Evaluation of in vitro functional activity of ANB4 drug for the treatment of spinal muscular atrophy

Abstract

BACKGROUND: Spinal muscular atrophy (SMA) is a monogenic neurodegenerative disease. SMA is caused by a deficiency of the functional survival motor neuron protein (SMN) as a result of a mutation in the SMN1 gene. BIOCAD is developing a domestic gene therapy drug for the treatment of SMA based on recombinant adeno-associated virus serotype 9 (rAAV9) carrying the SMN1 gene (ANB4). In vitro evaluation of the functional activity of ANB4 will allow a more complete characterization of the drug.

AIM: Development of an accurate and reproducible in vitro functional test that reflects the clinical mechanism of action of ANB4.

METHODS: To model SMA, the SMN1 gene was knocked down by transfection with small interfering RNA. Amount of the SMN protein was measured by enzyme-linked immunosorbent assay. The functional activity of the drug was evaluated by analysis of Gemin2 protein level using the western blot analysis.

RESULTS: Analytical method has been developed to assess the functional activity of the ANB4 drug for the treatment of SMA type 1 (rAAV9 carrying the SMN1 gene). The developed technique made it possible to obtain accurate and reproducible results. Production of Gemin2 after knockdown of SMN1 and the introduction of exogenous SMN1 gene was restored to control values, comparable with the restoration of the level of the SMN protein.

CONCLUSION: The developed technique closely reflects the clinical mechanism of action of the ANB4 drug for the treatment of SMA. By evaluating the functional activity in vitro, accurate and reproducible results were obtained in accordance with the required standards, including the principles of 3R.

Keywords

SMA / SMN1 / SMN2 / snRNP / Gemin2 / AAV9 / gene therapy

References

[1] https://www.ema.europa.eu/ [Internet]. Guideline on the quality, non-clinical and clinical aspects of gene therapy medicinal products. In: European medical agency. Scientific guideline [cited: 18.12.2022]. Available from: https://www.ema.europa.eu/en/quality-preclinical-clinical-aspects-gene-therapy-medicinal-products-scientific-guideline

[2] https://www.ema.europa.eu/ [Internet]. Guideline on the quality, non-clinical and clinical aspects of gene therapy medicinal products. In: European medical agency. Scientific guideline [дата обращения: 18.12.2022]. Доступ по ссылке: https://www.ema.europa.eu/en/quality-preclinical-clinical-aspects-gene-therapy-medicinal-products-scientific-guideline

[3] Potency tests for cellular and gene therapy products. In: U.S. Food and Drug Administration. Final guidance for industry: January 2011 [cited: 18.12.2022]. Available from: https://www.fda.gov/regulatory-information/search-fda-guidance-documents/potency-tests-cellular-and-gene-therapy-products

[4] https://www.fda.gov/ [Internet]. Potency tests for cellular and gene therapy products. In: U.S. Food and Drug Administration. Final guidance for industry: January 2011 [дата обращения: 18.12.2022]. Доступ по ссылке: https://www.fda.gov/regulatory-information/search-fda-guidance-documents/potency-tests-cellular-and-gene-therapy-products

[5] Chemistry, manufacturing, and control (CMC) information for human gene therapy investigational new drug applications (INDs). In: U.S. Food and Drug Administration. Guidance for Industry: January 2020 [cited: 18.12.2022]. Available from: https://www.fda.gov/regulatory-information/search-fda-guidance-documents/chemistry-manufacturing-and-control-cmc-information-human-gene-therapy-investigational-new-drug

[6] https://www.fda.gov/ [Internet]. Chemistry, manufacturing, and control (CMC) information for human gene therapy investigational new drug applications (INDs). In: U.S. Food and Drug Administration. Guidance for Industry: January 2020 [дата обращения: 18.12.2022]. Доступ по ссылке: https://www.fda.gov/regulatory-information/search-fda-guidance-documents/chemistry-manufacturing-and-control-cmc-information-human-gene-therapy-investigational-new-drug

