rAAV-compatible MiniPromoters for restricted expression in the brain and eye

Abstract

BackgroundSmall promoters that recapitulate endogenous gene expression patterns are important for basic, preclinical, and now clinical research. Recently, there has been a promising revival of gene therapy for diseases with unmet therapeutic needs. To date, most gene therapies have used viral-based ubiquitous promoters–however, promoters that restrict expression to target cells will minimize off-target side effects, broaden the palette of deliverable therapeutics, and thereby improve safety and efficacy. Here, we take steps towards filling the need for such promoters by developing a high-throughput pipeline that goes from genome-based bioinformatic design to rapid testing in vivo.MethodsFor much of this work, therapeutically interesting Pleiades MiniPromoters (MiniPs; ~4 kb human DNA regulatory elements), previously tested in knock-in mice, were “cut down” to ~2.5 kb and tested in recombinant adeno-associated virus (rAAV), the virus of choice for gene therapy of the central nervous system. To evaluate our methods, we generated 29 experimental rAAV2/9 viruses carrying 19 different MiniPs, which were injected intravenously into neonatal mice to allow broad unbiased distribution, and characterized in neural tissues by X-gal immunohistochemistry for icre, or immunofluorescent detection of GFP.ResultsThe data showed that 16 of the 19 (84 %) MiniPs recapitulated the expression pattern of their design source. This included expression of: Ple67 in brain raphe nuclei; Ple155 in Purkinje cells of the cerebellum, and retinal bipolar ON cells; Ple261 in endothelial cells of brain blood vessels; and Ple264 in retinal Müller glia.ConclusionsOverall, the methodology and MiniPs presented here represent important advances for basic and preclinical research, and may enable a paradigm shift in gene therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s13041-016-0232-4) contains supplementary material, which is available to authorized users.

Cerebellar dopamine D2 receptors regulate preference for social novelty

“…To further characterize the preference for social novelty, the amount of time that the test mouse spent sniffing the novel intruder S2 was evaluated by epochs of 2 minutes. Similar results were obtained using an AAV in which the Cre recombinase is driven selectively in PCs by the minipromoter Ple155(Pcp2) (AAVDJ-Pcp2-Cre) 45 , further confirming that reduced D2R levels in PCs promote preference for social novelty (Figures S11 and S12).…”

“…Addgene). We selected the minipromoter Ple155 because it selectively transduces PCs as it was previously described 45 . The promoter fragment was isolated via restriction with EcoRI and HindIII enzymes.…”

Adeno-Associated Virus Technologies and Methods for Targeted Neuronal Manipulation

“…In contrast, the regulatory elements that control mammalian gene transcription are often distributed over thousands to hundreds of thousands of bases. Due to the limited packaging capacity of the AAV genome, identifying AAV-compatible promoters has been challenging and the development of shortened promoters is an area of active study (de Leeuw et al, 2016;Nathanson, 2009). A list of cell type-specific promoters compatible with AAV vectors is provided in Table 3.…”

“…Many recombinant AAV genomes also include a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), which can dramatically enhance expression. For several examples of how inclusion of a WPRE affects expression from AAV vectors please see (de Leeuw et al, 2016).…”

Gene vector ‘magic bullet’: targeted expression in the central nervous system after peripheral delivery using the synapsin promoter

“…Specific pools of neuronal populations could be efficiently targeted throughout the nervous system without having to perturb the brain or spinal cord with a cannula. The field is working toward finer and finer pinpoint targeting of neurons, neuronal populations, and their subtypes [12]. However, our imagination may be beyond the ultimate precision of resolution and targeting capability necessary to affect or restore one CNS function.…”