Login Register Cart 0
Generic placeholder image

Current Biotechnology

Editor-in-Chief

ISSN (Print): 2211-5501
ISSN (Online): 2211-551X

Back
Journal Home About Journal Editorial Board Journal Insight Current Issue Volumes/Issues
Subscribe
Research Article

Investigation on Cross-correction of Cystinosis through Genetically Engineered Cells Secreting Cystinosin

Author(s): Valeria Graceffa*

Volume 13, Issue 1, 2024

Published on: 04 December, 2023

Page: [46 - 57] Pages: 12

DOI: 10.2174/0122115501271925231130074832

Price: $65

Abstract

Background: Cystinosis is a rare inherited lysosomal storage disease (LSD), caused by a mutation in the Cystinosin Lysosomal Cystine Transporter (CTNS). Novel therapies and strategies are needed to improve patients' clinical conditions and quality of life.

Objectives and Methods: This study assessed whether CTNS can be secreted, and investigated a method to enhance its secretion, by adding a secretion signal to the N-terminus. Human Embryonic Kidney (HEK) 293 cells were transfected with the resulting construct. The amount of protein secreted was then measured. Uptake by monolayer cultures of cystinotic cells and enzyme activity were also assessed.

Results: The recombinant protein could effectively be secreted, and the secretion signal slightly further increased its secretion. The secreted recombinant protein was taken up by cystinotic cells, and, after internalization, still retained its biological activity.

Conclusion: Optimization of the proposed method to increase the secretion of CTNS would provide new insights into the production of recombinant proteins for medical and industrial use. Further identification and screening of alternative signalling peptides and cell types can maximise the secretion and production of recombinant CNTS, to be used as a therapeutic agent in human healthcare.

Keywords: Cystinosis, cross-correction, recombinant protein secretion, cystinosin (CTNS), lysosomal storage diseases, lysosomes.

