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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

Two CLDN5 RRs-based MiniPromoters (MiniPs) showed brain endothelial cell-specific expression related to their source gene, when virus was introduced into the adult circulatory system. a The Ple34 MiniP drove expression of the miniSOG fluorescent green reporter in blood vessels in the brain. Co-labelling was observed with CD31, a marker of endothelial cells. No miniSOG expression was detected in the heart where CD31 stains endocardial endothelium, or liver, where CD16/32 stains endothelial cells. b Ple261, a “cut down” of Ple34, also drove expression of EGFP in blood vessels in the brain. Again, co-labelling was observed with CD31, a marker of endothelial cells, but no expression was detected in the heart or liver. bp, base pairs; Ctx, cortex; EGFP, enhanced green fluorescent protein; miniSOG, mini Singlet Oxygen Generator; OB, olfactory bulb; RRs, regulatory regions. [Scale bars = 50 μm]
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Fig5: Two CLDN5 RRs-based MiniPromoters (MiniPs) showed brain endothelial cell-specific expression related to their source gene, when virus was introduced into the adult circulatory system. a The Ple34 MiniP drove expression of the miniSOG fluorescent green reporter in blood vessels in the brain. Co-labelling was observed with CD31, a marker of endothelial cells. No miniSOG expression was detected in the heart where CD31 stains endocardial endothelium, or liver, where CD16/32 stains endothelial cells. b Ple261, a “cut down” of Ple34, also drove expression of EGFP in blood vessels in the brain. Again, co-labelling was observed with CD31, a marker of endothelial cells, but no expression was detected in the heart or liver. bp, base pairs; Ctx, cortex; EGFP, enhanced green fluorescent protein; miniSOG, mini Singlet Oxygen Generator; OB, olfactory bulb; RRs, regulatory regions. [Scale bars = 50 μm]

Mentions: Figure 5 presents two examples where the MiniP has been taken beyond screening, to modeling delivery applicable to gene therapy. For this, we evaluated the ability of Ple34 and Ple261 (CLDN5 RRs) to drive expression in the endothelial cells of the blood brain barrier, an important therapeutic target. Ple34 was previously positive driving lacZ in a KI mouse [24], and the “cut down” version, Ple261 (Fig. 2), was tested driving icre by temporal vein injection at P0. However, tail vein injection in adult mice models a possible therapeutic delivery, and allows evaluation of the expression with a mature blood-brain barrier. Figure 5a and b respectively, show consistent overlap of Ple34-miniSOG (a fluorescent reporter chosen for its small size [53]) and Ple261-EGFP, with the endothelial cell marker CD31. Importantly, MiniP-driven miniSOG or EGFP co-labeling with endothelial markers in the liver (CD16/CD32) and heart (CD31) was absent.Fig. 5


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)

Two CLDN5 RRs-based MiniPromoters (MiniPs) showed brain endothelial cell-specific expression related to their source gene, when virus was introduced into the adult circulatory system. a The Ple34 MiniP drove expression of the miniSOG fluorescent green reporter in blood vessels in the brain. Co-labelling was observed with CD31, a marker of endothelial cells. No miniSOG expression was detected in the heart where CD31 stains endocardial endothelium, or liver, where CD16/32 stains endothelial cells. b Ple261, a “cut down” of Ple34, also drove expression of EGFP in blood vessels in the brain. Again, co-labelling was observed with CD31, a marker of endothelial cells, but no expression was detected in the heart or liver. bp, base pairs; Ctx, cortex; EGFP, enhanced green fluorescent protein; miniSOG, mini Singlet Oxygen Generator; OB, olfactory bulb; RRs, regulatory regions. [Scale bars = 50 μm]
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig5: Two CLDN5 RRs-based MiniPromoters (MiniPs) showed brain endothelial cell-specific expression related to their source gene, when virus was introduced into the adult circulatory system. a The Ple34 MiniP drove expression of the miniSOG fluorescent green reporter in blood vessels in the brain. Co-labelling was observed with CD31, a marker of endothelial cells. No miniSOG expression was detected in the heart where CD31 stains endocardial endothelium, or liver, where CD16/32 stains endothelial cells. b Ple261, a “cut down” of Ple34, also drove expression of EGFP in blood vessels in the brain. Again, co-labelling was observed with CD31, a marker of endothelial cells, but no expression was detected in the heart or liver. bp, base pairs; Ctx, cortex; EGFP, enhanced green fluorescent protein; miniSOG, mini Singlet Oxygen Generator; OB, olfactory bulb; RRs, regulatory regions. [Scale bars = 50 μm]
Mentions: Figure 5 presents two examples where the MiniP has been taken beyond screening, to modeling delivery applicable to gene therapy. For this, we evaluated the ability of Ple34 and Ple261 (CLDN5 RRs) to drive expression in the endothelial cells of the blood brain barrier, an important therapeutic target. Ple34 was previously positive driving lacZ in a KI mouse [24], and the “cut down” version, Ple261 (Fig. 2), was tested driving icre by temporal vein injection at P0. However, tail vein injection in adult mice models a possible therapeutic delivery, and allows evaluation of the expression with a mature blood-brain barrier. Figure 5a and b respectively, show consistent overlap of Ple34-miniSOG (a fluorescent reporter chosen for its small size [53]) and Ple261-EGFP, with the endothelial cell marker CD31. Importantly, MiniP-driven miniSOG or EGFP co-labeling with endothelial markers in the liver (CD16/CD32) and heart (CD31) was absent.Fig. 5

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