Human Galectin 3 Binding Protein Interacts with Recombinant Adeno-Associated Virus Type 6

Abstract

i Recombinant adeno-associated viruses (rAAVs) hold enormous potential for human gene therapy. Despite the well-established safety and efficacy of rAAVs for in vivo gene transfer, there is still little information concerning the fate of vectors in blood following systemic delivery. We screened for serum proteins interacting with different AAV serotypes in humans, macaques, dogs, and mice. We report that serotypes rAAV-1, -5, and -6 but not serotypes rAAV-2, -7, -8, -9, and -10 interact in human sera with galectin 3 binding protein (hu-G3BP), a soluble scavenger receptor. Among the three serotypes, rAAV-6 has the most important capacities for binding to G3BP. rAAV-6 also bound G3BP in dog sera but not in macaque and mouse sera. In mice, rAAV-6 interacted with another protein of the innate immune system, C-reactive protein (CRP). Furthermore, interaction of hu-G3BP with rAAV-6 led to the formation of aggregates and hampered transduction when the two were codelivered into the mouse. Based on these data, we propose that species-specific interactions of AAVs with blood proteins may differentially impact vector distribution and efficacy in different animal models.T he recombinant adeno-associated vector (rAAV) platform, derived from a nonpathogenic dependovirus, has many attributes suitable for in vivo gene transfer: rAAV vectors are capable of transducing a wide range of cell types, including dividing and nondividing cells; rAAV genomes persist as episomal chromatin in the nucleus of transduced cells (38); and stable, persistent expression has been reported for many transgenes in different tissues and species (6,12,36,39). rAAVs have proven to be efficient in preclinical studies in animal models (16,28), and results from clinical trials are promising (7,47).In the case of systemic diseases, clinical relevance requires widespread distribution of the vector in order to target entire organs. This is particularly true for myopathies, where all striated muscles of the skeletal musculature and, frequently, cardiac muscles have to be treated. In this case, vascular delivery would be the optimal route for rAAV administration. Intravascular injection of a number of rAAV serotypes has proven efficient in murine models of muscular dystrophies (11,17,18,34,35). However, translating this approach to large animal models and humans is still challenging. Acquired immunity and neutralizing antibodies present in a large fraction of the human population might obviously be restrictive for rAAV gene delivery (5,20,27,29,30). Moreover, recent studies have demonstrated that serum might also contain other factors neutralizing rAAV vectors (40), indicating that detailed characterization of rAAV's molecular interactions in the bloodstream is obviously important in order to improve vector efficacy.We looked for serum proteins, other than immunoglobulins, which could interact with rAAVs in the bloodstreams of different species. By using a multidisciplinary approach involving proteomics, binding assays, electron microscopy (EM), and in vivo stud...

C-Reactive Protein (CRP) Is Essential for Efficient Systemic Transduction of Recombinant Adeno-Associated Virus Vector 1 (rAAV-1) and rAAV-6 in Mice

“…This difference is most probably due to the lysineto-glutamate change (K531E), which suppresses the heparin binding ability of AAV6 (22). Interestingly, while this change was important for human G3BP (huG3BP) interaction with the respective serotypes (10), no difference was found in the binding of rAAV-1 or rAAV-6 to mCRP, thus indicating that mCRP and huG3BP interact with different epitopes on the rAAV surface.…”

“…Thus, adeno-associated virus vector 3 (rAAV-3), which efficiently transduces human hepatocytes through the hepatocyte growth factor receptor (HGFR), failed to transduce murine hepatocytes, suggesting that AAV-3 specifically uses human HGFR, but not murine HGFR, as a cellular coreceptor for transduction (7)(8)(9). In human and dog blood, but neither mouse nor monkey blood, galectin 3 binding protein (G3BP) interacts with and decreases rAAV-6 efficiency (10). In this study, we also observed that C-reactive protein (CRP) interacts with rAAV-6 in mouse but not human sera.…”

An Emerging Adeno-Associated Viral Vector Pipeline for Cardiac Gene Therapy

“…More recently, both AAV1 and AAV6 have been shown to interact with canine and human galectin 3 binding protein (G3BP), a soluble scavenger receptor (Denard et al, 2012). This interaction is potentiated by the K531 residue unique to AAV6.…”

Duchenne muscular dystrophy gene therapy in the canine model

“…101,102,124 We believe that the difference is likely because of (1) the high-level preexisting AAV-6 neutralization antibody in the canine circulation, 36,125 or (2) the presence of galectin 3 binding protein (G3BP) in dog serum. 126 It has been shown that G3BP can cause AAV-6 particle aggregation and compromise transduction. 126 Few studies have evaluated bodywide gene transfer in the cDMD model (Fig.…”

“…126 It has been shown that G3BP can cause AAV-6 particle aggregation and compromise transduction. 126 Few studies have evaluated bodywide gene transfer in the cDMD model (Fig. 3B).…”