An efficient microcarrier based Adeno-associated virus production method

Adeno-associated viruses (AAV) are reported to have a great potential for gene therapy. However, a major bottleneck for these kinds of therapies are efficient methods for viral production. The purpose of the present study was to explore how a production-cell culture attached to microcarriers in a stirred tank bioreactor could provide a solution for efficient AAV production. Microcarriers can provide an accelerated process development platform where an already high product yielding adherent cell clone can be directly transitioned to suspension production. Higher cell specific AAV yields are often reported for adherent cell systems compared to cells in suspension, and microcarrier-based culturing is well established for anchored cells in larger scale. Taken together, using microcarriers as an AAV production platform has the potential to increase cell specific yields and increase volumetric productivity thus lowering the total cost of goods for AAV based therapies. Please click Additional Files below to see the full abstract

The effect of cell density on the plasmid utilization for the production of adeno-associated virus via the triple-transfection method

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Suspension like scalability of rAAV9 production in adherent cells

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NMR solution structure and position of transportan in neutral phospholipid bicelles

AbstractTransportan is a chimeric cell-penetrating peptide constructed from the peptides galanin and mastoparan, which has the ability to internalize living cells carrying a hydrophilic load. In this study, we have determined the NMR solution structure and investigated the position of transportan in neutral bicelles. The structure revealed a well-defined α-helix in the C-terminal mastoparan part of the peptide and a weaker tendency to form an α-helix in the N-terminal domain. The position of the peptide in relation to the membrane, as studied by adding paramagnetic probes, shows that the peptide lies parallel to, and in the head-group region of the membrane surface. This result is supported by amide proton secondary chemical shifts

Mechanisms of Cellular Uptake of Cell-Penetrating Peptides

Recently, much attention has been given to the problem of drug delivery through the cell-membrane in order to treat and manage several diseases. The discovery of cell penetrating peptides (CPPs) represents a major breakthrough for the transport of large-cargo molecules that may be useful in clinical applications. CPPs are rich in basic amino acids such as arginine and lysine and are able to translocate over membranes and gain access to the cell interior. They can deliver large-cargo molecules, such as oligonucleotides, into cells. Endocytosis and direct penetration have been suggested as the two major uptake mechanisms, a subject still under debate. Unresolved questions include the detailed molecular uptake mechanism(s), reasons for cell toxicity, and the delivery efficiency of CPPs for different cargoes. Here, we give a review focused on uptake mechanisms used by CPPs for membrane translocation and certain experimental factors that affect the mechanism(s)

Relevance of the N-terminal NLS-like sequence of the prion protein for membrane perturbation effects

AbstractWe investigated the nuclear localization-like sequence KKRPKP, corresponding to the residues 23–28 in the mouse prion protein (mPrP), for its membrane perturbation activity, by comparing effects of two mPrP-derived peptides, corresponding to residues 1–28 (mPrPp(1–28)) and 23–50 (mPrPp(23–50)), respectively. In erythrocytes, mPrPp(1–28) induced ∼60% haemoglobin leakage after 30 min, whereas mPrPp(23–50) had negligible effects. In calcein-entrapping, large unilamellar vesicles (LUVs), similar results were obtained. Cytotoxicity estimated by lactate dehydrogenase leakage from HeLa cells, was found to be ∼12% for 50 μM mPrPp(1–28), and ∼1% for 50 μM mPrPp(23–50). Circular dichroism spectra showed structure induction of mPrPp(1–28) in the presence of POPC:POPG (4:1) and POPC LUVs, while mPrPp(23–50) remained a random coil. Membrane translocation studies on live HeLa cells showed mPrPp(1–28) co-localizing with dextran, suggesting fluid-phase endocytosis, whereas mPrPp(23–50) hardly translocated at all. We conclude that the KKRPKP-sequence is not sufficient to cause membrane perturbation or translocation but needs a hydrophobic counterpart