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

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

Different protein composition and functional properties of adeno-associated virus-6 vector manufactured from the culture medium and cell lysates

International audienceVectors based on recombinant adeno-associated viruses (rAAV) attract a growing interest for human gene therapy. Recently, it was shown that many rAAV serotypes produced by transient transfection of human embryonic kidney 293 cell line (HEK293) are efficiently released into culture medium and functionally equivalent to those purified from cell lysates. Here, we report that HEK293 cells produce and secrete Galectin 3-binding protein (huG3BP), a protein that efficiently binds rAAV6 in vivo. Importantly, intracellular G3BP and secreted G3BP have different properties: while the secreted protein had the same electrophoretic mobility as serum huG3BP and interacted with rAAV6, intracellular protein migrated faster and did not bind rAAV6. Consequently, rAAV6 purified from culture medium (secreted, rAAV6-S) was physically associated with huG3BP while rAAV6 harvested from cell lysates (cellular, rAAV6-C) was huG3BP-free. After systemic injections, rAAV6-S bound to huG3BP was 3 times less efficient compared to rAAV6-C and induced an immune response against huG3BP protein. Our findings show that protein content of rAAVs purified from culture medium or from cell lysates can be different and these differences may impact vector efficacy and/or immune response

Evaluation of the serum matrix metalloproteinase-9 as a biomarker for monitoring disease progression in Duchenne muscular dystrophy

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Evaluation of the serum matrix metalloproteinase-9 as a biomarker for monitoring disease progression in Duchenne muscular dystrophy

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C-reactive protein (CRP) is essential for efficient rAAV-1 and rAAV-6 systemic transduction in mice

International audienceClinical relevance of gene therapy using the recombinant adeno-associated vectors (rAAV) often requires widespread distribution of the vector and in this case systemic delivery is the optimal route of administration. Humoral blood factors such as antibodies or complement are the first barriers met by the vectors administrated systemically. We have found that other blood proteins, galectin 3 binding protein (G3BP) and C-reactive protein (CRP) can interact with different AAV serotypes in a species-specific manner. While interactions of rAAVs with G3BP, antibodies or complement lead to a decrease in vector efficacy, systemic transduction of CRP-deficient mouse and its respective control clearly established that binding to mCRP boosts rAAV-1 and rAAV-6 transduction efficiency in skeletal muscles over ten times. Notably, the high efficacy of rAAV-6 in CRP-deficient mice can be restored by reconstitution of the CRP-deficient mouse with mCRP. Human CRP does not interact neither with rAAV-1 nor rAAV-6 and, consequently, the high efficiency of mCRP-mediated muscle transduction by these serotypes in mice cannot be translated to human. No interaction of mCRP or hCRP was observed with rAAV-8 and rAAV-9. We show, for the first time, that serum components can significantly enhance rAAV-mediated tissue transduction in a serotype- and species-specific manner. Bio-processing in body fluids should be considered when a transfer of preclinical proof of concept for AAV-based gene therapy to the human is planned

Human galectin 3 binding protein interacts with recombinant adeno-associated virus type 6

International audienceRecombinant 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

High urinary ferritin reflects myoglobin iron evacuation in DMD patients

Duchenne muscular dystrophy (DMD) is an X-linked disease caused by mutations in the dystrophin gene leading to the absence of the normal dystrophin protein. The efforts of many laboratories brought new treatments of DMD to the reality, but ongoing and forthcoming clinical trials suffer from absence of valuable biomarkers permitting to follow the outcome of the treatment day by day and to adjust the treatment if needed. In the present study the levels of 128 urinary proteins including growth factors, cytokines and chemokines were compared in urine of DMD patients and age related control subjects by antibody array approach. Surprisingly, statistically significant difference was observed only for urinary ferritin whose level was 50 times higher in young DMD patients. To explain the observed high urinary ferritin content we analysed the levels of iron, iron containing proteins and proteins involved in regulation of iron metabolism in serum and urine of DMD patients and their age-matched healthy controls. Obtained data strongly suggest that elevated level of urinary ferritin is functionally linked to the renal management of myoglobin iron derived from leaky muscles of DMD patients. This first observation of the high level of ferritin in urine of DMD patients permits to consider this protein as a new urinary biomarker in muscular dystrophies and sheds light on the mechanisms of iron metabolism and kidney functioning in DMD

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

International audienceRecombinant 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