Skip to main content
Log in

Optimized human factor IX expression cassettes for hepatic-directed gene therapy of hemophilia B

  • Research Article
  • Published:
Frontiers of Medicine Aims and scope Submit manuscript

Abstract

Gene therapy provides a potential cure for hemophilia B, and significant progress has been achieved in liver-directed gene transfer mediated by adeno-associated viral vectors. Recent clinical trials involving the use of a self-complementary adeno-associated virus serotype 8-human codon-optimized factor IX (AAV8-hFIXco) vector demonstrated encouraging efficacy with hFIX expression stabilized at 1% to 6% of normal level in patients, but safety concerns related to high vector doses are still present. Thus, further improvement of AAV vectors and hFIX expression cassette may positively contribute to the ultimate success of hemophilia B gene therapy. In this study, to obtain a higher expression level of hFIX that potentiates the coagulant capacity of recipients, human FIX expression vector was optimized by upgrading the codon adaption index and adjusting the GC content, inserting a Kozak sequence (GCCACC), and introducing a gain-of-function mutation, R338L (FIX Padua). The efficiency of the published and the presently constructed cassettes was compared through in vivo screening. In addition, the regulatory elements that control the FIX gene expression in these cassettes were screened for liver-specific effectiveness. Among all the constructed cassettes, scAAV-Pre-hFIXco-SIH-R338L, which was the construct under the control of the prothrombin enhancer and prealbumin promoter, resulted in the highest level of coagulant activity, and the expression levels of two constructed cassettes (scAAV-Chi-hFIXco-SIH-R338L and scAAV-Pre-hFIXco-SIH-R338L) were also higher than that of the published cassette (scAAV-LP1-hFIXco-SJ). In summary, our strategies led to a substantial increase in hFIX expression at the protein level or a remarkably elevated coagulant activity. Thus, these reconstructs of hFIX with AAV vector may potentially contribute to the creation of an efficacious gene therapy of hemophilia B.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Similar content being viewed by others

References

  1. Thrombosis and Hemostasis Group, Chinese Society of Hematology, Chinese Medical Association/Hemophilia Treatment Center Collaborative Network of China. Consensus of Chinese experts on diagnosis and treatment of hemophilia(2013). Chin J Hematol (Zhonghua Xue Ye Xue Za Zhi) 2013; 34(5): 461–463 (in Chinese)

    Google Scholar 

  2. Nilsson IM, Berntorp E, Löfqvist T, Pettersson H. Twenty-five years’ experience of prophylactic treatment in severe haemophilia A and B. J Intern Med 1992; 232(1): 25–32

    Article  CAS  PubMed  Google Scholar 

  3. Manco-Johnson MJ, Abshire TC, Shapiro AD, Riske B, Hacker MR, Kilcoyne R, Ingram JD, Manco-Johnson ML, Funk S, Jacobson L, Valentino LA, Hoots WK, Buchanan GR, DiMichele D, Recht M, Brown D, Leissinger C, Bleak S, Cohen A, Mathew P, Matsunaga A, Medeiros D, Nugent D, Thomas GA, Thompson AA, McRedmond K, Soucie JM, Austin H, Evatt BL. Prophylaxis versus episodic treatment to prevent joint disease in boys with severe hemophilia. N Engl J Med 2007; 357(6): 535–544

    Article  CAS  PubMed  Google Scholar 

  4. Nathwani AC, Tuddenham EG, Rangarajan S, Rosales C, McIntosh J, Linch DC, Chowdary P, Riddell A, Pie AJ, Harrington C, O’Beirne J, Smith K, Pasi J, Glader B, Rustagi P, Ng CY, Kay MA, Zhou J, Spence Y, Morton CL, Allay J, Coleman J, Sleep S, Cunningham JM, Srivastava D, Basner-Tschakarjan E, Mingozzi F, High KA, Gray JT, Reiss UM, Nienhuis AW, Davidoff AM. Adenovirus-associated virus vector-mediated gene transfer in hemophilia B. N Engl J Med 2011; 365(25): 2357–2365

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  5. Nathwani AC, Gray JT, Ng CY, Zhou J, Spence Y, Waddington SN, Tuddenham EG, Kemball-Cook G, McIntosh J, Boon-Spijker M, Mertens K, Davidoff AM. Self-complementary adeno-associated virus vectors containing a novel liver-specific human factor IX expression cassette enable highly efficient transduction of murine and nonhuman primate liver. Blood 2006; 107(7): 2653–2661

