X-linked myotubular myopathy (XLMTM) results from MTM1 gene mutations and myotubularin deficiency. Most XLMTM patients develop severe muscle weakness leading to respiratory failure and death, typically within 2 years of age. Our objective was to evaluate the efficacy and safety of systemic gene therapy in the p.N155K canine model of XLMTM by performing a dose escalation study. A recombinant adeno-associated virus serotype 8 (rAAV8) vector expressing canine myotubularin (cMTM1) under the muscle-specific desmin promoter (rAAV8-cMTM1) was administered by simple peripheral venous infusion in XLMTM dogs at 10 weeks of age, when signs of the disease are already present. A comprehensive analysis of survival, limb strength, gait, respiratory function, neurological assessment, histology, vector biodistribution, transgene expression, and immune response was performed over a 9-month study period. Results indicate that systemic gene therapy was well tolerated, prolonged lifespan, and corrected the skeletal musculature throughout the body in a dose-dependent manner, defining an efficacious dose in this large-animal model of the disease. These results support the development of gene therapy clinical trials for XLMTM.

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...

eThe clinical relevance of gene therapy using the recombinant adeno-associated virus (rAAV) vectors 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 administered 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 speciesspecific manner. While interactions of rAAV vectors with G3BP, antibodies, or complement lead to a decrease in vector efficacy, systemic transduction of the CRP-deficient mouse and its respective control clearly established that binding to mouse CRP (mCRP) boosts rAAV vector 1 (rAAV-1) and rAAV-6 transduction efficiency in skeletal muscles over 10 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 (hCRP) does not interact with either rAAV-1 or rAAV-6, and, consequently, the high efficiency of mCRP-mediated muscle transduction by these serotypes in mice cannot be translated to humans. 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. Bioprocessing in body fluids should be considered when transfer of a preclinical proof of concept for AAV-based gene therapy to humans is planned.A deno-associated virus (AAV) vectors attract great attention as a promising tool for a wide range of applications in gene therapy. The process of cell transduction by recombinant AAVs (rAAVs) has been studied in detail, and cellular receptors responsible for the virus entry have been identified. Most of these studies were accomplished in cell culture (1-3) without taking into account the exposure of rAAVs to components of body fluids in the in vivo situation. Interestingly, in many cases, protein classes having specific posttranslational modifications, such as ␣-2,3 and ␣-2,6 sialic acids, N-linked glycoproteins, or heparan sulfate proteoglycan, were identified as primary cell receptors for efficient rAAV transduction (4-6). These posttranslational modifications are common between mammalian species, giving hope to the possibility that rAAV efficiency could be similar across species and that animal data are predictive of the human situation.Nevertheless, some recent data indicate that interactions of cellular receptors or blood proteins with rAAVs can be species specific. 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 (7-9). In human and dog blood, but neither mouse nor monkey blood, galectin 3 binding protein (G3BP) in...

Among the integrative gene therapy vectors developed to date, human immunodeficiency virus type 1 (HIV-1)-derived lentiviral vectors (LV) are distinguished by their capacity to infect both dividing and non-dividing cells. Recombinant LV particles contain viral proteins necessary for their packaging, infectious and integrating functions. Like the parental HIV-1 virus they are able to acquire various cellular proteins, but the number and localisation of these proteins are poorly characterised. In the present study we used 2-DE followed by MALDI-TOF to quantify the protein content of several types of vesicular stomatitis virus G-pseudotyped LV including those that were extensively purified in the perspective of clinical gene therapy studies. A proteinase K treatment was used to distinguish between cellular proteins incorporated into virions (I-proteins) and those co-purified with vectors (C-proteins). We found 10 C-proteins and 18 I-proteins associated with LV. Copy numbers for these core I-proteins varied from 5 (AIP-1/ALIX) to 280 (Cyclophilin A) per vector particle. Three novel I-proteins, guanine nucleotide-binding protein 2, L-lactate dehydrogenase B chain and hnRNP core protein A1, were found. This study defines for the first time, the protein stoichiometry of infectious HIV-1-derived LV particles.

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.