Delivery of BACE1 siRNA mediated by TARBP-BTP fusion protein reduces β-amyloid deposits in a transgenic mouse model of Alzheimer’s disease

  • Mohamed Mohamed Haroon
  • Kamal Saba
  • Venkata Harshavardhan Boddedda
  • Jerald Mahesh Kumar
  • Anant Bahadur PatelEmail author
  • Vijaya GopalEmail author


Systemic delivery of nucleic acids to the central nervous system (CNS) is a major challenge for the development of RNA interference-based therapeutics due to lack of stability, target specificity, non-permeability to the blood–brain barrier (BBB), and lack of suitable carriers. Using a designed bi-functional fusion protein TARBP-BTP in a complex with siRNA, we earlier demonstrated knockdown of target genes in the brain of both AβPP-PS1 (Alzheimer’s disease, AD) and wild-type C57BL/6 mice. In this report, we further substantiate the approach through an extended use in AβPP-PS1 mice, which upon treatment with seven doses of β-secretase AβPP cleaving Enzyme 1 (BACE1) TARBP-BTP:siRNA, led to target-specific effect in the mouse brain. Concomitant gene silencing of BACE1, and consequent reduction in plaque load in the cerebral cortex and hippocampus (>60%) in mice treated with TARBP-BTP:siRNA complex, led to improvement in spatial learning and memory. The study validates the efficiency of TARBP-BTP fusion protein as an efficient mediator of RNAi, giving considerable scope for future intervention in neurodegenerative disorders through the use of short nucleic acids as gene specific inhibitors.


Alzheimer’s disease amyloid-β gene silencing peptide-based delivery system RNAi therapeutics siRNA delivery 



The work was supported by BRNS (DAE) Grant No. 37(1)/14/51/2014-BRNS. The authors thank Mr Sairam for diligent help with animal dissections, and Ms Nandini Rangaraj for help with confocal microscopy. We acknowledge Ms Durga Jeyalakshmi Srinivasan’s inputs to the progress of this work, and timely assistance from Ms Haritha N with the in vivo experiments.

Supplementary material

12038_2018_9822_MOESM1_ESM.doc (822 kb)
Supplementary material 1 (DOC 822 kb)


