title: Gene silencing of EXTL2 and EXTL3 as a substrate deprivation therapy for heparan sulphate storing mucopolysaccharidoses.
abstract: Neurological pathology is characteristic of the mucopolysaccharidoses (MPSs) that store heparan sulphate (HS) glycosaminoglycan (gag) and has been proven to be refractory to systemic therapies. Substrate deprivation therapy (SDT) using general inhibitors of gag synthesis improves neurological function in mouse models of MPS, but is not specific to an MPS type. We have investigated RNA interference (RNAi) as a method of targeting SDT to the HS synthesising enzymes, EXTL2 and EXTL3. Multiple shRNA molecules specific to EXTL2 or EXTL3 were designed and validated in a reporter gene assay, with four out of six shRNA constructs reducing expression by over 90%. The three EXTL2-specific shRNA constructs reduced endogenous target gene expression by 68, 32 and 65%, and decreased gag synthesis by 46, 50 and 27%. One EXTL3-specific shRNA construct reduced endogenous target gene expression by 14% and gag synthesis by 39%. Lysosomal gag levels in MPS IIIA and MPS I fibroblasts were also reduced by EXTL2 and EXTL3-specific shRNA. Incorporation of shRNAs into a lentiviral expression system reduced gene expression, and one EXTL2-specific shRNA reduced gag synthesis. These results indicate that deprivation therapy through shRNA-mediated RNAi has potential as a therapy for HS-storing MPSs.
authors.affiliation: Department of Genetics and Molecular Pathology, SA Pathology (WCH site), North Adelaide, South Australia, Australia.
authors.forename: Xenia
authors.initials: X
authors.lastname: Kaidonis
authors.forename: Wan Chin
authors.initials: WC
authors.lastname: Liaw
authors.forename: Ainslie Derrick
authors.initials: AD
authors.lastname: Roberts
authors.forename: Marleesa
authors.initials: M
authors.lastname: Ly
authors.forename: Donald
authors.initials: D
authors.lastname: Anson
authors.forename: Sharon
authors.initials: S
authors.lastname: Byers
mesh_terms: D002648:Child; D017931:DNA Primers; D005786:Gene Expression Regulation; D015683:Gene Products, gag / Q000235:genetics; D020868:Gene Silencing; D017930:Genes, Reporter; D006497:Heparan Sulfate / Q000378:metabolism; D006801:Humans; D008247:Lysosomes / Q000502:physiology; D008565:Membrane Proteins / Q000235:genetics*; D009083:Mucopolysaccharidoses / Q000235:genetics*; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012313:RNA / Q000235:genetics; D013601:T-Lymphocytes / Q000502:physiology; D014162:Transfection
chemical_list: D017931:DNA Primers; D015683:Gene Products, gag; D006497:Heparan Sulfate; D008565:Membrane Proteins; D017351:N-Acetylglucosaminyltransferases; D012313:RNA; C110774:EXTL3 protein, human; C110834:EXTL2 protein, human
references.citation: J Biol Chem. 1999 Apr 9;274(15):10474-80
references.pmid: 10187838
references.citation: Pediatr Res. 2006 Sep;60(3):309-14
references.pmid: 16857766
references.citation: Anal Biochem. 1976 May 7;72:248-54
references.pmid: 942051
references.citation: Philos Trans R Soc Lond B Biol Sci. 2003 May 29;358(1433):955-60
references.pmid: 12803929
references.citation: J Biol Chem. 1996 Mar 29;271(13):7645-53
references.pmid: 8631801
references.citation: Eur J Hum Genet. 2006 Jul;14(7):846-52
references.pmid: 16670689
references.citation: J Pediatr. 2006 Apr;148(4):533-539
references.pmid: 16647419
references.citation: J Biol Chem. 1998 May 8;273(19):11902-7
references.pmid: 9565617
references.citation: Neurobiol Dis. 2006 Sep;23(3):578-86
references.pmid: 16857362
references.citation: Curr Ther Res Clin Exp. 2008 Apr;69(2):166-79
references.pmid: 24692796
references.citation: Mol Genet Metab. 2007 Sep-Oct;92(1-2):115-21
references.pmid: 17681480
references.citation: J Gene Med. 2005 Nov;7(11):1390-9
references.pmid: 16025547
references.citation: RNA. 2002 Jun;8(6):842-50
references.pmid: 12088155
references.citation: Eur J Hum Genet. 2010 Feb;18(2):200-5
references.pmid: 19690584
references.citation: Biochim Biophys Acta. 1998 Jul 30;1399(1):57-61
references.pmid: 9714738
references.citation: Glycobiology. 1996 Jan;6(1):51-7
references.pmid: 8991509
references.citation: Genet Vaccines Ther. 2007 Jan 16;5:1
references.pmid: 17227588
references.citation: J Biol Chem. 1999 Jan 22;274(4):2456-63
references.pmid: 9891016
references.citation: Mol Genet Metab. 2007 Mar;90(3):329-37
references.pmid: 17185020
references.citation: Philos Trans R Soc Lond B Biol Sci. 2003 May 29;358(1433):961-6
references.pmid: 12803930
references.citation: Proc Natl Acad Sci U S A. 2001 Jun 19;98(13):7176-81
references.pmid: 11390981
references.citation: Nat Med. 2003 Mar;9(3):347-51
references.pmid: 12579197
references.citation: Proc Natl Acad Sci U S A. 2005 Apr 19;102(16):5820-5
references.pmid: 15811941
references.citation: J Lipid Res. 1987 May;28(5):565-71
references.pmid: 2955067
references.citation: J Biol Chem. 1998 Oct 9;273(41):26265-8
references.pmid: 9756849
references.citation: Mol Genet Metab. 2007 Feb;90(2):171-80
references.pmid: 17011223
references.citation: J Biol Chem. 1999 May 14;274(20):13933-7
references.pmid: 10318803
references.citation: IUBMB Life. 2002 Oct;54(4):163-75
references.pmid: 12512855
references.citation: Methods. 2001 Dec;25(4):402-8
references.pmid: 11846609
references.citation: J Biol Chem. 1998 Apr 10;273(15):9208-13
references.pmid: 9535912
references.citation: Carbohydr Res. 2000 Jun 30;326(4):250-63
references.pmid: 10890273
references.citation: FASEB J. 1992 Feb 1;6(3):861-70
references.pmid: 1740236
references.citation: J Inherit Metab Dis. 2003;26(6):513-26
references.pmid: 14605497
references.citation: J Clin Invest. 2001 Jul;108(2):169-73
references.pmid: 11457867
references.citation: Proc Natl Acad Sci U S A. 2005 Oct 11;102(41):14777-82
references.pmid: 16162667
references.citation: Proc Natl Acad Sci U S A. 2008 Jul 22;105(29):10238-43
references.pmid: 18632556