[7] Human gene therapy for rare diseases. In: U.S. Food and Drug Administration. Guidance for industry: January 2020 [cited: 18.12.2022]. Available from: https://www.fda.gov/regulatory-information/search-fda-guidance-documents/human-gene-therapy-rare-diseases

[8] https://www.fda.gov/ [Internet]. Human gene therapy for rare diseases. In: U.S. Food and Drug Administration. Guidance for industry: January 2020 [дата обращения: 18.12.2022]. Доступ по ссылке: https://www.fda.gov/regulatory-information/search-fda-guidance-documents/human-gene-therapy-rare-diseases

[9] Burden N, Clift MJD, Jenkins GJS, et al. Opportunities and challenges for integrating new in vitro methodologies in hazard testing and risk assessment. Small. 2021;17(15):2006298. doi: 10.1002/smll.202006298

[10] Burden N., Clift M.J.D., Jenkins G.J.S., et al. Opportunities and challenges for integrating new in vitro methodologies in hazard testing and risk assessment // Small. 2021. Vol. 17, N 15. P. 2006298. doi: 10.1002/smll.202006298

[11] Hartung T, Daston G. Are in vitro tests suitable for regulatory use? Toxicol Sci. 2009;111(2):233–237. doi: 10.1093/toxsci/kfp149

[12] Hartung T., Daston G. Are in vitro tests suitable for regulatory use? // Toxicol Sci. 2009. Vol. 111, N 2. P. 233–237. doi: 10.1093/toxsci/kfp149

[13] Hartung T. Perspectives on in vitro to in vivo extrapolations. Appl In Vitro Toxicol. 2018;4(4):305–316. doi: 10.1089/aivt.2016.0026

[14] Hartung T. Perspectives on in vitro to in vivo extrapolations // Appl In Vitro Toxicol. 2018. Vol. 4, N 4. P. 305–316. doi: 10.1089/aivt.2016.0026

[15] Talbot K, Tizzano EF. The clinical landscape for SMA in a new therapeutic era. Gene Ther. 2017;24(9):529–533. doi: 10.1038/gt.2017.52

[16] Talbot K., Tizzano E.F. The clinical landscape for SMA in a new therapeutic era // Gene Ther. 2017. Vol. 24, N 9. P. 529–533. doi: 10.1038/gt.2017.52

[17] Al-Zaidy SA, Mendell JR. From clinical trials to clinical practice: practical considerations for gene replacement therapy in SMA type 1. Pediatr Neurol. 2019;100:3–11. doi 10.1016/j.pediatrneurol.2019.06.007

[18] Al-Zaidy S.A., Mendell J.R. From clinical trials to clinical practice: practical considerations for gene replacement therapy in SMA type 1 // Pediatr Neurol. 2019. Vol. 100. P. 3–11. doi: 10.1016/j.pediatrneurol.2019.06.007

[19] Cho S, Dreyfuss GA. A degron created by SMN2 exon 7 skipping is a principal contributor to spinal muscular atrophy severity. Genes Dev. 2010;24(5):438–442. doi: 10.1101/gad.1884910

[20] Cho S., Dreyfuss G.A. A degron created by SMN2 exon 7 skipping is a principal contributor to spinal muscular atrophy severity // Genes Dev. 2010. Vol. 24, N 5. P. 438–442. doi: 10.1101/gad.1884910

[21] Zhang R, So BR, Li P, et al. Structure of a key intermediate of the SMN complex reveals Gemin2’s crucial function in snRNP assembly. Cell. 2011;146(3):384–395. doi: 10.1016/j.cell.2011.06.043

[22] Zhang R., So B.R., Li P., et al. Structure of a key intermediate of the SMN complex reveals Gemin2’s crucial function in snRNP assembly // Cell. 2011. Vol. 146, N 3. P. 384–395. doi: 10.1016/j.cell.2011.06.043

[23] Raker VA, Plessel G, Lührmann R. The snRNP core assembly pathway: identification of stable core protein heteromeric complexes and an snRNP subcore particle in vitro. EMBO J. 1996;15(9):2256–2269. doi: 10.1002/j.1460-2075.1996.tb00579.x

[24] Raker V.A., Plessel G., Lührmann R. The snRNP core assembly pathway: identification of stable core protein heteromeric complexes and an snRNP subcore particle in vitro // EMBO J. 1996. Vol. 15, N 9. P. 2256–2269. doi: 10.1002/j.1460-2075.1996.tb00579.x

[25] Helmken C, Hofmann Y, Schoenen F, et al. Evidence for a modifying pathway in SMA discordant families: reduced SMN level decreases the amount of its interacting partners and Htra2-beta1. Hum Genet. 2003;114(1):11–21. doi: 10.1007/s00439-003-1025-2

[26] Helmken C., Hofmann Y., Schoenen F., et al. Evidence for a modifying pathway in SMA discordant families: reduced SMN level decreases the amount of its interacting partners and Htra2-beta1 // Hum Genet. 2003. Vol. 114, N 1. P. 11–21. doi: 10.1007/s00439-003-1025-2

[27] Yi H, Mu L, Shen C, et al. Negative cooperativity between Gemin2 and RNA provides insights into RNA selection and the SMN complex’s release in snRNP assembly. Nucleic Acids Res. 2020;48(2):895–911. doi: 10.1093/nar/gkz1135

[28] Yi H., Mu L., Shen C., et al. Negative cooperativity between Gemin2 and RNA provides insights into RNA selection and the SMN complex’s release in snRNP assembly // Nucleic Acids Res. 2020. Vol. 48, N 2. P. 895–911. doi: 10.1093/nar/gkz1135

[29] Friesen WJ, Paushkin S, Wyce A, et al. The methylosome, a 20S complex containing JBP1 and pICln, produces dimethylarginine-modified Sm proteins. Mol Cell Biol. 2001;21(24):8289–8300. doi: 10.1128/MCB.21.24.8289-8300.2001

[30] Friesen W.J., Paushkin S., Wyce A., et al. The methylosome, a 20S complex containing JBP1 and pICln, produces dimethylarginine-modified Sm proteins // Mol Cell Biol. 2001. Vol. 21, N 24. P. 8289–8300. doi: 10.1128/MCB.21.24.8289-8300.2001

[31] Friesen WJ, Wyce A, Paushkin S, et al. A novel WD repeat protein component of the methylosome binds Sm proteins. J Biol Chem. 2002;277(10):8243–8247. doi: 10.1074/jbc.M109984200

[32] Friesen W.J., Wyce A., Paushkin S., et al. A novel WD repeat protein component of the methylosome binds Sm proteins // J Biol Chem. 2002. Vol. 277, N 10. P. 8243–8247. doi: 10.1074/jbc.M109984200

[33] Chari A, Golas MM, Klingenhäger M, et al. An assembly chaperone collaborates with the SMN complex to generate spliceosomal SnRNPs. Cell. 2008;135(3):497–509. doi: 10.1016/j.cell.2008.09.020

[34] Chari A., Golas M.M., Klingenhäger M., et al. An assembly chaperone collaborates with the SMN complex to generate spliceosomal SnRNPs // Cell. 2008. Vol. 135, N 3. P. 497–509. doi: 10.1016/j.cell.2008.09.020

[35] Grimm C, Chari A, Pelz JP, et al. Structural basis of assembly chaperone- mediated snRNP formation. Mol Cell. 2013;49(4):692–703. doi: 10.1016/j.molcel.2012.12.009

[36] Grimm C., Chari A., Pelz J.P., et al. Structural basis of assembly chaperone- mediated snRNP formation // Mol Cell. 2013. Vol. 49, N 4. P. 692–703. doi: 10.1016/j.molcel.2012.12.009

[37] Sarachan KL, Valentine KG, Gupta K, et al. Solution structure of the core SMN-Gemin2 complex. Biochem J. 2012;445(3):361–370. doi: 10.1042/BJ20120241

[38] Sarachan K.L., Valentine K.G., Gupta K., et al. Solution structure of the core SMN-Gemin2 complex // Biochem J. 2012. Vol. 445, N 3. P. 361–370. doi: 10.1042/BJ20120241

[39] Kannan A, Bhatia K, Branzei D, Gangwani L. Combined deficiency of Senataxin and DNA-PKcs causes DNA damage accumulation and neurodegeneration in spinal muscular atrophy. Nucleic Acids Res. 2018;46(16):8326–8346. doi: 10.1093/nar/gky641

[40] Kannan A., Bhatia K., Branzei D., Gangwani L. Combined deficiency of Senataxin and DNA-PKcs causes DNA damage accumulation and neurodegeneration in spinal muscular atrophy // Nucleic Acids Res. 2018. Vol. 46, N 16. P. 8326–8346. doi: 10.1093/nar/gky641

[41] Sharma A, Singh K, Almasan A. Histone H2AX phosphorylation: a marker for DNA damage. Methods Mol Biol. 2012;920:613–626. doi: 10.1007/978-1-61779-998-3_40

[42] Sharma A., Singh K., Almasan A. Histone H2AX phosphorylation: a marker for DNA damage // Methods Mol Biol. 2012. Vol. 920. P. 613–626. doi: 10.1007/978-1-61779-998-3_40

[43] Caraballo-Miralles V, Cardona-Rossinyol A, Garcera A, et al. SMN deficiency attenuates migration of U87MG astroglioma cells through the activation of RhoA. Mol Cell Neurosci. 2012;49(3):282–289. doi: 10.1016/j.mcn.2011.12.003

[44] Caraballo-Miralles V., Cardona-Rossinyol A., Garcera A., et al. SMN deficiency attenuates migration of U87MG astroglioma cells through the activation of RhoA // Mol Cell Neurosci. 2012. Vol. 49, N 3. P. 282–289. doi: 10.1016/j.mcn.2011.12.003

[45] Coque E, Raoul C, Bowerman M. ROCK inhibition as a therapy for spinal muscular atrophy: understanding the repercussions on multiple cellular targets. Front Neurosci. 2014;8:271. doi: 10.3389/fnins.2014.00271

[46] Coque E., Raoul C., Bowerman M. ROCK inhibition as a therapy for spinal muscular atrophy: understanding the repercussions on multiple cellular targets // Front Neurosci. 2014. Vol. 8. P. 271. doi: 10.3389/fnins.2014.00271

[47] Messina S, Sframeli M. New treatments in spinal muscular atrophy: positive results and new challenges. J Clin Med. 2020;9(7):2222. doi: 10.3390/jcm9072222

[48] Messina S., Sframeli M. New treatments in spinal muscular atrophy: positive results and new challenges // J Clin Med. 2020. Vol. 9, N 7. P. 2222. doi: 10.3390/jcm9072222

[49] Shpargel KB, Matera AG. Gemin proteins are required for efficient assembly of Sm-class ribonucleoproteins. Proc Natl Acad Sci U S A. 2005;102(48):17372–17377. doi: 10.1073/pnas.0508947102

[50] Shpargel K.B., Matera A.G. Gemin proteins are required for efficient assembly of Sm-class ribonucleoproteins // Proc Natl Acad Sci U S A. 2005. Vol. 102, N 48. P. 17372–17377. doi: 10.1073/pnas.0508947102

[51] Wan L, Battle DJ, Yong J, et al. The survival of motor neurons protein determines the capacity for snRNP assembly: biochemical deficiency in spinal muscular atrophy. Mol Cell Biol. 2005;25(13):5543–5551. doi: 10.1128/MCB.25.13.5543-5551.2005

[52] Wan L., Battle D.J., Yong J., et al. The survival of motor neurons protein determines the capacity for snRNP assembly: biochemical deficiency in spinal muscular atrophy // Mol Cell Biol. 2005. Vol. 25, N 13. P. 5543–5551. doi: 10.1128/MCB.25.13.5543-5551.2005

[53] Wan L, Ottinger E, Cho S, Dreyfuss G. Inactivation of the SMN complex by oxidative stress. Mol Cell. 2008;31(2):244–254. doi: 10.1016/j.molcel.2008.06.004

[54] Wan L., Ottinger E., Cho S., Dreyfuss G. Inactivation of the SMN complex by oxidative stress // Mol Cell. 2008. Vol. 31, N 2. P. 244–254. doi: 10.1016/j.molcel.2008.06.004

[55] Kye MJ, Niederst ED, Wertz MH, et al. SMN regulates axonal local translation via miR-183/mTOR pathway. Hum Mol Genet. 2014;23(23):6318–6331. doi: 10.1093/hmg/ddu350

[56] Kye M.J., Niederst E.D., Wertz M.H., et al. SMN regulates axonal local translation via miR-183/mTOR pathway // Hum Mol Genet. 2014. Vol. 23, N 23. P. 6318–6331. doi: 10.1093/hmg/ddu350

[57] Querfurth H, Lee HK. Mammalian/mechanistic target of rapamycin (mTOR) complexes in neurodegeneration. Mol Neurodegener. 2021;16:44. doi: 10.1186/s13024-021-00428-5

[58] Querfurth H., Lee H.K. Mammalian/mechanistic target of rapamycin (mTOR) complexes in neurodegeneration // Mol Neurodegener. 2021. Vol. 16. P. 44. doi: 10.1186/s13024-021-00428-5

[59] Feschenko M, Bergelson S, Leyme AC, inventors. Potency assays for viral vector production. United States patent US 2022/0267798A1. 2022.

[60] Feschenko M., Bergelson S., Leyme A.C., inventors. Potency assays for viral vector production. United States patent US 2022/0267798A1. 2022.

[61] Staněk D, Fox AH. Nuclear bodies: news insights into structure and function. Curr Opin Cell Biol. 2017;46:94–101. doi: 10.1016/j.ceb.2017.05.001

[62] Staněk D., Fox A.H. Nuclear bodies: news insights into structure and function // Curr Opin Cell Biol. 2017. Vol. 46. P. 94–101. doi: 10.1016/j.ceb.2017.05.001

[63] Morimoto M, Boerkoel CF. The role of nuclear bodies in gene expression and disease. Biology (Basel). 2013;2(3):976–1033. doi: 10.3390/biology2030976

[64] Morimoto M., Boerkoel C.F. The role of nuclear bodies in gene expression and disease // Biology (Basel). 2013. Vol. 2, N 3. P. 976–1033. doi: 10.3390/biology2030976

[65] Jablonka S, Holtmann B, Meister G, et al. Gene targeting of Gemin2 in mice reveals a correlation between defects in the biogenesis of U snRNPs and motoneuron cell death. Proc Natl Acad Sci USA. 2002;99(15):10126–10131. doi: 10.1073/pnas.152318699

[66] Jablonka S., Holtmann B., Meister G., et al. Gene targeting of Gemin2 in mice reveals a correlation between defects in the biogenesis of U snRNPs and motoneuron cell death // Proc Natl Acad Sci USA. 2002. Vol. 99, N 15. P. 10126–10131. doi: 10.1073/pnas.152318699

[67] Helmken C, Hofmann Y, Schoenen F, et al. Evidence for a modifying pathway in SMA discordant families: reduced SMN level decreases the amount of its interacting partners and Htra2-beta1. Hum Genet. 2003;114:11–21. doi: 10.1007/s00439-003-1025-2

[68] Helmken C., Hofmann Y., Schoenen F., et al. Evidence for a modifying pathway in SMA discordant families: reduced SMN level decreases the amount of its interacting partners and Htra2-beta1 // Hum Genet. 2003. Vol. 114. P. 11–21. doi: 10.1007/s00439-003-1025-2

[69] Wang J, Dreyfuss G. A cell system with targeted disruption of the SMN gene: functional conservation of the SMN protein and dependence of Gemin2 on SMN. J Biol Chem. 2001;276(13):9599–9605. doi: 10.1074/jbc.M009162200

[70] Wang J., Dreyfuss G. A cell system with targeted disruption of the SMN gene: functional conservation of the SMN protein and dependence of Gemin2 on SMN // J Biol Chem. 2001. Vol. 276, N 13. P. 9599–9605. doi: 10.1074/jbc.M009162200

RIGHTS & PERMISSIONS

Eco-Vector