  1. Atwal JK, Chen Y, Chiu C, Mortensen DL, Meilandt WJ, Liu Y, Heise CE, Hoyte K, et al. 2011 A therapeutic antibody targeting BACE1 inhibits amyloid-beta production in vivo. Sci. Trans. Med. 3 84ra43Google Scholar
  2. Barao S, Moechars D, Lichtenthaler SF and De Strooper B 2016 BACE1 Physiological functions may limit its use as therapeutic target for Alzheimer’s disease. Trends Neurosci. 39 158–169CrossRefGoogle Scholar
  3. Bartlett DW and Davis ME 2006 Insights into the kinetics of siRNA-mediated gene silencing from live-cell and live-animal bioluminescent imaging. Nucleic Acids Res. 34 322–333CrossRefGoogle Scholar
  4. Citron M 2004a Strategies for disease modification in Alzheimer’s disease. Nat. Rev. Neurosci. 5 677–685CrossRefGoogle Scholar
  5. Citron M 2004b Beta-secretase inhibition for the treatment of Alzheimer’s disease–promise and challenge. Trends Pharmacol. Sci. 25 92–97CrossRefGoogle Scholar
  6. Dar GH, Gopal V and Rao M 2015 Conformation-dependent binding and tumor-targeted delivery of siRNA by a designed TRBP2: Affibody fusion protein. Nanomed. Nanotechnol. Biol. Med. 11 1455–1466CrossRefGoogle Scholar
  7. El-Andaloussi S, Johansson HJ, Holm T and Langel U 2007 A novel cell-penetrating peptide, M918, for efficient delivery of proteins and peptide nucleic acids. Mol. Therapy 15 1820–1826CrossRefGoogle Scholar
  8. Elbashir SM, Lendeckel W and Tuschl T 2001 RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 15 188–200CrossRefGoogle Scholar
  9. Fiedel BA, Rent R, Myhrman R and Gewurz H 1976 Complement activation by interaction of polyanions and polycations. Immunology 30 161–169PubMedPubMedCentralGoogle Scholar
  10. Haroon MM, Dar GH, Jeyalakshmi D, Venkatraman U, Saba K, Rangaraj N, Patel AB and Gopal V 2016 A designed recombinant fusion protein for targeted delivery of siRNA to the mouse brain. J. Control. Release 228 120–131CrossRefGoogle Scholar
  11. Haass C 2004 Take five—BACE and the gamma-secretase quartet conduct Alzheimer’s amyloid beta-peptide generation. EMBO J. 23 483–488CrossRefGoogle Scholar
  12. Hawkins BT and Davis TP 2005 The blood-brain barrier/neurovascular unit in health and disease. Pharmacol. Rev. 57 173–185CrossRefGoogle Scholar
  13. He G, Luo W, Li P, Remmers C, Netzer WJ, Hendrick J, Bettayeb K, Flajolet M, Gorelick F, Wennogle LP and Greengard P 2010 Gamma-secretase activating protein is a therapeutic target for Alzheimer’s disease. Nature 467 95–98CrossRefGoogle Scholar
  14. Jankowsky JL, Fadale DJ, Anderson J, Xu GM, Gonzales V, Jenkins NA, Copeland NG, Lee MK, Younkin LH, Wagner SL, Younkin SG and Borchelt DR 2004 Mutant presenilins specifically elevate the levels of the 42 residue beta-amyloid peptide in vivo: evidence for augmentation of a 42-specific gamma secretase. Hum. Mol. Genet. 13 159–170CrossRefGoogle Scholar
  15. Kennedy ME, et al. 2016 The BACE1 inhibitor verubecestat (MK-8931) reduces CNS beta-amyloid in animal models and in Alzheimer’s disease patients. Sci. Trans. Med. 8 363ra150Google Scholar
  16. Kim DH and Rossi JJ 2007 Strategies for silencing human disease using RNA interference. Nat. Rev. Genet. 8 173–184CrossRefGoogle Scholar
  17. Kumar P, Wu H, McBride JL, Jung KE, Kim MH, Davidson BL, Lee SK, Shankar P and Manjunath N 2007 Transvascular delivery of small interfering RNA to the central nervous system. Nature 448 39–43CrossRefGoogle Scholar
  18. Markwell MA, Haas SM, Bieber LL and Tolbert NE 1978 A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal. Biochem. 87 206–210CrossRefGoogle Scholar
  19. Mohamed MH, Kamal S, Venkata HB, Jerald MK, Patel AB and Vijaya G 2016 Delivery of BACE1 siRNA mediated by TARBP-BTP fusion protein reduces b-amyloid deposits in a transgenic mouse model of Alzheimer’s disease. bioRxiv Scholar
  20. Neuwelt E, Abbott NJ, Abrey L, Banks WA, Blakley B, Davis T, Engelhardt B, Grammas P, Nedergaard M, Nutt J, Pardridge W, Rosenberg GA, Smith Q and Drewes LR 2008 Strategies to advance translational research into brain barriers. Lancet Neurol. 7 84–96CrossRefGoogle Scholar