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  6. Li YM, Li DJ, Xu XJ, Cui M, Zhen HH, Wang Q. Effect of codon optimization on expression levels of human cystatin C in Pichia pastoris. Genet Mol Res 2014; 13(3): 4990–5000

    Article  CAS  PubMed  Google Scholar 

  7. Burgess-Brown NA, Sharma S, Sobott F, Loenarz C, Oppermann U, Gileadi O. Codon optimization can improve expression of human genes in Escherichia coli: a multi-gene study. Protein Expr Purif 2008; 59(1): 94–102

    Article  CAS  PubMed  Google Scholar 

  8. Ward NJ, Buckley SM, Waddington SN, Vandendriessche T, Chuah MK, Nathwani AC, McIntosh J, Tuddenham EG, Kinnon C, Thrasher AJ, McVey JH. Codon optimization of human factor VIII cDNAs leads to high-level expression. Blood 2011; 117(3): 798–807

    Article  CAS  PubMed  Google Scholar 

  9. Sack BK, Merchant S, Markusic DM, Nathwani AC, Davidoff AM, Byrne BJ, Herzog RW. Transient B cell depletion or improved transgene expression by codon optimization promote tolerance to factor VIII in gene therapy. PLoS ONE 2012; 7(5): e37671

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  10. Miao CH, Thompson AR, Loeb K, Ye X. Long-term and therapeutic-level hepatic gene expression of human factor IX after naked plasmid transfer in vivo. Mol Ther 2001; 3(6): 947–957

    Article  CAS  PubMed  Google Scholar 

  11. Zhang G, Song YK, Liu D. Long-term expression of human α1-antitrypsin gene in mouse liver achieved by intravenous administration of plasmid DNA using a hydrodynamics-based procedure. Gene Ther 2000; 7(15): 1344–1349

    Article  CAS  PubMed  Google Scholar 

  12. Aliño SF, Crespo A, Dasí F. Long-term therapeutic levels of human α1-antitrypsin in plasma after hydrodynamic injection of nonviral DNA. Gene Ther 2003; 10(19): 1672–1679

    Article  PubMed  Google Scholar 

  13. Nguyen AT, Dow AC, Kupiec-Weglinski J, Busuttil RW, Lipshutz GS. Evaluation of gene promoters for liver expression by hydrodynamic gene transfer. J Surg Res 2008; 148(1): 60–66

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  14. Al-Dosari M, Zhang G, Knapp JE, Liu D. Evaluation of viral and mammalian promoters for driving transgene expression in mouse liver. Biochem Biophys Res Commun 2006; 339(2): 673–678

    Article  CAS  PubMed  Google Scholar 

  15. Xu ZL, Mizuguchi H, Ishii-Watabe A, Uchida E, Mayumi T, Hayakawa T. Optimization of transcriptional regulatory elements for constructing plasmid vectors. Gene 2001; 272(1–2): 149–156

    Article  CAS  PubMed  Google Scholar 

  16. Simioni P, Tormene D, Tognin G, Gavasso S, Bulato C, Iacobelli NP, Finn JD, Spiezia L, Radu C, Arruda VR. X-linked thrombophilia with a mutant factor IX (factor IX Padua). N Engl J Med 2009; 361(17): 1671–1675

    Article  CAS  PubMed  Google Scholar 

  17. Minghetti PP, Ruffner DE, Kuang WJ, Dennison OE, Hawkins JW, Beattie WG, Dugaiczyk A. Molecular structure of the human albumin gene is revealed by nucleotide sequence within q11-22 of chromosome 4. J Biol Chem 1986; 261(15): 6747–6757

    CAS  PubMed  Google Scholar 

  18. Frain M, Hardon E, Ciliberto G, Sala-Trepat JM. Binding of a liverspecific factor to the human albumin gene promoter and enhancer. Mol Cell Biol 1990; 10(3): 991–999

    PubMed Central  CAS  PubMed  Google Scholar 

  19. Hayashi Y, Chan J, Nakabayashi H, Hashimoto T, Tamaoki T. Identification and characterization of two enhancers of the human albumin gene. J Biol Chem 1992; 267(21): 14580–14585

    CAS  PubMed  Google Scholar 

  20. Costa RH, Grayson DR, Darnell JE Jr. Multiple hepatocyte-enriched nuclear factors function in the regulation of transthyretin and α 1-antitrypsin genes. Mol Cell Biol 1989; 9(4): 1415–1425

    PubMed Central  CAS  PubMed  Google Scholar 

  21. Costa RH, Lai E, Darnell JE Jr. Transcriptional control of the mouse prealbumin (transthyretin) gene: both promoter sequences and a distinct enhancer are cell specific. Mol Cell Biol 1986; 6(12): 4697–4708

    PubMed Central  CAS  PubMed  Google Scholar 

  22. Tsuzuki T, Mita S, Maeda S, Araki S, Shimada K. Structure of the human prealbumin gene. J Biol Chem 1985; 260(22): 12224–12227

    CAS  PubMed  Google Scholar 

  23. Monaci P, Nicosia A, Cortese R. Two different liver-specific factors stimulate in vitro transcription from the human α1-antitrypsin promoter. EMBO J 1988; 7(7): 2075–2087

    PubMed Central  CAS  PubMed  Google Scholar 

  24. De Simone V, Ciliberto G, Hardon E, Paonessa G, Palla F, Lundberg L, Cortese R. Cis- and trans-acting elements responsible for the cell-specific expression of the human α1-antitrypsin gene. EMBO J 1987; 6(9): 2759–2766

    PubMed Central  PubMed  Google Scholar 

  25. Bancroft JD, McDowell SA, Degen SJ. The human prothrombin gene: transcriptional regulation in HepG2 cells. Biochemistry 1992; 31(49): 12469–12476

    Article  CAS  PubMed  Google Scholar 

  26. Chen Y, Schroeder JA, Kuether EL, Zhang G, Shi Q. Platelet gene therapy by lentiviral gene delivery to hematopoietic stem cells restores hemostasis and induces humoral immune tolerance in FIX (null) mice. Mol Ther 2014; 22(1): 169–177

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  27. Song YK, Liu F, Zhang G, Liu D. Hydrodynamics-based transfection: simple and efficient method for introducing and expressing transgenes in animals by intravenous injection of DNA. Methods Enzymol 2002; 346: 92–105

    Article  CAS  PubMed  Google Scholar 

  28. Liu F, Song Y, Liu D. Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA. Gene Ther 1999; 6(7): 1258–1266

    Article  CAS  PubMed  Google Scholar 

  29. Nathwani AC, Davidoff AM, Hanawa H, Hu Y, Hoffer FA, Nikanorov A, Slaughter C, Ng CY, Zhou J, Lozier JN, Mandrell TD, Vanin EF, Nienhuis AW. Sustained high-level expression of human factor IX (hFIX) after liver-targeted delivery of recombinant adeno-associated virus encoding the hFIX gene in rhesus macaques. Blood 2002; 100(5): 1662–1669

    Article  CAS  PubMed  Google Scholar 

  30. Gutman GA, Hatfield GW. Nonrandom utilization of codon pairs in Escherichia coli. Proc Natl Acad Sci U S A 1989; 86(10): 3699–3703

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  31. Papadakis ED, Nicklin SA, Baker AH, White SJ. Promoters and control elements: designing expression cassettes for gene therapy. Curr Gene Ther 2004; 4(1): 89–113

    Article  CAS  PubMed  Google Scholar 

  32. Crooks GE, Hon G, Chandonia JM, Brenner SE. WebLogo: a sequence logo generator. Genome Res 2004; 14(6): 1188–1190

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  33. Tokuoka M, Tanaka M, Ono K, Takagi S, Shintani T, Gomi K. Codon optimization increases steady-state mRNA levels in Aspergillus oryzae heterologous gene expression. Appl Environ Microbiol 2008; 74(21): 6538–6546

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  34. Hu S, Li L, Qiao J, Guo Y, Cheng L, Liu J. Codon optimization, expression, and characterization of an internalizing anti-ErbB2 single-chain antibody in Pichia pastoris. Protein Expr Purif 2006; 47(1): 249–257

    Article  CAS  PubMed  Google Scholar 

  35. Xia X. How optimized is the translational machinery in Escherichia coli, Salmonella typhimurium and Saccharomyces cerevisiae? Genetics 1998; 149(1): 37–44

    PubMed Central  CAS  PubMed  Google Scholar 

  36. Gvritishvili AG, Leung KW, Tombran-Tink J. Codon preference optimization increases heterologous PEDF expression. PLoS One 2010; 5(11): e15056

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  37. Wang L, Morizono H, Lin J, Bell P, Jones D, McMenamin D, Yu H, Batshaw ML, Wilson JM. Preclinical evaluation of a clinical candidate AAV8 vector for ornithine transcarbamylase (OTC) deficiency reveals functional enzyme from each persisting vector genome. Mol Genet Metab 2012; 105(2): 203–211

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  38. Kumar S, Tamura K, Nei M. MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 2004; 5(2): 150–163

    Article  CAS  PubMed  Google Scholar 

  39. Xia X, Xie Z. DAMBE: software package for data analysis in molecular biology and evolution. J Hered 2001; 92(4): 371–373

    Article  CAS  PubMed  Google Scholar 

  40. Xiao W, Berta SC, Lu MM, Moscioni AD, Tazelaar J, Wilson JM. Adeno-associated virus as a vector for liver-directed gene therapy. J Virol 1998; 72(12): 10222–10226

    PubMed Central  CAS  PubMed  Google Scholar 

  41. Kay MA, Baley P, Rothenberg S, Leland F, Fleming L, Ponder KP, Liu T, Finegold M, Darlington G, Pokorny W. Expression of human α1-antitrypsin in dogs after autologous transplantation of retroviral transduced hepatocytes. Proc Natl Acad Sci U S A 1992; 89(1): 89–93

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  42. Kay MA, Li Q, Liu TJ, Leland F, Toman C, Finegold M, Woo SL. Hepatic gene therapy: persistent expression of human α1-antitrypsin in mice after direct gene delivery in vivo. Hum Gene Ther 1992; 3(6): 641–647

    Article  CAS  PubMed  Google Scholar 

  43. Gehrke S, Jérôme V, Müller R. Chimeric transcriptional control units for improved liver-specific transgene expression. Gene 2003; 322: 137–143

    Article  CAS  PubMed  Google Scholar 

  44. Wu KJ, Wilson DR, Shih C, Darlington GJ. The transcription factor HNF1 acts with C/EBPα to synergistically activate the human albumin promoter through a novel domain. J Biol Chem 1994; 269(2): 1177–1182

    CAS  PubMed  Google Scholar 

  45. Godbout R, Ingram R, Tilghman SM. Multiple regulatory elements in the intergenic region between the α-fetoprotein and albumin genes. Mol Cell Biol 1986; 6(2): 477–487

    PubMed Central  CAS  PubMed  Google Scholar 

  46. Costa RH, Lai E, Grayson DR, Darnell JE Jr. The cell-specific enhancer of the mouse transthyretin (prealbumin) gene binds a common factor at one site and a liver-specific factor(s) at two other sites. Mol Cell Biol 1988; 8(1): 81–90

    PubMed Central  CAS  PubMed  Google Scholar 

  47. Costa RH, Grayson DR, Xanthopoulos KG, Darnell JE Jr. A liverspecific DNA-binding protein recognizes multiple nucleotide sites in regulatory regions of transthyretin, α1-antitrypsin, albumin, and simian virus 40 genes. Proc Natl Acad Sci U S A 1988; 85(11): 3840–3844

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  48. Grayson DR, Costa RH, Xanthopoulos KG, Darnell JE Jr. A cellspecific enhancer of the mouse α1-antitrypsin gene has multiple functional regions and corresponding protein-binding sites. Mol Cell Biol 1988; 8(3): 1055–1066

    PubMed Central  CAS  PubMed  Google Scholar 

  49. Lemken ML, Wybranietz WA, Schmidt U, Graepler F, Armeanu S, Bitzer M, Lauer UM. Expression liver-directed genes by employing synthetic transcriptional control units. World J Gastroenterol 2005; 11(34): 5295–5302

    CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Saijuan Chen.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhang, R., Wang, Q., Zhang, L. et al. Optimized human factor IX expression cassettes for hepatic-directed gene therapy of hemophilia B. Front. Med. 9, 90–99 (2015). https://doi.org/10.1007/s11684-015-0390-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11684-015-0390-2

Keywords

Navigation