Enhancement of Adeno-Associated Virus-Mediated Gene Therapy Using Hydroxychloroquine in Murine and Human Tissues

Laurel C Chandler,Alun R Barnard,Sarah L Caddy,Maria I Patrício,Michelle E Mcclements,Howell Fu,Cristina Rada,Robert E Maclaren,Kanmin Xue

doi:10.1016/j.omtm.2019.05.012

Abstract

The therapeutic effects of gene therapy using adeno-associated virus (AAV) vectors are dependent on the efficacy of viral transduction. Currently, we have reached the safe limits of AAV vector dose, beyond which damaging inflammatory responses are seen. To improve the efficacy of AAV transduction, we treated mouse embryonic fibroblasts, primate retinal pigment epithelial cells, and human retinal explants with hydroxychloroquine (HCQ) 1 h prior to transduction with an AAV2 vector encoding GFP driven by a ubiquitous CAG promoter. This led to a consistent increase in GFP expression, up to 3-fold, compared with vector alone. Comparing subretinal injections of AAV2.CAG.GFP vector alone versus co-injection with 18.75 μM HCQ in paired eyes in mice, mean GFP expression was 4.6-fold higher in retinae co-treated with HCQ without retinal toxicity. A comparative 5.9-fold effect was seen with an AAV8(Y733F).GRK1.GFP vector containing the photoreceptor-specific rhodopsin kinase promoter. While the mechanism of action remains to be fully elucidated, our data suggest that a single pulse of adjunctive HCQ could safely improve AAV transduction in vivo, thus providing a novel strategy for enhancing the clinical effects of gene therapy.

Highlights

Gene therapies based on adeno-associated virus (AAV) vector-mediated gene transfer are at advanced stages of clinical development to treat a broad range of monogenic disorders, including inherited retinal dystrophies,[1,2,3,4] hemophilia B,5 hemophilia A,6 muscular dystrophies,[7] and inherited neurodegenerations.[8]
AAV Retinal Gene Therapy Activates Intracellular Innate Immune Responses In Vivo To assess the activation of innate immune responses to AAV following retinal gene therapy, we first performed subretinal injections of an AAV serotype 2 vector encoding GFP driven by a ubiquitous CAG promoter (AAV2.CAG.GFP) and sham injections of diluent in paired eyes of 4-week-old female C57BL/6J mice
Significant upregulation of the intracellular viral sensors Tlr[9], Rig-I, Cgas (Mb21d1), Sting (Tmem173), and Trim[21] (Figure 1A) and anti-viral effectors Ifn-g, Tnf-a, Cxcl[10], Isg[1], and Apobec[3] (Figure 1B) was detected in eyes treated with AAV compared to those sham injected with PBS

Summary

Introduction

Gene therapies based on adeno-associated virus (AAV) vector-mediated gene transfer are at advanced stages of clinical development to treat a broad range of monogenic disorders, including inherited retinal dystrophies,[1,2,3,4] hemophilia B,5 hemophilia A,6 muscular dystrophies,[7] and inherited neurodegenerations.[8] The approval of voretigene neparvovec by the US Food and Drug Administration (FDA) for the treatment of Leber congenital amaurosis caused by mutations in RPE65 has paved the way for similar AAV-based gene therapies in the near future.[9] Recombinant AAV vectors are considered ideal vehicles for transgene delivery compared with lentiviral or adenoviral vectors due to their low immunogenicity; broad tissue tropism; and the ability of the delivered therapeutic transgenes to persist as episomes, which carry low mutagenic potential while allowing sustained transgene expression. While transduction can be increased to some extent by vector dosage, host inflammatory and immune responses to AAV become limiting factors at high doses, and they may compromise the persistence of transgene expression and therapeutic effects.[10,11,12,13] despite promising results seen with retinal gene therapy, cases of intraocular inflammation have been encountered.[14,15,16] one of the major challenges of gene therapy for retinal diseases is how to achieve sufficient levels of gene replacement at a safe vector dose

Methods

Results

Discussion

Conclusion