JSRV Intragenic Enhancer Element Increases Expression from a Heterologous Promoter and Promotes High Level AAV-mediated Transgene Expression in the Lung and Liver of Mice.

Darrick L Yu,Natalie Chow,Sarah K Wootton

doi:10.3390/v12111266

Abstract

Jaagsiekte sheep retrovirus (JSRV) induces tumors in the distal airways of sheep and goats. A putative intragenic enhancer, termed JE, localized to the 3′ end of the JSRV env gene, has been previously described. Herein we provide further evidence that the JE functions as a transcriptional enhancer, as it was able to enhance gene expression when placed in either forward or reverse orientation when combined with a heterologous chicken beta actin promoter. We then generated novel composite promoters designed to improve transgene expression from adeno-associated virus (AAV) gene therapy vectors. A hybrid promoter consisting of the shortest JE sequence examined (JE71), the U3 region of the JSRV long terminal repeat (LTR), and the chicken beta actin promoter, demonstrated robust expression in vitro and in vivo, when in the context of AAV vectors. AAV-mediated transgene expression in vivo from the hybrid promoter was marginally lower than that observed for AAV vectors encoding the strong CAG promoter, but greatly reduced in the heart, making this promoter/enhancer combination attractive for non-cardiac applications, particularly respiratory tract or liver directed therapies. Replacement of the murine leukemia virus intron present in the original vector construct with a modified SV40 intron reduced the promoter/enhancer/intron cassette size to 719 bp, leaving an additional ~4 kb of coding capacity when packaged within an AAV vector. Taken together, we have developed a novel, compact promoter that is capable of directing high level transgene expression from AAV vectors in both the liver and lung with diminished transgene expression in the heart.

Highlights

Adeno-associated virus (AAV) is a small, non-pathogenic, non-enveloped, single stranded DNA virus belonging to the Parvoviridae family [1,2]
Constructs bearing short, medium, or long length JE sequences on their own demonstrated little or no expression when transfected into human embryonic kidney (HEK 293), human fibrosarcoma (HTX), or rat fibroblast (208F) cells (Figure 2)
There was a low level of activity in HEK 293T cells, but not 293 cells for forward and reverse JE constructs, suggesting that the SV40 large T antigen was able to promote transcription from the AAV2 inverted terminal repeats (ITR) present on the 50 flanking end of the JE

Summary

Introduction

Adeno-associated virus (AAV) is a small, non-pathogenic, non-enveloped, single stranded DNA virus belonging to the Parvoviridae family [1,2]. In 2017, Luxturna (voretigene neparvovec-rzyl), a recombinant vector based on AAV was approved by the U.S Food and Drug Administration for the treatment of biallelic RPE65 gene mutation-associated retinal dystrophy, a rare form of inherited vision loss that may result [3]. AAV offers many advantages over other gene delivery vectors such as adenovirus vectors due to its superior transducing efficiency in vivo, its ability to promoter sustained transgene expression, its low immunogenicity and the fact that it can and is being used in a wide range of clinical applications [4]. The limited packaging capacity of AAV vectors (~4.7 kb) necessitates the selection of promoter/enhancer elements that are as small as possible, yet retain a high degree of expression, in scenarios where the transgene is of a considerable size, such as in the case of cystic fibrosis transmembrane conductance regulator (CFTR) (4.4 kb). The optimal promoter/enhancer combination for AAV vectored gene therapy applications would be one that has high activity in the target cell population, but minimal to low activity in non-target cells

Methods

Results

Conclusion