Abstract
Rett syndrome (RTT) is a severe X-linked neurodevelopmental disorder that is primarily caused by mutations in the methyl CpG binding protein 2 gene (MECP2). RTT is the second most prevalent genetic cause of intellectual disability in girls, and there is currently no cure for the disease. We have previously shown that gene therapy using a self-complementary AAV9 viral vector expressing a codon-optimized Mecp2 version (AAV9-MCO) significantly improved symptoms and increased survival in male Mecp2-deficient mice. Here, we pursued our studies and investigated the safety and efficacy of long-term gene therapy in the genetically relevant RTT mouse model: the heterozygous (HET) Mecp2 deficient female mouse. These mice were injected with the AAV9-MCO vector through the tail vein and an array of behavioral tests was performed. At 16- and 30-weeks post-injection, this treatment was able to rescue apneas and improved the spontaneous locomotor deficits and circadian locomotor activity in Mecp2 HET mice treated with AAV9-MCO at a dose of 5 × 1011 vg/mouse. To examine whether a higher dose of vector could result in increased improvements, we injected Mecp2 HET mice with a higher MCO vector dose (1012 vg/mouse), which resulted in some severe, sometimes lethal, side effects. In order to confirm these effects, a new cohort of Mecp2 HET mice were administered increasing doses of MCO vector (1011, 5 × 1011 and 1012 vg/mouse). Again, two weeks after vector administration, some Mecp2 HET mice were found dead while others displayed severe side effects and had to be euthanized. These deleterious effects were not observed in Mecp2 HET mice injected with a high dose of AAV9-GFP and were directly proportionate to vector dosage (0, 23 or 54% mortality at an AAV9-MCO dose of 1011, 5 × 1011, 1012 vg/mouse, respectively), and no such lethality was observed in wild-type (WT) mice. In the Mecp2 HET mice treated with the high and medium AAV9-MCO doses, blood chemistry analysis and post-mortem histology showed liver damage with drastically elevated levels of liver transaminases and disorganized liver architecture. Apoptosis was confirmed by the presence of TUNEL- and cleaved-caspase 3-positive cells in the Mecp2 HET mice treated with the higher doses of AAV9-MCO. We then studied the involvement of the unfolded protein response (UPR) in triggering apoptosis since it can be activated by AAV vectors. Increased expression of the C/EBP homologous protein (CHOP), one of UPR downstream effectors, was confirmed in Mecp2 HET mice after vector administration. The toxic reaction seen in some treated mice indicates that, although gene therapy for RTT improved breathing deficits observed in Mecp2 HET mice, further studies are needed to better understand the underlying mechanisms and caution must be exercised before similar attempts are undertaken in female Rett patients.