AAV Gene Transfer with Tandem Promoter Design Prevents Anti-transgene Immunity and Provides Persistent Efficacy in Neonate Pompe Mice

Abstract

Hepatocyte-restricted, AAV-mediated gene transfer is being used to provide sustained, tolerogenic transgene expression in gene therapy. However, given the episomal status of the AAV genome, this approach cannot be applied to pediatric disorders when hepatocyte proliferation may result in significant loss of therapeutic efficacy over time. In addition, many multi-systemic diseases require widespread expression of the therapeutic transgene that, when provided with ubiquitous or tissue-specific non-hepatic promoters, often results in anti-transgene immunity. Here we have developed tandem promoter monocistronic expression cassettes that, packaged in a single AAV, provide combined hepatic and extra-hepatic tissue-specific transgene expression and prevent anti-transgene immunity. We validated our approach in infantile Pompe disease, a prototype disease caused by lack of the ubiquitous enzyme acid-alpha-glucosidase (GAA), presenting multi-systemic manifestations and detrimental anti-GAA immunity. We showed that the use of efficient tandem promoters prevents immune responses to GAA following systemic AAV gene transfer in immunocompetent Gaa−/− mice. Then we demonstrated that neonatal gene therapy with either AAV8 or AAV9 in Gaa−/− mice resulted in persistent therapeutic efficacy when using a tandem liver-muscle promoter (LiMP) that provided high and persistent transgene expression in non-dividing extra-hepatic tissues. In conclusion, the tandem promoter design overcomes important limitations of AAV-mediated gene transfer and can be beneficial when treating pediatric conditions requiring persistent multi-systemic transgene expression and prevention of anti-transgene immunity.

Advancements in AAV-mediated Gene Therapy for Pompe Disease

“…Ultimately, Colella et al showed that LiMP and LiNeuP tandem promoters resulted in specific hGAA transgene co-transcription in the liver, muscle and CNS, respectively. Moreover, this approach prevented anti-hGAA immunity in GAA −/− mice [81]. However, this approach is not suitable for pediatric patients.…”

“…Other promoters include desmin (DES) which shows preferential expression in motor neurons, skeletal and cardiac muscles [77,79], the neuro-targeted synapsin promoter, the liver-specific promoter (LSP) used to induce tolerance [80], and the liver-muscle promoter (LiMP) that provides expression in nondividing extra-hepatic tissues [81].…”

“…Using the liver as a target for gene therapy takes advantage of the high biodistribution of AAV vectors to the liver and also allows for lower doses of AAV. The liver is able to secrete high levels of many serum proteins and this is an advantage for high-level expression of GAA, especially when engineered to enhance secretion [81]. Additionally, liver-directed expression can lead to immune tolerance induction.…”

“…Similarly, Colella et al developed a hepatocyterestricted, AAV-mediated gene transfer to provide sustained transgene expression that in tandem prevents immune response to GAA [81]. They hypothesized the AAV delivery using cassettes controlled by liver-muscle (LiMP, ApoE-hAAT-C5.12) and liver-neuron (LiNeuP, ApoE-hAAT-hSYN) tandem promoters could express the transgene in both hepatic and extra-hepatic target tissues.…”

“…However, this approach is not suitable for pediatric patients. In fact, following AAV vector-mediated gene transfer, therapeutic efficacy can be lost as hepatocytes proliferate and the liver matures [72,81].…”