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

de Leeuw CN, Korecki AJ, Berry GE, Hickmott JW, Lam SL, Lengyell TC, Bonaguro RJ, Borretta LJ, Chopra V, Chou AY, D'Souza CA, Kaspieva O, Laprise S, McInerny SC, Portales-Casamar E, Swanson-Newman MI, Wong K, Yang GS, Zhou M, Jones SJ, Holt RA, Asokan A, Goldowitz D, Wasserman WW, Simpson EM - Mol Brain (2016)

Bottom Line: 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.The 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.

View Article: PubMed Central - PubMed

Affiliation: Centre for Molecular Medicine and Therapeutics at the Child & Family Research Institute, University of British Columbia, 950 W 28 Ave, Vancouver, BC, V5Z 4H4, Canada.

ABSTRACT

Background: Small 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.

Methods: For 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.

Results: The 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.

Conclusions: Overall, the methodology and MiniPs presented here represent important advances for basic and preclinical research, and may enable a paradigm shift in gene therapy.

No MeSH data available.


Related in: MedlinePlus

“Plug and Play” rAAV2 genome plasmid used to clone MiniPromoters (MiniPs) upstream of icre or EmGFP enables high throughput pipeline testing. a Plasmids were generated containing either the icre or the EmGFP reporter. An AsiSI site flanks a removable WPRE. MiniPs were cloned at the MCS using a combination of the four available cut sites. The plasmids are subsequently used to generate single stranded rAAV. b Screening step one, an historical-indirect reporter system. MiniPs drive expression of icre, which in turn recombines the endogenous loxP sites and removes the stop sequence 5ʹ of the lacZ gene, thus driving expression of β-galactosidase from the strong ubiquitous ROSA26 promoter. c Screening step two, a direct reporter system. MiniPs drive direct expression of EmGFP, which can be imaged by epifluorescence or signal amplified using antibodies. bp, base pairs; ITR, inverted terminal repeat; MCS, multiple cloning site; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element
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Fig1: “Plug and Play” rAAV2 genome plasmid used to clone MiniPromoters (MiniPs) upstream of icre or EmGFP enables high throughput pipeline testing. a Plasmids were generated containing either the icre or the EmGFP reporter. An AsiSI site flanks a removable WPRE. MiniPs were cloned at the MCS using a combination of the four available cut sites. The plasmids are subsequently used to generate single stranded rAAV. b Screening step one, an historical-indirect reporter system. MiniPs drive expression of icre, which in turn recombines the endogenous loxP sites and removes the stop sequence 5ʹ of the lacZ gene, thus driving expression of β-galactosidase from the strong ubiquitous ROSA26 promoter. c Screening step two, a direct reporter system. MiniPs drive direct expression of EmGFP, which can be imaged by epifluorescence or signal amplified using antibodies. bp, base pairs; ITR, inverted terminal repeat; MCS, multiple cloning site; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element

Mentions: In establishing this pipeline, we wanted a system that transduced as many cells as possible and showed expression with a ubiquitous promoter in all relevant cell types, such that any observed restriction would be attributable to the promoter. Thus, we tested 15 control rAAVs including: self-complementary and single-stranded genomes; AAV9 and AAVrh.10 serotypes; CMV, smCBA, CBA, CAGGS, and promoterless constructs; hGFP, GFP, cre, icre, and EmGFP reporters; and the 3′-UTR woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). We used WPRE mut6, a DNA element known to substantially increase expression levels but without promoter activity [32, 33]. Figure 1a depicts the “plug and play” rAAV2 genome plasmid we developed, with AAV2-based inverted terminal repeats (ITRs), an intron, either icre or EmGFP reporters, with or without WPRE. Restriction enzyme sites allowed for the rapid exchange of promoters, reporters, or removal of the intron and/or WPRE. Figure 1b depicts the first evaluation step of the pipeline, in which a MiniP drove icre, which permanently removed the “stop” from a mouse genomic locus where the ubiquitous ROSA26 promoter drove a “lox-stop-lox-lacZ” allele, resulting in high-level expression of lacZ wherever and whenever icre was expressed. Thus, this step used an historical-indirect reporter of cre activity. Figure 1c depicts the second evaluation step of the pipeline, in which a positive MiniP was retested driving EmGFP, a direct reporter of MiniP expression, detected via epifluorescence or immunofluorescence staining.Fig. 1


