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Systemic low-molecular weight drug delivery to pre-selected neuronal regions.

Campbell M, Humphries MM, Kiang AS, Nguyen AT, Gobbo OL, Tam LC, Suzuki M, Hanrahan F, Ozaki E, Farrar GJ, Kenna PF, Humphries P - EMBO Mol Med (2011)

Bottom Line: We describe a procedure for controlled, periodic, reversible modulation of selected regions of the blood-brain barrier (BBB) or the inner-blood-retina barrier (iBRB) based on incorporation into an AAV-2/9 vector of a doxycycline-inducible gene encoding shRNA targeting claudin-5, one of 30 or so proteins constituting the BBB and iBRB.The vector may be introduced stereotaxically into pre-selected regions of the brain or into the retina, rendering these regions permeable to low-molecular weight compounds up to approximately 1 kDa for the period of time during which the inducing agent, doxycycline, is administered in drinking water, but excluding potentially toxic higher molecular weight materials.We report on the use of barrier modulation in tandem with systemic drug therapy to prevent retinal degeneration and to suppress laser-induced choroidal neovascularization (CNV), the latter being the hallmark pathology associated with the exudative, or wet, form of age-related macular degeneration (AMD).

View Article: PubMed Central - PubMed

Affiliation: Ocular Genetics Unit, Department of Genetics, Trinity College Dublin, Dublin 2, Ireland. matthew.campbell@tcd.ie

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Assessment of the efficacy of CLDN5 AAV-2/9A,B. The plasmid incorporating the inducible system with claudin-5 shRNA (A) or a NT luciferase shRNA (B) was cloned into the plasmid pAAV-MCS, such as to incorporate L-ITR and R-ITR. Abbreviations: tTS, tetracycline-inducible transcriptional suppressor; β-globpA, beta-globin promoter; pTRE-U6, Tet-responsive U6 promoter; f1 ori, f1 origin of replication; Amp, ampicillin selection; pUC, pUC origin of replication. AAV-2/9 was then generated using a triple transfection system in a stably transfected HEK-293 cell line for the generation of high-titre viruses. tTS is a fusion of the Tet repressor and the Kid-1 KRAB-AB silencing domain. In the absence of doxycycline, tTS repressor binds to Tet operator (TetO) elements in a modified polIII promoter (pTRE-U6), inhibiting expression of claudin-5 (or NT luciferase) shRNAs. In the presence of doxycycline, tTS no longer binds to the promoter, allowing expression of shRNA.C. 3 weeks post-sub-retinal inoculation of 3 µl of 5 × 1011 viral particles/ml of the NT AAV-2/9 or CLDN5 AAV-2/9 and subsequent supplementation in the drinking water of mice of 2 mg/ml doxycycline with 5% sucrose.D,E. Strong and significant suppression of claudin-5 was observed at both the protein (***p = 0.0005) and transcript level (*p = 0.0218).F,G. Qualitative assessment of claudin-5 expression in retinal flatmounts showed a continuous and strong pattern of staining at the endothelial cell margins in the microvasculature of the retinas of mice injected with the NT AAV-2/9. However, following sub-retinal inoculation of the CLDN5 AAV-2/9, this pattern of staining, while not completely ablated, was discontinuous and fragmented, with large immunoreactive precipitates of claudin-5 manifesting in the microvasculature.H. Analysis of claudin-5 expression in retinal cryosections of mice injected with the NT AAV showed claudin-5 expression associated with the microvessels of the retina, permeating as far as the OPL (Arrows). Mice injected with the CLDN5 AAV-2/9 showed a significant decrease in expression and localization of claudin-5 in each layer permeated by microvessels. Outer nuclear layer (ONL), inner nuclear layer (INL), IPL and GCL.
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fig01: Assessment of the efficacy of CLDN5 AAV-2/9A,B. The plasmid incorporating the inducible system with claudin-5 shRNA (A) or a NT luciferase shRNA (B) was cloned into the plasmid pAAV-MCS, such as to incorporate L-ITR and R-ITR. Abbreviations: tTS, tetracycline-inducible transcriptional suppressor; β-globpA, beta-globin promoter; pTRE-U6, Tet-responsive U6 promoter; f1 ori, f1 origin of replication; Amp, ampicillin selection; pUC, pUC origin of replication. AAV-2/9 was then generated using a triple transfection system in a stably transfected HEK-293 cell line for the generation of high-titre viruses. tTS is a fusion of the Tet repressor and the Kid-1 KRAB-AB silencing domain. In the absence of doxycycline, tTS repressor binds to Tet operator (TetO) elements in a modified polIII promoter (pTRE-U6), inhibiting expression of claudin-5 (or NT luciferase) shRNAs. In the presence of doxycycline, tTS no longer binds to the promoter, allowing expression of shRNA.C. 3 weeks post-sub-retinal inoculation of 3 µl of 5 × 1011 viral particles/ml of the NT AAV-2/9 or CLDN5 AAV-2/9 and subsequent supplementation in the drinking water of mice of 2 mg/ml doxycycline with 5% sucrose.D,E. Strong and significant suppression of claudin-5 was observed at both the protein (***p = 0.0005) and transcript level (*p = 0.0218).F,G. Qualitative assessment of claudin-5 expression in retinal flatmounts showed a continuous and strong pattern of staining at the endothelial cell margins in the microvasculature of the retinas of mice injected with the NT AAV-2/9. However, following sub-retinal inoculation of the CLDN5 AAV-2/9, this pattern of staining, while not completely ablated, was discontinuous and fragmented, with large immunoreactive precipitates of claudin-5 manifesting in the microvasculature.H. Analysis of claudin-5 expression in retinal cryosections of mice injected with the NT AAV showed claudin-5 expression associated with the microvessels of the retina, permeating as far as the OPL (Arrows). Mice injected with the CLDN5 AAV-2/9 showed a significant decrease in expression and localization of claudin-5 in each layer permeated by microvessels. Outer nuclear layer (ONL), inner nuclear layer (INL), IPL and GCL.