« Previous Next »
Graphical Abstract

[1]
Graceffa V. Clinical development of cell therapies to halt lysosomal storage diseases: results and lessons learned. Curr Gene Ther 2022; 22(3): 191-213.
[http://dx.doi.org/10.2174/1566523221666210728141924] [PMID: 34323185]
[2]
Ariceta G, Giordano V, Santos F. Effects of long-term cysteamine treatment in patients with cystinosis. Pediatr Nephrol 2019; 34(4): 571-8.
[http://dx.doi.org/10.1007/s00467-017-3856-4] [PMID: 29260317]
[3]
Cherqui S. Cysteamine therapy: A treatment for cystinosis, not a cure. Kidney Int 2012; 81(2): 127-9.
[http://dx.doi.org/10.1038/ki.2011.301] [PMID: 22205430]
[4]
Castro-Balado A, Mondelo-García C, Varela-Rey I, et al. Recent research in ocular cystinosis: Drug delivery systems, cysteamine detection methods and future perspectives. Pharmaceutics 2020; 12(12): 1177.
[5]
Elmonem MA, Veys KRP, Prencipe G. Nephropathic cystinosis: Pathogenic roles of inflammation and potential for new therapies. Cells 2022; 11(2): 190.
[6]
Graceffa V. Development of a fibrin-mediated gene delivery system for the treatment of cystinosis via design of experiment. Sci Rep 2022; 12(1): 3752.
[http://dx.doi.org/10.1038/s41598-022-07750-y] [PMID: 35260693]
[7]
De Brabander P, Uitterhaegen E, Delmulle T, De Winter K, Soetaert W. Challenges and progress towards industrial recombinant protein production in yeasts: A review. Biotechnol Adv 2023; 64: 108121.
[http://dx.doi.org/10.1016/j.biotechadv.2023.108121] [PMID: 36775001]
[8]
Pollet J, Chen WH, Strych U. Recombinant protein vaccines, a proven approach against coronavirus pandemics. Adv Drug Deliv Rev 2021; 170: 71-82.
[http://dx.doi.org/10.1016/j.addr.2021.01.001] [PMID: 33421475]
[9]
Neef J, van Dijl JM, Buist G. Recombinant protein secretion by Bacillus subtilis and Lactococcus lactis: pathways, applications, and innovation potential. Essays Biochem 2021; 65(2): 187-95.
[http://dx.doi.org/10.1042/EBC20200171] [PMID: 33955475]
[10]
Chen X, Li C, Liu H. Enhanced recombinant protein production under special environmental stress. Front Microbiol 2021; 12: 630814.
[http://dx.doi.org/10.3389/fmicb.2021.630814] [PMID: 33935992]
[11]
Huang M, Wang G, Qin J, Petranovic D, Nielsen J. Engineering the protein secretory pathway of Saccharomyces cerevisiae enables improved protein production. Proc Natl Acad Sci USA 2018; 115(47): E11025-32.
[http://dx.doi.org/10.1073/pnas.1809921115] [PMID: 30397111]
[12]
Wang JY. Improving the secretion of designed protein assemblies through negative design of cryptic transmembrane domains. In: Daniel WK, Ed. Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery. La Jolla, CA: The Scripps Research Institute 2023.
[13]
Xue S, Liu X, Pan Y, et al. Comprehensive analysis of signal peptides in Saccharomyces cerevisiae reveals features for efficient secretion. Adv Sci (Weinh) 2023; 10(2): 2203433.
[http://dx.doi.org/10.1002/advs.202203433] [PMID: 36478443]
[14]
Ehsan A, Mahmood K, Khan YD, Khan SA, Chou KC. A novel modeling in mathematical biology for classification of signal peptides. Sci Rep 2018; 8(1): 1039.
[http://dx.doi.org/10.1038/s41598-018-19491-y] [PMID: 29348418]
[15]
Hui X, Chen Z, Zhang J, et al. Computational prediction of secreted proteins in gram-negative bacteria. Comput Struct Biotechnol J 2021; 19: 1806-28.
[http://dx.doi.org/10.1016/j.csbj.2021.03.019] [PMID: 33897982]
[16]
