Development of a Novel AAV Gene Therapy Cassette with Improved Safety Features and Efficacy in a Mouse Model of Rett Syndrome

Abstract

Rett syndrome (RTT), caused by loss-of-function mutations in the MECP2 gene, is a neurological disorder characterized by severe impairment of motor and cognitive functions. The aim of this study was to investigate the impact of vector design, dosage, and delivery route on the efficacy and safety of gene augmentation therapy in mouse models of RTT. Our results show that AAV-mediated delivery of MECP2 to Mecp2 null mice by systemic administration, and utilizing a minimal endogenous promoter, was associated with a narrow therapeutic window and resulted in liver toxicity at higher doses. Lower doses of this vector significantly extended the survival of mice lacking MeCP2 or expressing a mutant T158M allele but had no impact on RTT-like neurological phenotypes. Modifying vector design by incorporating an extended Mecp2 promoter and additional regulatory 3′ UTR elements significantly reduced hepatic toxicity after systemic administration. Moreover, direct cerebroventricular injection of this vector into neonatal Mecp2-null mice resulted in high brain transduction efficiency, increased survival and body weight, and an amelioration of RTT-like phenotypes. Our results show that controlling levels of MeCP2 expression in the liver is achievable through modification of the expression cassette. However, it also highlights the importance of achieving high brain transduction to impact the RTT-like phenotypes.

Improved MECP2 Gene Therapy Extends the Survival of MeCP2-Null Mice without Apparent Toxicity after Intracisternal Delivery

“…25, 26, 27, 28, 29, 30 Numerous other vector design variations to the hMECP2 (v1) cassette have been carefully tested by our colleagues, leading to the development of the hMECP2 (v2) cassette. These other vector designs are described in a companion paper by Gadalla et al 31 . in this issue of Molecular Therapy – Methods & Clinical Development .…”

Whole brain delivery of an instability-proneMecp2transgene improves behavioral and molecular pathological defects in mouse models of Rett syndrome

“…Given that the therapeutic range of Mecp2 gene expression is very limited, earlier studies employed a viral cassette with a minimal Mecp2 promoter with the attempt to reconstitute endogenous gene expression levels. However, the regulatory regions of Mecp2 remain poorly characterized and the use of self-complementary AAV9 with a limited cargo capacity has obliged to include only very short fragments of the proximal promoter [15][16][17]21 . With these conditions, it remains very unlikely to recapitulate the endogenous Mecp2 expression pattern, whereas the risk of insufficient expression become higher.…”

“…Given the systemic delivery of the virus in Mecp2 deficient mice, we set out to assess whether the immune response to the transgene could explain the health deterioration observed in the treated mice. This was unanticipated at least since none of the previous attempts of systemic gene therapy have considered or described such response to happen 15,16,21 . However, Mecp2 has been implicated in the regulation of immunity 25 and of FoxP3 expression, a transcription factor required for the generation of regulatory T (Treg) cells, during inflammation 26 .…”

“…Thus, a successful gene therapy for RTT has to deliver the correct MeCP2 dosage in a tight range that overlaps with endogenous levels. Different studies have recently provided encouraging results showing that intravenous administration of an Adeno-associated virus serotype 9 (AAV9) expressing the wild-type (WT) MECP2 attenuated neurological dysfunctions and extended lifespan in RTT mice [14][15][16][17] . However, the AAV9 does not cross efficiently the blood-brain barrier in adult mice while having a preferential tropism for peripheral organs.…”

“…However, the AAV9 does not cross efficiently the blood-brain barrier in adult mice while having a preferential tropism for peripheral organs. In fact, intravenous delivery of high doses of AAV9 can lead to severe liver toxicity and sudden death due to ectopic MeCP2 expression 15 . Moreover, the limited brain transduction obtained in these studies was not sufficient to determine a correlation between the viral dose, transduction efficiency and therapeutic benefits.…”