Please wait a minute...
Frontiers of Medicine

ISSN 2095-0217

ISSN 2095-0225(Online)

CN 11-5983/R

Postal Subscription Code 80-967

2018 Impact Factor: 1.847

Front. Med.    2015, Vol. 9 Issue (1) : 90-99     DOI: 10.1007/s11684-015-0390-2
RESEARCH ARTICLE |
Optimized human factor IX expression cassettes for hepatic-directed gene therapy of hemophilia B
Ru Zhang,Qiang Wang,Lin Zhang,Saijuan Chen()
State Key Laboratory for Medical Genomics, Shanghai Institute of Hematology, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
Download: PDF(793 KB)   HTML
Export: BibTeX | EndNote | Reference Manager | ProCite | RefWorks
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.

Keywords factor IX      hemophilia B      liver-specific regulatory elements      hydrodynamic gene transfer     
Corresponding Authors: Saijuan Chen   
Just Accepted Date: 16 January 2015   Online First Date: 10 February 2015    Issue Date: 02 March 2015
URL:  
http://academic.hep.com.cn/fmd/EN/10.1007/s11684-015-0390-2     OR     http://academic.hep.com.cn/fmd/EN/Y2015/V9/I1/90
Fig.1  Construction of hFIX expression cassettes. (A) Diagram of human FIX expression cassette: a liver-specific FIX expression cassette comprises FIX cDNA and liver-specific regulatory elements, including enhancer, promoter, intron, and polyA flanked by the AAV internal terminal repeats. The AAV internal terminal repeats were necessary for packaging the virion. (B) Each cassette had intact 5′ and 3′ ITRs. scAAV-LP1-hFIXco-SJ contained the LP1 promoter, which comprised core liver-specific elements from HCR and hAAT, modified SV40 intron, hFIXco-SJ, and late SV40 polyA [5]. scAAV-LP1-hFIXco-SJ-R338L, scAAV-LP1-hFIXco-SIH, scAAV-LP1-hFIXco-SIH-R338L, and scAAV-LP1-hFIXwt contained the same control elements as scAAV-LP1-hFIXco-SJ, and those cassettes had different hFIX as shown in the Figure above. scAAV-Alb-hFIXco-SIH-R338L, scAAV-Pre-hFIXco-SIH-R338L, and scAAV-Chi-hFIXco-SIH-R338L contained the same intron (Chimeric intron), polyA (Synthetic polyA), and hFIXco-SIH, which were driven by albumin enhancer (221 bp) and albumin promoter (292 bp), prothrombin/prealbumin enhancer (180 bp) and prealbumin promoter (216 bp), chimeric albumin enhancer (228 bp) and prealbumin/albumin promoter (215 bp), respectively.
Plasmid name Enhancer Promoter Intron hFIX PolyA
LP1-hFIXwt ApoE-HCR hAAT Mod-SV40 hFIXwt Late SV40
LP1-hFIXco-SJ ApoE-HCR hAAT Mod-SV40 hFIXco-SJ Late SV40
LP1-hFIXco-SIH ApoE-HCR hAAT Mod-SV40 hFIXco-SIH Late SV40
LP1-hFIXco-SJ-R338L ApoE-HCR hAAT Mod-SV40 hFIXco-SJ-R338L Late SV40
LP1-hFIXco-SIH-R338L ApoE-HCR hAAT Mod-SV40 hFIXco-SIH-R338L Late SV40
Alb-hFIXco-SIH-R338L Alb Alb Chimeric hFIXco-SIH-R338L Synthetic
Pre-hFIXco-SIH-R338L Pro/Prea Prealbumin Chimeric hFIXco-SIH-R338L Synthetic
Chi-hFIXco-SIH-R338L Alb/Albb Pre/Albc Chimeric hFIXco-SIH-R338L Synthetic
Tab.1  Structures of different FIX expression cassettes applied in this study
Fig.2  Codon optimization of FIX: adjustment of the CAI, GC content and the frequency of codon usage for improving FIX expression. (A) A CAI of 1.0 was considered as perfect, whereas a CAI of>0.8 was good, for high-level gene expression in the desired organism [30]. (B) The ideal range of GC content was between 30% and 70%. Peaks of GC content percentage in a 60 bp window were removed. (C) Frequency of optimal codons was the percentage distribution of codons in computed codon quality groups. The value of 100 was set for the codon with the highest usage frequency for a given amino acid in the desired expression organism.