Wang X, Li F, Xu J, et al. ASPIRER: a new computational approach for identifying non-classical secreted proteins based on deep learning. Brief Bioinform 2022; 23(2): bbac031.
[http://dx.doi.org/10.1093/bib/bbac031] [PMID: 35176756]
[17]
Raschmanová H, Weninger A, Knejzlík Z, Melzoch K, Kovar K. Engineering of the unfolded protein response pathway in Pichia pastoris: Enhancing production of secreted recombinant proteins. Appl Microbiol Biotechnol 2021; 105(11): 4397-414.
[http://dx.doi.org/10.1007/s00253-021-11336-5] [PMID: 34037840]
[18]
Cho JS, Oh HJ, Jang YE, et al. Synthetic pro‐peptide design to enhance the secretion of heterologous proteins by Saccharomyces cerevisiae. MicrobiologyOpen 2022; 11(3): e1300.
[http://dx.doi.org/10.1002/mbo3.1300] [PMID: 35765186]
[19]
Peng C, Guo Y, Ren S, Li C, Liu F, Lu F. SPSED: A Signal Peptide Secretion Efficiency Database. Front Bioeng Biotechnol 2022; 9: 819789.
[http://dx.doi.org/10.3389/fbioe.2021.819789] [PMID: 35118058]
[20]
Silva AKA, Sagné C, Gazeau F, Abasolo I. Enzyme replacement therapy: current challenges and drug delivery prospects via extracellular vesicles. Rare Disease and Orphan Drugs Journal 2022; 1(3): 13.
[http://dx.doi.org/10.20517/rdodj.2022.09]
[21]
Massaro G, Geard AF, Liu W. Gene therapy for lysosomal storage disorders: Ongoing studies and clinical development. Biomolecules 2021; 11(4): 611.
[22]
Sevin C, Deiva K. Clinical trials for gene therapy in lysosomal diseases with CNS involvement. Front Mol Biosci 2021; 8: 624988.
[http://dx.doi.org/10.3389/fmolb.2021.624988] [PMID: 34604300]
[23]
Naphade S, Sharma J, Gaide Chevronnay HP, et al. Brief reports: Lysosomal cross-correction by hematopoietic stem cell-derived macrophages via tunneling nanotubes. Stem Cells 2015; 33(1): 301-9.
[http://dx.doi.org/10.1002/stem.1835] [PMID: 25186209]
[24]
Syres K, Harrison F, Tadlock M, et al. Successful treatment of the murine model of cystinosis using bone marrow cell transplantation. Blood 2009; 114(12): 2542-52.
[http://dx.doi.org/10.1182/blood-2009-03-213934] [PMID: 19506297]
[25]
Yeagy BA, Harrison F, Gubler MC, Koziol JA, Salomon DR, Cherqui S. Kidney preservation by bone marrow cell transplantation in hereditary nephropathy. Kidney Int 2011; 79(11): 1198-206.
[http://dx.doi.org/10.1038/ki.2010.537] [PMID: 21248718]
[26]
Coulson-Thomas VJ, Caterson B, Kao WWY. Transplantation of human umbilical mesenchymal stem cells cures the corneal defects of mucopolysaccharidosis VII mice. Stem Cells 2013; 31(10): 2116-26.
[http://dx.doi.org/10.1002/stem.1481] [PMID: 23897660]
[27]
Iglesias DM, El-Kares R, Taranta A, et al. Stem cell microvesicles transfer cystinosin to human cystinotic cells and reduce cystine accumulation in vitro. PLoS One 2012; 7(8): e42840.
[http://dx.doi.org/10.1371/journal.pone.0042840] [PMID: 22912749]
[28]
Leone DA, Peschel A, Brown M, et al. Surface LAMP-2 is an endocytic receptor that diverts antigen internalized by human dendritic cells into highly immunogenic exosomes. J Immunol 2017; 199(2): 531-46.
[http://dx.doi.org/10.4049/jimmunol.1601263] [PMID: 28607115]
[29]
Zhang L, Leng Q, Mixson AJ. Alteration in the IL‐2 signal peptide affects secretion of proteins in vitro and in vivo. J Gene Med 2005; 7(3): 354-65.
[http://dx.doi.org/10.1002/jgm.677] [PMID: 15619290]
[30]
Graceffa V. Physical and mechanical cues affecting biomaterial-mediated plasmid DNA delivery: insights into non-viral delivery systems. J Genet Eng Biotechnol 2021; 19(1): 90.
