Abstract
Under intravenous delivery, recombinant adeno-associated vectors (rAAVs) interact with blood-borne components in ways that can critically alter their therapeutic efficiencies. We have previously shown that interaction with human galectin 3 binding protein dramatically reduces rAAV-6 efficacy, whereas binding of mouse C-reactive protein improves rAAV-1 and rAAV-6 transduction effectiveness. Herein we have assessed, through qualitative and quantitative studies, the proteins from mouse and human sera that bind with rAAV-8 and rAAV-9, two vectors that are being considered for clinical trials for patients with neuromuscular disorders. We show that, in contrast to rAAV-1 and rAAV-6, there was a substantial similarity in protein binding patterns between mouse and human sera for these vector serotypes. To establish an in vivo role for the vector binding of these sera proteins, we chose to study platelet factor 4 (PF4), which interacts with both vectors in both mouse and human sera. Experiments using PF4-knockout mice showed that a complete lack of PF4 did not alter skeletal muscle transduction for these vectors, whereas heart transduction was moderately improved. Our results strongly support our position that the impact of serum proteins on the transduction properties of rAAV-8 and rAAV-9, already observed in mouse models, should be similar in human preclinical trials.
Highlights
Adeno-associated virus (AAV)-derived recombinant vectors are attracting significant attention as promising tools for a wide range of applications in the field of gene therapy
Identification of Serum Proteins Interacting with recombinant adeno-associated vectors (rAAVs) To accurately identify the proteins interacting with rAAV-8 and -9, we adapted the technology of a vector-protein binding assay, in which serum proteins bound to immobilized rAAV particles are digested with trypsin and the resulting peptides are identified on an Orbitrap mass spectrometry (MS) instrument
Human galectin 3 binding protein (G3BP) and murine C-reactive protein (CRP) were the major proteins bound to rAAV-6, representing more than 90% of the quantity of bound proteins, our assay made it possible to identify additional proteins not detected using previous assays.[9,10]
Summary
Adeno-associated virus (AAV)-derived recombinant vectors (rAAV) are attracting significant attention as promising tools for a wide range of applications in the field of gene therapy. Protein classes having specific post-translational modifications, such as alpha-2,3 and alpha-2,6 sialic acids, N-linked glycoproteins, or heparin sulfate proteoglycans, are the primary cell receptors for rAAV uptake.[1,2,3] These post-translational modifications are so common among mammals that researchers initially assumed that rAAV efficiency would be similar across species lines, such that data obtained from animal models would be predictive of the human situation This optimism, was tempered by subsequent studies showing that rAAV-3 could efficiently transduce human hepatocytes through the human hepatocyte growth factor receptor (HGFR), but had no such uptake mechanism in murine hepatocytes.[4,5] many studies have been completed using cells grown in culture,[6,7,8] without taking into account the likely disruptive interactions of rAAV with actual components of more complex human body fluids
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