  21. Nishitomi K, Sakaguchi G, Horikoshi Y, Gray AJ, Maeda M, Hirata-Fukae C, Becker AG, Hosono M, et al. 2006 BACE1 inhibition reduces endogenous Abeta and alters APP processing in wild-type mice. J. Neurochem. 99 1555–1563CrossRefGoogle Scholar
  22. Ohno M, Sametsky EA, Younkin LH, Oakley H, Younkin SG, Citron M, Vassar R and Disterhoft JF 2004 BACE1 deficiency rescues memory deficits and cholinergic dysfunction in a mouse model of Alzheimer’s disease. Neuron 41 27–33CrossRefGoogle Scholar
  23. Pardridge WM 2005 The blood-brain barrier: bottleneck in brain drug development. NeuroRx 2 3–14CrossRefGoogle Scholar
  24. Perl DP 2010 Neuropathology of Alzheimer’s disease. Mount Sinai J. Med. 77 32–42CrossRefGoogle Scholar
  25. Reichelt P, Schwarz C and Donzeau M 2006 Single step protocol to purify recombinant proteins with low endotoxin contents. Protein Expression Purification 46 483–488CrossRefGoogle Scholar
  26. Sankaranarayanan S, Price EA, Wu G, Crouthamel MC, Shi XP, Tugusheva K, Tyler KX, Kahana J, Ellis J, Jin L, Steele T, Stachel S, Coburn C and Simon AJ 2008 In vivo beta-secretase 1 inhibition leads to brain Abeta lowering and increased alpha-secretase processing of amyloid precursor protein without effect on neuregulin-1. J. Pharmacol. Exp. Therapeutics 324 957–969CrossRefGoogle Scholar
  27. Schneider B, et al. 2012 Targeted siRNA delivery and mRNA knockdown mediated by bispecific digoxigenin-binding antibodies. Mol. Therapy Nucleic Acids 1 e46CrossRefGoogle Scholar
  28. Selkoe DJ and Schenk D 2003 Alzheimer’s disease: molecular understanding predicts amyloid-based therapeutics. Annu. Rev. Pharmacol. Toxicol. 43 545–584CrossRefGoogle Scholar
  29. Singer O, Marr RA, Rockenstein E, Crews L, Coufal NG, Gage FH, Verma IM and Masliah E 2005 Targeting BACE1 with siRNAs ameliorates Alzheimer disease neuropathology in a transgenic model. Nat. Neurosci. 8 1343–1349CrossRefGoogle Scholar
  30. Tanzi RE and Bertram L 2005 Twenty years of the Alzheimer’s disease amyloid hypothesis: a genetic perspective. Cell 120 545–555CrossRefGoogle Scholar
  31. Vassar R 2014 BACE1 inhibitor drugs in clinical trials for Alzheimer’s disease. Alzheimer’s Res. Therapy 6 89CrossRefGoogle Scholar
  32. Vorhees CV and Williams MT 2006 Morris water maze: procedures for assessing spatial and related forms of learning and memory. Nat. Protocols 1 848–858CrossRefGoogle Scholar
  33. Walsh DM and Selkoe DJ 2007 A beta oligomers - a decade of discovery. J. Neurochem. 101 1172–1184CrossRefGoogle Scholar
  34. Weber M, Wu T, Meilandt WJ, Dominguez SL, Solanoy HO, Maloney JA, Ngu H, Baca M, et al. 2017 BACE1 across species: a comparison of the in vivo consequences of BACE1 deletion in mice and rats. Sci. Rep. 7 44249CrossRefGoogle Scholar
  35. Wilcock DM, Gordon MN and Morgan D 2006 Quantification of cerebral amyloid angiopathy and parenchymal amyloid plaques with Congo red histochemical stain. Nat. Protocols 1 1591–1595CrossRefGoogle Scholar
  36. Vassar R, Kovacs DM, Yan R and Wong PC 2009 The beta-secretase enzyme BACE in health and Alzheimer’s disease: regulation, cell biology, function, and therapeutic potential. J. Neurosci. 29 12787–12794CrossRefGoogle Scholar
  37. Willem M, Garratt AN, Novak B, Citron M, Kaufmann S, Rittger A, DeStrooper B, Saftig P, Birchmeier C and Haass C 2006 Control of peripheral nerve myelination by the beta-secretase BACE1. Science 314 664–666CrossRefGoogle Scholar
  38. Yan R and Vassar R 2014 Targeting the β secretase BACE1 for Alzheimer’s disease therapy. Lancet Neurol. 13 319–329CrossRefGoogle Scholar

Copyright information

© Indian Academy of Sciences 2019

Authors and Affiliations

  • Mohamed Mohamed Haroon
    • 1
    • 3
  • Kamal Saba
    • 1
    • 2
  • Venkata Harshavardhan Boddedda
    • 1
  • Jerald Mahesh Kumar
    • 1
  • Anant Bahadur Patel
    • 1
    • 2
    Email author
  • Vijaya Gopal
    • 1
    Email author
  1. 1.CSIR–Centre for Cellular and Molecular BiologyHyderabadIndia
  2. 2.Academy of Scientific and Innovative Research (AcSIR)GhaziabadIndia
  3. 3.Institute for Stem Cell Biology and Regenerative Medicine (inStem), National Centre for Biological SciencesBengaluruIndia

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