[http://dx.doi.org/10.1186/s43141-021-00194-3] [PMID: 34142237]
[31]
Gaffen S, Liu K. Overview of interleukin-2 function, production and clinical applications. Cytokine 2004; 28(3): 109-23.
[http://dx.doi.org/10.1016/j.cyto.2004.06.010] [PMID: 15473953]
[32]
Zhu X, Yin J, Liang S, et al. The Multivesicular Bodies (MVBs)-Localized AAA ATPase LRD6-6 Inhibits Immunity and Cell Death Likely through Regulating MVBs-Mediated Vesicular Trafficking in Rice. PLoS Genet 2016; 12(9): e1006311.
[http://dx.doi.org/10.1371/journal.pgen.1006311] [PMID: 27618555]
[33]
Meyer C, Losacco J, Stickney Z, Li L, Marriott G, Lu B. Pseudotyping exosomes for enhanced protein delivery in mammalian cells. Int J Nanomedicine 2017; 12: 3153-70.
[http://dx.doi.org/10.2147/IJN.S133430] [PMID: 28458537]
[34]
Felsheim RF, Chávez ASO, Palmer GH, et al. Transformation of Anaplasma marginale. Vet Parasitol 2010; 167(2-4): 167-74.
[http://dx.doi.org/10.1016/j.vetpar.2009.09.018] [PMID: 19837516]
[35]
Chiaverini C, Sillard L, Flori E, et al. Cystinosin is a melanosomal protein that regulates melanin synthesis. FASEB J 2012; 26(9): 3779-89.
[http://dx.doi.org/10.1096/fj.11-201376] [PMID: 22649030]
[36]
Freudl R. Signal peptides for recombinant protein secretion in bacterial expression systems. Microb Cell Fact 2018; 17(1): 52.
[http://dx.doi.org/10.1186/s12934-018-0901-3] [PMID: 29598818]
[37]
Cheng KW, Wang F, Lopez GA, et al. Evaluation of artificial signal peptides for secretion of two lysosomal enzymes in CHO cells. Biochem J 2021; 478(12): 2309-19.
[http://dx.doi.org/10.1042/BCJ20210015] [PMID: 34032266]
[38]
Elliger SS, Elliger CA, Lang C, Watson GL. Enhanced secretion and uptake of beta-glucuronidase improves adeno-associated viral-mediated gene therapy of mucopolysaccharidosis type VII mice. Mol Ther 2002; 5(5): 617-26.
[http://dx.doi.org/10.1006/mthe.2002.0594] [PMID: 11991753]
[39]
Sun B, Zhang H, Benjamin DK Jr, et al. Enhanced efficacy of an AAV vector encoding chimeric, highly secreted acid alpha-glucosidase in glycogen storage disease type II. Mol Ther 2006; 14(6): 822-30.
[http://dx.doi.org/10.1016/j.ymthe.2006.08.001] [PMID: 16987711]
[40]
Trabulo S, Cardoso AL, Mano M, et al. Cell-penetrating peptides-mechanisms of cellular uptake and generation of delivery systems. Pharmaceuticals 2010; 3(4): 961-93.
[http://dx.doi.org/10.3390/ph3040961]
[41]
Orii KO, Grubb JH, Vogler C, et al. Defining the pathway for Tat-mediated delivery of beta-glucuronidase in cultured cells and MPS VII mice. Mol Ther 2005; 12(2): 345-52.
[http://dx.doi.org/10.1016/j.ymthe.2005.02.031] [PMID: 16043103]
[42]
Xia H, Mao Q, Davidson BL. The HIV Tat protein transduction domain improves the biodistribution of β-glucuronidase expressed from recombinant viral vectors. Nat Biotechnol 2001; 19(7): 640-4.
[http://dx.doi.org/10.1038/90242] [PMID: 11433275]
[43]
Lee KO, Luu N, Kaneski CR, Schiffmann R, Brady RO, Murray GJ. Improved intracellular delivery of glucocerebrosidase mediated by the HIV-1 TAT protein transduction domain. Biochem Biophys Res Commun 2005; 337(2): 701-7.
[http://dx.doi.org/10.1016/j.bbrc.2005.05.207] [PMID: 16223608]
[44]
Higuchi K, Yoshimitsu M, Fan X, et al. Alpha-galactosidase A-Tat fusion enhances storage reduction in hearts and kidneys of Fabry mice. Mol Med 2010; 16(5-6): 216-21.
