Endoplasmic reticulum MHC: Major histocompatibility complex H 2 O 2 : Hydrogen peroxide ERp57: A member of PDI family also know as glucose-regulated protein or 58-kD (GRP58) NF-kB: Factor nuclear kappa B AP-1: Activator protein 1 STAT-3: Signal transducer and activator of transcription 3. Improved Immunological Tolerance Following Combination Therapy with CTLA-4/Ig and AAV-Mediated PD-L1/2 Muscle Gene Transfer Initially thought as being non-immunogenic, recombinant AAVs have emerged as efficient vector candidates for treating monogenic diseases. It is now clear however that they induce potent immune responses against transgene products which can lead to destruction of transduced cells. Therefore, developing strategies to circumvent these immune responses and facilitate long-term expression of transgenic therapeutic proteins is a main challenge in gene therapy. We evaluated herein a strategy to inhibit the undesirable immune activation that follows muscle gene transfer by administration of CTLA-4/Ig to block the costimulatory signals required early during immune priming and by using gene transfer of PD-1 ligands to inhibit T cell functions at the tissue sites. We provide the proof of principle that this combination immunoregulatory therapy targeting two non-redundant checkpoints of the immune response, i.e., priming and effector functions, can improve persistence of transduced cells in experimental settings where cytotoxic T cells escape initial blockade. Therefore, CTLA-4/Ig plus PD-L1/2 combination therapy represents a candidate approach to circumvent the bottleneck of immune responses directed toward transgene products. Since the original reports describing the use of adeno-associated virus (AAV) vectors for transfer of β-galactosidase gene to muscle (Kessler et al., 1996; Xiao et al., 1996; Fisher et al., 1997) , recombinant AAVs (rAAV) have emerged as very efficient and potentially non-immunogenic vector candidates for delivering therapeutic genes to a variety of tissues and treating monogenic diseases. As they poorly activate innate immunity and weakly transduce dendritic cells, rAAV appear as far less immunogenic than adenoviral vectors (Zhang et al., 2000; Zaiss et al., 2002; McCaffrey et al., 2008) . Nevertheless, it has rapidly become clear that rAAV vectors carrying various transgenes can, under different conditions, induce potent immune responses that could ultimately lead to destruction of transduced cells in vivo and consecutive disappearance of transgene expression (Manning et al., 1997 (Manning et al., , 1998 Halbert et al., 1998; Brockstedt et al., 1999) . Not surprisingly therefore, their use has even been proposed in genetic vaccination protocols aimed at eliciting cellular and humoral immune responses against different microorganisms (Kuck et al., 2006; Du et al., 2008) . In primates and humans, rAAV administration has also been documented to elicit significant cytotoxic CD8 + T cell responses directed against the viral as well as the transgenic "exogenous" proteins, resulting in the destruction of transduced cells and complete loss of transgene expression (Manno et al., 2006; Mingozzi et al., 2007; Gao et al., 2009) . Additionally, on the side of the humoral immunity, production of neutralizing antibodies targeting capsid proteins may also prevent vector readministration and accelerate the loss of the therapeutic protein through the formation of immune complexes. Such immune complexes may further sensitize the cellular immune response by enhancing cross-presentation of the transgenic protein by the antigen-presenting cells (APC). Therefore, developing strategies to circumvent immune responses and facilitate long-term expression of transgenic therapeutic proteins has been identified as one of today's main challenges for the translation of rAAV vectors into the clinic (Mingozzi and High, 2011a,b; Nayak and Herzog, 2011) .