Fig.3  Effects of codon optimization on hFIX expression under the control of the same elements. ELISA was used to detect plasma hFIX antigen at 36 h after tail-vein hydrodynamic administration of saline (CON, n = 3), scAAV-LP1-hFIX (FIX-WT, n = 5), scAAV-LP1-hFIXco-SIH (FIX-SIH, n = 8), and scAAV-LP1-hFIXco-SJ (FIX-SJ, n = 8). (A+ B) hFIX antigen levels in the plasma of individual mice are indicated in (A) and presented as mean±SD in (B). Saline-injected mice produced nearly undetectable hFIX antigen (0.2% of normal). FIX-WT, FIX-SIH, and FIX-SJ had a mean hFIX antigen levels of 47%±7% (2.35±0.14 μg/ml), 162%±15% (8.1±0.3 μg/ml), and 157%±19% (7.85±0.95 μg/ml) of normal, respectively.
Fig.4  Comparison of hFIX coagulant activity and expression level mediated by transgene cassettes in HB mice after hydrodynamic injection. FIX-SJ: scAAV-LP1-hFIXco-SJ; FIX-SJ-M: scAAV-LP1-hFIXco-SJ-R338L; FIX-SIH: scAAV-LP1-hFIXco-SIH; and FIX-SIH-M: scAAV-LP1-hFIXco-SIH-R338L. (A+ B) Chromogenic assay to determine hFIX activity in individual mice in (A) is presented as mean±SD in (B). The activity is calculated as a percentage of normal hFIX activity in the pooled plasma. The average hFIX activities mediated by FIX-SJ, FIX-SJ-M, FIX-SIH, and FIX-SIH-M were about 60%±24%, 406%±32%, 66%±16%, and 479%±37%, respectively. (C+ D) ELISA results of hFIX expression in individual mice in (C) are presented as mean±SD in (D). The expression of codon-optimized hFIX with or without R338L mutation showed no significant difference.
Fig.5  Comparison of liver-specific regulatory elements driving hFIX expressionin vivo after hydrodynamic delivery. (A+ B) Chromogenic assay to determine hFIX coagulant activity, which was calculated as percentage of normal hFIX activity in pooled human plasma. The activities in individual mice are indicated in (A) and presented as mean±SD in (B). LP1-SJ: LP1-hFIXco-SJ (n = 4); LP1-SIH-M: LP1-hFIXco-SIH-M (n = 5); Alb-SIH-M: Alb-hFIXco-SIH-M (n = 5); Chi-SIH-M: Chi-hFIXco-SIH-M (n = 4); and Pre-SIH-M: Pre-hFIXco-SIH-M (n = 5).
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)
pmid: 23688764
2 Nilsson IM, Berntorp E, L?fqvist T, Pettersson H. Twenty-five years’ experience of prophylactic treatment in severe haemophilia A and B. J Intern Med 1992; 232(1): 25–32
doi: 10.1111/j.1365-2796.1992.tb00546.x pmid: 1640190
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
doi: 10.1056/NEJMoa067659 pmid: 17687129
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
doi: 10.1056/NEJMoa1108046 pmid: 22149959
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
doi: 10.1182/blood-2005-10-4035 pmid: 16322469
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
doi: 10.4238/2014.July.4.14 pmid: 25062487
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
doi: 10.1016/j.pep.2008.01.008 pmid: 18289875
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
doi: 10.1182/blood-2010-05-282707 pmid: 21041718
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
doi: 10.1371/journal.pone.0037671 pmid: 22655063
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
doi: 10.1006/mthe.2001.0333 pmid: 11407909
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
doi: 10.1038/sj.gt.3301229 pmid: 10918507
12 Ali?o 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
doi: 10.1038/sj.gt.3302065 pmid: 12923566
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
doi: 10.1016/j.jss.2008.02.016 pmid: 18570932
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
doi: 10.1016/j.bbrc.2005.11.063 pmid: 16316630
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
doi: 10.1016/S0378-1119(01)00550-9 pmid: 11470520
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
doi: 10.1056/NEJMoa0904377 pmid: 19846852
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
pmid: 3009475
18 Frain M, Hardon E, Ciliberto G, Sala-Trepat JM. Binding of a liver-specific factor to the human albumin gene promoter and enhancer. Mol Cell Biol 1990; 10(3): 991–999