[http://dx.doi.org/10.2119/molmed.2009.00163] [PMID: 20454522]
[45]
Meng XL, Eto Y, Schiffmann R, Shen JS. HIV tat domain improves cross-correction of human galactocerebrosidase in a gene- and flanking sequence-dependent manner. Mol Ther Nucleic Acids 2013; 2(10): e130.
[http://dx.doi.org/10.1038/mtna.2013.57] [PMID: 24150577]
[46]
Matsuoka K, Tsuji D, Aikawa S, Matsuzawa F, Sakuraba H, Itoh K. Introduction of an N-glycan sequon into HEXA enhances human beta-hexosaminidase cellular uptake in a model of Sandhoff disease. Mol Ther 2010; 18(8): 1519-26.
[http://dx.doi.org/10.1038/mt.2010.113] [PMID: 20571546]
[47]
Do MA, Levy D, Brown A, Marriott G, Lu B. Targeted delivery of lysosomal enzymes to the endocytic compartment in human cells using engineered extracellular vesicles. Sci Rep 2019; 9(1): 17274.
[http://dx.doi.org/10.1038/s41598-019-53844-5] [PMID: 31754156]
[48]
LeBowitz JH, Grubb JH, Maga JA, Schmiel DH, Vogler C, Sly WS. Glycosylation-independent targeting enhances enzyme delivery to lysosomes and decreases storage in mucopolysaccharidosis type VII mice. Proc Natl Acad Sci USA 2004; 101(9): 3083-8.
[http://dx.doi.org/10.1073/pnas.0308728100] [PMID: 14976248]
[49]
Li Z, Zhou X, Gao X, et al. Fusion protein engineered exosomes for targeted degradation of specific RNAs in lysosomes: a proof‐of‐concept study. J Extracell Vesicles 2020; 9(1): 1816710.
[http://dx.doi.org/10.1080/20013078.2020.1816710] [PMID: 33133429]
[50]
Shen B, Wu N, Yang JM, Gould SJ. Protein targeting to exosomes/microvesicles by plasma membrane anchors. J Biol Chem 2011; 286(16): 14383-95.
[http://dx.doi.org/10.1074/jbc.M110.208660] [PMID: 21300796]
[51]
Thoene J, Goss T, Witcher M, et al. In vitro correction of disorders of lysosomal transport by microvesicles derived from baculovirus-infected Spodoptera cells. Mol Genet Metab 2013; 109(1): 77-85.
[http://dx.doi.org/10.1016/j.ymgme.2013.01.014] [PMID: 23465695]
[52]
Benedikter BJ, Bouwman FG, Vajen T, et al. Ultrafiltration combined with size exclusion chromatography efficiently isolates extracellular vesicles from cell culture media for compositional and functional studies. Sci Rep 2017; 7(1): 15297.
[http://dx.doi.org/10.1038/s41598-017-15717-7] [PMID: 29127410]
[53]
Shu SL, Allen CL, Benjamin-Davalos S, et al. A Rapid Exosome Isolation Using Ultrafiltration and Size Exclusion Chromatography (REIUS) Method for Exosome Isolation from Melanoma Cell Lines. Methods Mol Biol 2021; 2265: 289-304.
[http://dx.doi.org/10.1007/978-1-0716-1205-7_22] [PMID: 33704723]
[54]
Carnino JM, Lee H, Jin Y. Isolation and characterization of extracellular vesicles from Broncho-alveolar lavage fluid: a review and comparison of different methods. Respir Res 2019; 20(1): 240.
[http://dx.doi.org/10.1186/s12931-019-1210-z] [PMID: 31666080]
[55]
Lobb RJ, Becker M, Wen Wen S, et al. Optimized exosome isolation protocol for cell culture supernatant and human plasma. J Extracell Vesicles 2015; 4(1): 27031.
[http://dx.doi.org/10.3402/jev.v4.27031] [PMID: 26194179]
[56]
Campello E, Radu CM, Simion C, et al. Longitudinal Trend of Plasma Concentrations of Extracellular Vesicles in Patients Hospitalized for COVID-19. Front Cell Dev Biol 2022; 9: 770463.
[http://dx.doi.org/10.3389/fcell.2021.770463] [PMID: 35111751]
[57]
Tumne A, Prasad VS, Chen Y, et al. Noncytotoxic suppression of human immunodeficiency virus type 1 transcription by exosomes secreted from CD8+ T cells. J Virol 2009; 83(9): 4354-64.