Mentions: The system described here allows for reversible modulation of the iBRB with the exclusion of the BBB, and vice versa. To achieve this, we incorporated shRNA targeting claudin-5 into a doxycycline-inducible plasmid system. Subsequently, this was incorporated into the genome of an AAV-2/9 vector (these viruses have recently been shown to transduce endothelial cells of the neuronal microvasculature with high efficiency; Fig 1A and B; Foust et al, 2009, 2010). Viral purity was assessed using SDS–PAGE (Supplementary Fig 1).


Systemic low-molecular weight drug delivery to pre-selected neuronal regions.

Campbell M, Humphries MM, Kiang AS, Nguyen AT, Gobbo OL, Tam LC, Suzuki M, Hanrahan F, Ozaki E, Farrar GJ, Kenna PF, Humphries P - EMBO Mol Med (2011)

Assessment of the efficacy of CLDN5 AAV-2/9A,B. The plasmid incorporating the inducible system with claudin-5 shRNA (A) or a NT luciferase shRNA (B) was cloned into the plasmid pAAV-MCS, such as to incorporate L-ITR and R-ITR. Abbreviations: tTS, tetracycline-inducible transcriptional suppressor; β-globpA, beta-globin promoter; pTRE-U6, Tet-responsive U6 promoter; f1 ori, f1 origin of replication; Amp, ampicillin selection; pUC, pUC origin of replication. AAV-2/9 was then generated using a triple transfection system in a stably transfected HEK-293 cell line for the generation of high-titre viruses. tTS is a fusion of the Tet repressor and the Kid-1 KRAB-AB silencing domain. In the absence of doxycycline, tTS repressor binds to Tet operator (TetO) elements in a modified polIII promoter (pTRE-U6), inhibiting expression of claudin-5 (or NT luciferase) shRNAs. In the presence of doxycycline, tTS no longer binds to the promoter, allowing expression of shRNA.C. 3 weeks post-sub-retinal inoculation of 3 µl of 5 × 1011 viral particles/ml of the NT AAV-2/9 or CLDN5 AAV-2/9 and subsequent supplementation in the drinking water of mice of 2 mg/ml doxycycline with 5% sucrose.D,E. Strong and significant suppression of claudin-5 was observed at both the protein (***p = 0.0005) and transcript level (*p = 0.0218).F,G. Qualitative assessment of claudin-5 expression in retinal flatmounts showed a continuous and strong pattern of staining at the endothelial cell margins in the microvasculature of the retinas of mice injected with the NT AAV-2/9. However, following sub-retinal inoculation of the CLDN5 AAV-2/9, this pattern of staining, while not completely ablated, was discontinuous and fragmented, with large immunoreactive precipitates of claudin-5 manifesting in the microvasculature.H. Analysis of claudin-5 expression in retinal cryosections of mice injected with the NT AAV showed claudin-5 expression associated with the microvessels of the retina, permeating as far as the OPL (Arrows). Mice injected with the CLDN5 AAV-2/9 showed a significant decrease in expression and localization of claudin-5 in each layer permeated by microvessels. Outer nuclear layer (ONL), inner nuclear layer (INL), IPL and GCL.
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Related In: Results  -  Collection