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

de Leeuw CN, Korecki AJ, Berry GE, Hickmott JW, Lam SL, Lengyell TC, Bonaguro RJ, Borretta LJ, Chopra V, Chou AY, D'Souza CA, Kaspieva O, Laprise S, McInerny SC, Portales-Casamar E, Swanson-Newman MI, Wong K, Yang GS, Zhou M, Jones SJ, Holt RA, Asokan A, Goldowitz D, Wasserman WW, Simpson EM - Mol Brain (2016)

“Plug and Play” rAAV2 genome plasmid used to clone MiniPromoters (MiniPs) upstream of icre or EmGFP enables high throughput pipeline testing. a Plasmids were generated containing either the icre or the EmGFP reporter. An AsiSI site flanks a removable WPRE. MiniPs were cloned at the MCS using a combination of the four available cut sites. The plasmids are subsequently used to generate single stranded rAAV. b Screening step one, an historical-indirect reporter system. MiniPs drive expression of icre, which in turn recombines the endogenous loxP sites and removes the stop sequence 5ʹ of the lacZ gene, thus driving expression of β-galactosidase from the strong ubiquitous ROSA26 promoter. c Screening step two, a direct reporter system. MiniPs drive direct expression of EmGFP, which can be imaged by epifluorescence or signal amplified using antibodies. bp, base pairs; ITR, inverted terminal repeat; MCS, multiple cloning site; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4862195&req=5

Fig1: “Plug and Play” rAAV2 genome plasmid used to clone MiniPromoters (MiniPs) upstream of icre or EmGFP enables high throughput pipeline testing. a Plasmids were generated containing either the icre or the EmGFP reporter. An AsiSI site flanks a removable WPRE. MiniPs were cloned at the MCS using a combination of the four available cut sites. The plasmids are subsequently used to generate single stranded rAAV. b Screening step one, an historical-indirect reporter system. MiniPs drive expression of icre, which in turn recombines the endogenous loxP sites and removes the stop sequence 5ʹ of the lacZ gene, thus driving expression of β-galactosidase from the strong ubiquitous ROSA26 promoter. c Screening step two, a direct reporter system. MiniPs drive direct expression of EmGFP, which can be imaged by epifluorescence or signal amplified using antibodies. bp, base pairs; ITR, inverted terminal repeat; MCS, multiple cloning site; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element
Mentions: In establishing this pipeline, we wanted a system that transduced as many cells as possible and showed expression with a ubiquitous promoter in all relevant cell types, such that any observed restriction would be attributable to the promoter. Thus, we tested 15 control rAAVs including: self-complementary and single-stranded genomes; AAV9 and AAVrh.10 serotypes; CMV, smCBA, CBA, CAGGS, and promoterless constructs; hGFP, GFP, cre, icre, and EmGFP reporters; and the 3′-UTR woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). We used WPRE mut6, a DNA element known to substantially increase expression levels but without promoter activity [32, 33]. Figure 1a depicts the “plug and play” rAAV2 genome plasmid we developed, with AAV2-based inverted terminal repeats (ITRs), an intron, either icre or EmGFP reporters, with or without WPRE. Restriction enzyme sites allowed for the rapid exchange of promoters, reporters, or removal of the intron and/or WPRE. Figure 1b depicts the first evaluation step of the pipeline, in which a MiniP drove icre, which permanently removed the “stop” from a mouse genomic locus where the ubiquitous ROSA26 promoter drove a “lox-stop-lox-lacZ” allele, resulting in high-level expression of lacZ wherever and whenever icre was expressed. Thus, this step used an historical-indirect reporter of cre activity. Figure 1c depicts the second evaluation step of the pipeline, in which a positive MiniP was retested driving EmGFP, a direct reporter of MiniP expression, detected via epifluorescence or immunofluorescence staining.Fig. 1

Bottom Line: 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.The 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.

View Article: PubMed Central - PubMed

Affiliation: Centre for Molecular Medicine and Therapeutics at the Child & Family Research Institute, University of British Columbia, 950 W 28 Ave, Vancouver, BC, V5Z 4H4, Canada.

ABSTRACT

Background: Small 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.

Methods: For 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.

Results: The 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.

Conclusions: Overall, the methodology and MiniPs presented here represent important advances for basic and preclinical research, and may enable a paradigm shift in gene therapy.

No MeSH data available.


Related in: MedlinePlus