AAV8 locoregional delivery induces long-term expression of an immunogenic transgene in macaques despite persisting local inflammation

Label‐free 3D characterization of cardiac fibrosis in muscular dystrophy using SHG imaging of cleared tissue

“…In our study, we selected the CUBIC protocol for tissue clearing, and demonstrated the compatibility of this method with collagen SHG imaging. Unlike other clearing methods, the CUBIC protocol preserves GFP fluorescence (Kolesová et al., 2016; Matryba et al., 2018), which is widely used to track protein expression and implanted cells following gene and cell‐based therapy approaches, particularly in the context of DMD (Gernoux et al., 2021; Li et al., 2006; Yang et al., 2009). Furthermore, the CUBIC clearing protocol has been described as compatible with wholemount fluorescent immunolabeling.…”

Durability of transgene expression after rAAV gene therapy

“…Interestingly, analysis showed both a humoral and cellular immune response to the transgenic GFP protein that did not appear to affect expression levels. 133 Two important conclusions may be drawn from this pre-clinical study: first, the route of administration appears to affect the potential for a deleterious immune response following gene therapy, perhaps due to the increased risk of an inflammatory response; and second, Tregs may play a role in regulating the CTL response to the AAV capsid and transgene product, helping to maintain stable transgene expression. 133 Even though CTL responses to transgenes have been observed in NHPs following the systemic delivery of liver-directed AAV gene therapy, no immune responses against transgenic proteins have been reported to date in clinical trials of liver-directed gene therapy, 103,106,118 potentially due to the tolerizing effects of expression in this organ.…”

“…133 Two important conclusions may be drawn from this pre-clinical study: first, the route of administration appears to affect the potential for a deleterious immune response following gene therapy, perhaps due to the increased risk of an inflammatory response; and second, Tregs may play a role in regulating the CTL response to the AAV capsid and transgene product, helping to maintain stable transgene expression. 133 Even though CTL responses to transgenes have been observed in NHPs following the systemic delivery of liver-directed AAV gene therapy, no immune responses against transgenic proteins have been reported to date in clinical trials of liver-directed gene therapy, 103,106,118 potentially due to the tolerizing effects of expression in this organ. 83 In fact, in FVIII or FIX antibody-prone dog models of hemophilia A and B, liver-directed rAAV gene therapy has been shown to have a tolerizing effect, with the ability to eradicate preexisting antibodies against the therapeutic protein (inhibitors).…”

“…79,150 Locoregional intravenous delivery has been shown to reduce inflammatory and subsequent T cell responses compared with intramuscular delivery, with the potential for improved durability versus direct tissue administration. 133 Dose is also an important consideration since higher vector doses are more likely to be detected by the immune system, resulting in the loss of expression due to the elimination of transduced cells, as well as the potential for serious adverse events, including death, likely due to the innate immune response. 83,107,151…”

“… 132 More recently, durable expression was observed in NHPs 5 years after intravenous locoregional infusion of an rAAV2/1 vector encoding an inducible erythropoietin reporter gene, with no evidence of an immune response to the transgene product. 133 , 134 The same group further tested the potential of a T cell response to the transgenic protein delivering a high-dose self-complementary (rather than the less immunogenic single-stranded) rAAV8 vector encoding green fluorescent protein (GFP) via locoregional intravenous injection in an immunogenic NHP model. Despite the group using both an immunogenic recipient and transgene product, long-term transgene expression was achieved.…”