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fig01: Assessment of the efficacy of CLDN5 AAV-2/9A,B. The plasmid incorporating the inducible system with claudin-5 shRNA (A) or a NT luciferase shRNA (B) was cloned into the plasmid pAAV-MCS, such as to incorporate L-ITR and R-ITR. Abbreviations: tTS, tetracycline-inducible transcriptional suppressor; β-globpA, beta-globin promoter; pTRE-U6, Tet-responsive U6 promoter; f1 ori, f1 origin of replication; Amp, ampicillin selection; pUC, pUC origin of replication. AAV-2/9 was then generated using a triple transfection system in a stably transfected HEK-293 cell line for the generation of high-titre viruses. tTS is a fusion of the Tet repressor and the Kid-1 KRAB-AB silencing domain. In the absence of doxycycline, tTS repressor binds to Tet operator (TetO) elements in a modified polIII promoter (pTRE-U6), inhibiting expression of claudin-5 (or NT luciferase) shRNAs. In the presence of doxycycline, tTS no longer binds to the promoter, allowing expression of shRNA.C. 3 weeks post-sub-retinal inoculation of 3 µl of 5 × 1011 viral particles/ml of the NT AAV-2/9 or CLDN5 AAV-2/9 and subsequent supplementation in the drinking water of mice of 2 mg/ml doxycycline with 5% sucrose.D,E. Strong and significant suppression of claudin-5 was observed at both the protein (***p = 0.0005) and transcript level (*p = 0.0218).F,G. Qualitative assessment of claudin-5 expression in retinal flatmounts showed a continuous and strong pattern of staining at the endothelial cell margins in the microvasculature of the retinas of mice injected with the NT AAV-2/9. However, following sub-retinal inoculation of the CLDN5 AAV-2/9, this pattern of staining, while not completely ablated, was discontinuous and fragmented, with large immunoreactive precipitates of claudin-5 manifesting in the microvasculature.H. Analysis of claudin-5 expression in retinal cryosections of mice injected with the NT AAV showed claudin-5 expression associated with the microvessels of the retina, permeating as far as the OPL (Arrows). Mice injected with the CLDN5 AAV-2/9 showed a significant decrease in expression and localization of claudin-5 in each layer permeated by microvessels. Outer nuclear layer (ONL), inner nuclear layer (INL), IPL and GCL.
Mentions: The system described here allows for reversible modulation of the iBRB with the exclusion of the BBB, and vice versa. To achieve this, we incorporated shRNA targeting claudin-5 into a doxycycline-inducible plasmid system. Subsequently, this was incorporated into the genome of an AAV-2/9 vector (these viruses have recently been shown to transduce endothelial cells of the neuronal microvasculature with high efficiency; Fig 1A and B; Foust et al, 2009, 2010). Viral purity was assessed using SDS–PAGE (Supplementary Fig 1).

Bottom Line: We describe a procedure for controlled, periodic, reversible modulation of selected regions of the blood-brain barrier (BBB) or the inner-blood-retina barrier (iBRB) based on incorporation into an AAV-2/9 vector of a doxycycline-inducible gene encoding shRNA targeting claudin-5, one of 30 or so proteins constituting the BBB and iBRB.The vector may be introduced stereotaxically into pre-selected regions of the brain or into the retina, rendering these regions permeable to low-molecular weight compounds up to approximately 1 kDa for the period of time during which the inducing agent, doxycycline, is administered in drinking water, but excluding potentially toxic higher molecular weight materials.We report on the use of barrier modulation in tandem with systemic drug therapy to prevent retinal degeneration and to suppress laser-induced choroidal neovascularization (CNV), the latter being the hallmark pathology associated with the exudative, or wet, form of age-related macular degeneration (AMD).

View Article: PubMed Central - PubMed

Affiliation: Ocular Genetics Unit, Department of Genetics, Trinity College Dublin, Dublin 2, Ireland. matthew.campbell@tcd.ie

Show MeSH
Related in: MedlinePlus