn(2+)-dependent metalloprotease of the insulinase family, regulates the plasma membrane expression of mK44 and its response to increases in intracellular Ca(2+). We show that nardilysin convertase isoform 1 is present in human myometrium and colocalizes with mK44. Studies indicate that nardilysin convertase regulates 1) retention of the mK44 COOH-terminal fragment in the endoplasmic reticulum in quiescent myometrial smooth muscle and 2) Ca(2+)-induced translocation of mK44 to the plasma membrane. In mouse fibroblasts, nardilysin convertase significantly attenuates mK44-dependent current. In human myometrial smooth muscle cells, inhibition of nardilysin convertase promotes membrane localization of mK44 and an increase in maxi-K current. Overall, our data indicate that, in human myometrium, nardilysin convertase and mK44 channels are a part of the molecular mechanism that regulates the excitability of smooth muscle cells.