Abstract
The inner blood–retina barrier (iBRB) is essential in restricting the movement of systemic components such as enzymes, anaphylatoxins, or pathogens that could otherwise enter the neural retina and cause extensive damage. The barrier has evolved to confer protection to the delicate microenvironment of the retina, and the tight junctions located between adjacent microvascular endothelial cells can restrict the passage of up to 98% of clinically validated low-molecular-weight therapeutics which could hold significant promise for a range of degenerative retinal conditions. Here, we describe a method for the selective RNAi-mediated targeting of one component of the tight junction, claudin-5. We outline the generation of a doxycycline inducible adeno-associated viral vector for the localized, inducible, and size-selective modulation of the iBRB and describe how this vector can be used in ophthalmology research.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Pardridge WM (2005) Molecular biology of the blood–brain barrier. Mol Biotechnol 30(1):57–70
Campbell M, Humphries MM, Kiang A-S, Nguyen ATH, Gobbo OL, Tam LCS, Suzuki M, Hanrahan F, Ozaki E, Farrar G-J, Kenna PF, Humphries P (2011) Systemic low molecular weight drug delivery to pre-selected neuronal regions. EMBO Mol Med 3:235–245
Campbell M, Nguyen ATH, Kiang AS, Tam LCS, Gobbo OL, Kerskens C, Ni Dhubhghaill S, Humphries MM, Farrar GJ, Kenna PF, Humphries P (2009) An experimental platform for systemic drug delivery to the retina. Proc Natl Acad Sci U S A 106(42):17817–17822
Campbell M, Kiang AS, Kenna PF, Kerskens C, Blau C, O’Dwyer L, Tivnan A, Kelly JA, Brankin B, Farrar GJ, Humphries P (2008) RNAi-mediated reversible opening of the blood–brain barrier. J Gene Med 10(8):930–947
Tam LC, Kiang AS, Campbell M, Keaney J, Farrar GJ, Humphries MM, Kenna PF, Humphries P (2010) Prevention of autosomal dominant retinitis pigmentosa by systemic drug therapy targeting heat shock protein 90 (Hsp90). Hum Mol Genet 19(22):4421–4436
Hammond SM, Caudy AA, Hammon GJ (2001) Post-transcriptional gene silencing by double-sranded RNA. Nat Rev Genet 2:110–119
Sharp PA (2001) RNA interference—2001. Genes Dev 15(5):485–490
Hutvangner G, Zamore PD (2002) RNAi: nature abhors a double-strand. Curr Opin Genet Dev 12(2):225–232
Paddison PJ, Caudy AA, Bernstein E, Hannon GJ, Conklin DS (2002) Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev 16(8):948–958
Paul CP, Good PD, Winer I, Engelke DR (2002) Effective expression of small interfering RNA in human cells. Nat Biotechnol 20(5):505–508
Yu J-Y, DeRuiter SL, Turner DL (2002) RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc Natl Acad Sci U S A 99(9):6047–6052
Lee NS, Dohjima T, Bauer G, Li H, Li MJ, Ehsani A, Salvaterra P, Rossi J (2002) Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nat Biotechnol 20(5):500–505
Brummelkamp TR, Bernards R, Agami R (2002) A system for stable expression of short interfering RNAs in mammalian cells. Science 296:550–553
Kunkel GR, Pederson T (1989) Transcription of a human U6 small nuclear RNA gene in vivo withstands deletion of intragenic sequences but not of an up-stream TATATA box. Nucleic Acid Res 17:7371–7379
Gossen M, Bujard H (1992) Tight control of gene expression in mammalian cells by tetracycline responsive promoters. Proc Natl Acad Sci U S A 89(12):5547–5551
Gossen M, Bonin AL, Bujard H (1993) Control of gene activity in higher eukaryotic cells by prokaryotic regulatory elements. Trends Biochem Sci 18:471–475
Gossen M, Bonin AL, Freundlieb S, Bujard H (1994) Inducible gene expression systems for higher eukaryotic cells. Curr Opin Biotechnol 5:516–520
Wigzgall R, O’Leary E, Leaf A, Onaldi D, Bonventre JV (1994) The Kruppel-associated box-A (KRAB-A) domain of zinc finger proteins mediates transcriptional repression. Proc Natl Acad Sci U S A 91(10):4514–4518
Gossen M, Bujard H (1995) Efficacy of tetracycline-controlled gene expression is influenced by cell type. Bio Techniques 19(2):213–215
Freundlieb S, Schirra-Muller C, Bujard H (1999) A tetracycline controlled activation/repression system with increased potential for gene transfer into mammalian cells. J Gene Med 1:4–12
pTet-tTS Vector (1999) Clontechniques XIV(2):10–11
Wiznerowicz M, Trono D (2003) Conditional suppression of cellular genes: lentivirus vector-mediated drug-inducible RNA interference. J Virol 77(16):8957–8961
pTRE-Tight Vectors (2003) Clontechniques XVIII(2):10–11
Knockout Single Vector System for Inducible RNAi (2006) Clontechniques XXI(3):2–3
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Campbell, M., Humphries, M.M., Humphries, P. (2012). Barrier Modulation in Drug Delivery to the Retina. In: Weber, B., LANGMANN, T. (eds) Retinal Degeneration. Methods in Molecular Biology, vol 935. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-080-9_26
Download citation
DOI: https://doi.org/10.1007/978-1-62703-080-9_26
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-079-3
Online ISBN: 978-1-62703-080-9
eBook Packages: Springer Protocols