[http://dx.doi.org/10.1128/JVI.02629-08] [PMID: 19193788]
[58]
Sung MS, Jung JH, Jeong C, Yoon TY, Park JH. Single‐molecule co‐immunoprecipitation reveals functional inheritance of EGFRs in extracellular vesicles. Small 2018; 14(42): 1802358.
[http://dx.doi.org/10.1002/smll.201802358] [PMID: 30239124]
[59]
Dehghani M, Gaborski TR. Fluorescent labeling of extracellular vesicles. Methods Enzymol United States 2020; 645: 15-42.
[PMID: 33565969]
[60]
Guevara-Morales JM, Echeverri-Peña OY. Implementation of a method to quantify white blood cell cystine as a diagnostic support for cystinosis. Nefrologia 2020; 40(1): 99-103. [English Edition]
[PMID: 31740152]
[61]
Gowda ASP, Schaefer AD, Schuck TK. A simple RP-HPLC method for the stability-indicating determination of N-acetyl-L-cysteine and N,N′-diacetyl-L-cystine in cell culture media. Cell Gene Ther Insights 2020; 6(2): 303-23.
[http://dx.doi.org/10.18609/cgti.2020.041]
[62]
Wang Y, Kang X-J, Ge WH, Sun X-Z, Peng J. Simple, Rapid, and Accurate RP-HPLC method for determination of cystine in human urine after derivatization with dansyl chloride. Chromatographia 2007; 65(9-10): 527-32.
[http://dx.doi.org/10.1365/s10337-007-0210-1]
[63]
Kalaiyarasan G, Hemlata C, Joseph J. Fluorescence turn-on, specific detection of cystine in human blood plasma and urine samples by nitrogen-doped carbon quantum dots. ACS Omega 2019; 4(1): 1007-14.
[http://dx.doi.org/10.1021/acsomega.8b03187] [PMID: 31459376]
[64]
Sekar A, Santhanam A, Sandhu C. Area under the curve spectrophotometric method for determination of irbesatran in pharmaceutical formulation. IRA-Int J Appl Sci 2017; 7(2): 95.
[65]
Attia KAM, Elabasawy NM, Abolmagd E. Simultaneous equation and area under the curve spectrophotometric methods for estimation of cefaclor in presence of its acid induced degradation product; A comparative study. Fut J Pharm Sci 2017; 3(2): 163-7.
[http://dx.doi.org/10.1016/j.fjps.2017.06.001]
[66]
Dagdu KD, Gadhave M, Bhujbal S. Area under curve by UV spectrophotometric method for determination albendazole in bulk. JDDT 2019; 9(6): 47.
[http://dx.doi.org/10.22270/jddt.v9i6.3667]
[67]
Gadhave M, Jadhav S, Gaikwad D, et al. Area under curve by UV spectrophotometric method for determination of ascorbic acid in bulk. Bull Env Pharmacol Life Sci 2020; 9(8): 35-9.
[68]
Dhumane S, Patel S, Kedar V. Area under curve by uv spectrophotometric method for determination aloe vera gel powder in bulk. SSRN 2020; 7(1)
[69]
Karpova S, Blazheyevskiy M, Mozgova O. Development and validation of UV Spectrophotometric area under curve method quantitative estimation of piperacillin. Int J Pharm Sci Res 2018; 9: 3556-60.
[70]
Tagaya Y, Kurys G, Thies TA, et al. Generation of secretable and nonsecretable interleukin 15 isoforms through alternate usage of signal peptides. Proc Natl Acad Sci USA 1997; 94(26): 14444-9.
[http://dx.doi.org/10.1073/pnas.94.26.14444] [PMID: 9405632]
[71]
Cherqui S, Kalatzis V, Trugnan G, Antignac C. The targeting of cystinosin to the lysosomal membrane requires a tyrosine-based signal and a novel sorting motif. J Biol Chem 2001; 276(16): 13314-21.
[http://dx.doi.org/10.1074/jbc.M010562200] [PMID: 11150305]

Rights & Permissions Print Cite
Article Metrics
12
2
Wayfinder Image
© 2024 Bentham Science Publishers | Privacy Policy