pmid: 2304474
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
pmid: 1321810
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
pmid: 2786140
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
pmid: 3025666
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
pmid: 2995367
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
pmid: 3262058
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
pmid: 2824193
25 Bancroft JD, McDowell SA, Degen SJ. The human prothrombin gene: transcriptional regulation in HepG2 cells. Biochemistry 1992; 31(49): 12469–12476
doi: 10.1021/bi00164a025 pmid: 1463733
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
doi: 10.1038/mt.2013.197 pmid: 24042561
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
doi: 10.1016/S0076-6879(02)46050-8 pmid: 11883099
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
doi: 10.1038/sj.gt.3300947 pmid: 10455434
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
doi: 10.1182/blood-2002-02-0589 pmid: 12176886
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
doi: 10.1073/pnas.86.10.3699 pmid: 2657727
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
doi: 10.2174/1566523044578077 pmid: 15032617
32 Crooks GE, Hon G, Chandonia JM, Brenner SE. WebLogo: a sequence logo generator. Genome Res 2004; 14(6): 1188–1190
doi: 10.1101/gr.849004 pmid: 15173120
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
doi: 10.1128/AEM.01354-08 pmid: 18791013
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
doi: 10.1016/j.pep.2005.11.014 pmid: 16403645
35 Xia X. How optimized is the translational machinery in Escherichia coli, Salmonella typhimurium and Saccharomyces cerevisiae? Genetics 1998; 149(1): 37–44
pmid: 9584084
36 Gvritishvili AG, Leung KW, Tombran-Tink J. Codon preference optimization increases heterologous PEDF expression. PLoS One 2010; 5(11): e15056
doi: 10.1371/journal.pone.0015056 pmid: 21152082
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
doi: 10.1016/j.ymgme.2011.10.020 pmid: 22133298
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
doi: 10.1093/bib/5.2.150 pmid: 15260895
39 Xia X, Xie Z. DAMBE: software package for data analysis in molecular biology and evolution. J Hered 2001; 92(4): 371–373
doi: 10.1093/jhered/92.4.371 pmid: 11535656
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
pmid: 9811765
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
doi: 10.1073/pnas.89.1.89 pmid: 1729724
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
doi: 10.1089/hum.1992.3.6-641 pmid: 1482704
43 Gehrke S, Jér?me V, Müller R. Chimeric transcriptional control units for improved liver-specific transgene expression. Gene 2003; 322: 137–143
doi: 10.1016/j.gene.2003.08.010 pmid: 14644505
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
pmid: 8288579
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
pmid: 2431269
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
pmid: 3336368
47 Costa RH, Grayson DR, Xanthopoulos KG, Darnell JE Jr. A liver-specific 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
doi: 10.1073/pnas.85.11.3840 pmid: 2836860
48 Grayson DR, Costa RH, Xanthopoulos KG, Darnell JE Jr. A cell-specific enhancer of the mouse α1-antitrypsin gene has multiple functional regions and corresponding protein-binding sites. Mol Cell Biol 1988; 8(3): 1055–1066
pmid: 2835657
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
pmid: 16149135
[1] Supplementary Material Download
[1] Wei Lu,Qingzhang Zhou,Hao Yang,Hao Wang,Yexing Gu,Qi Shen,Jinglun Xue,Xiaoyan Dong,Jinzhong Chen. Gene therapy for hemophilia B mice with scAAV8-LP1-hFIX[J]. Front. Med., 2016, 10(2): 212-218.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed