iBET

Viral vectors for gene therapy

Although the first successful treatments are just know being demonstrated in clinical trials, gene therapy will have a huge impact on many life-threatening diseases. Viral vectors are the most efficient gene delivery vehicles; however their manufacturing is complex, requiring the use of animal cell cultures. Currently, they are the basis for numerous gene therapy clinical trials. Our group has a track record in the development of several viral vectors for gene therapy namely, Retrovirus and Lentivirus, Adenovirus, Adeno Associated virus and more recently Baculovirus, each presenting several advantages and disadvantages from the safety, efficacy and manufacturing perspective. These issues are being addressed by our group to further improve them.

Retrovirus and Lentivirus

Retroviral vectors, based on Murine leukaemia virus, were the first viral vectors to become available and used in clinical trials. One of the characteristics of retroviral vectors is their ability to integrate the genetic material transferred into the host-cell resulting in long-term transgene expression. More recently another class of retrovirus was developed from HIV into gene therapy vectors, these were denominated lentiviral vectors. They present the additional advantage of being able to transduce quiescent cells. Both vectors present however several difficulties in their manufacturing, as they are very labile and the cell productivities are low. Current research in the group focuses on the development of enhanced cell lines and analysis of the metabolic requirements for vector production. Additionally, we develop production and purification processes for both vectors.

Adenovirus

Adenoviral vectors are presently the most widely used vectors in gene therapy clinical trials to target pathologies of different origins, such as cancers, infection diseases or neurological disorders. Human adenoviruses (hAv) are generally the chosen prototype vector backbone. A platform for production, purification and storage was developed in our group where the constraints on viral production and the so called ‘cell density effect’ was thoroughly analyzed and elucidated in HEK 293 cells. Presently alternative human cell lines are being study.

One of the major hurdles regarding the use of hAv is the pre-existent immunity memory that may limit the efficiency of transgene expression in humans. To circumvent this, non-human adenovirus vectors such as Canine Adenovirus Vectors type 2 are being developed and studied by our group. The restrict experience in the field with these vectors requires the establishment of an entire process development suitable for GMP manufacturing. Current objectives include the development of canine transcomplementing cell lines that support the amplification of the vectors (E1 deleted and helper dependent vectors) and also the development of upstream and downstream processes.

Adeno Associated Virus (AAV)

Along with their strong clinical safety profile, AAVs can transduce both dividing and non-dividing cells and achieve long term transgene expression. Nonetheless, AAV show a wide tissue tropism, which constitutes the major limitation for their use for some applications, especially for systemic application. Our group is investing on new approaches towards targeting AAVs to cells of therapeutic interest with the final goal of using them as improved vehicles for gene therapy.

Baculovirus

For several decades, the baculovirus-insect cell system has been used for industrial and research purposes, maturing into a versatile technology for producing biopesticides, recombinant proteins and vaccines. Given their safety and large genomic capacity, the baculovirus (BV) itself is beginning to be addressed as a gene therapy vector. However, from a bioprocessing viewpoint, a critical bottleneck is the decreased specific productivities obtainable from high cell density culture, with implications for overall process costs. Current research by our group on this subject includes a combination of experimental and computational tools to assess the metabolic determinants of BV production. Furthermore, we have been developing processes for BV purification. The distinct properties of this virus in particular surface heterogeneity and non-spherical, rod-shaped structure represent major challenges in the development of robust downstream strategies to produce clinical grade preparations.

Selected Publications

 

  1. Bandeira VS, Tomas HÁ, Alice E, Carrondo MJT, Coroadinha AS. 2017. Disclosing the Parameters Leading to High Productivity of Retroviral Producer Cells Lines: Evaluating Random Versus Targeted Integration. HUMAN GENE THERAPY METHODS. 28, 78-90.  
  2. Soares HR, Castro R, Tomas HA, Rodrigues AF, Gomes-Alves P, Bellier B, Klatzmann D, Carrondo MJ, Alves PM, Coroadinha AS. 2016. Tetraspanins displayed in retrovirus-derived virus-like particles and their immunogenicity. Vaccine. 
  3. Fernandes CS, Castro R, Coroadinha AS, Roque AC. 2016. Small synthetic ligands for the enrichment of viral particles pseudotyped with amphotropic murine leukemia virus envelope. J Chromatogr A. pii: S0021-9673(16)30125-X. 
  4. Silva R, Mota JP, Peixoto C, Alves PM, Carrondo MJT. 2015. Improving the downstream processing of vaccine and gene therapy vectors with continuous chromatography. Pharmaceutical Bioprocessing 3(8): 489-505
  5. Rodrigues A F, Guerreiro M R, Formas-Oliveira A S, Fernandes P, Blechert A-K, Genzel Y, Alves P M, Hu W S, Coroadinha A S. 2015. Increased titer and reduced lactate accumulation in recombinant retrovirus production through the down-regulation of HIF1 and PDK. Biotechnol Bioeng. Jul 1 
  6. Silva AC, Simão D, Sousa MFQ, Küppers C, Lucas T, Cruz P, Carrondo MJ,  Kochanek S, Alves PM 2015 Human Amniocyte-derived Cells for the Production of Adenoviral Vectors. Biotechnology Journal, May;10(5):760-71. 
  7. Rodrigues AF, Soares HR, Guerreiro MR, Alves PM, Coroadinha AS. Viral vaccines and their manufacturing cell substrates: New trends and designs in modern vaccinology. Biotechnol J. 2015 Sep;10(9):1329-44 
  8. Rodrigues AF, Formas-Oliveira AS, Guerreiro M, Tomas H, Alves PM and Coroadinha AS. 2015. Single step cloning-screening method:  a new tool for developing and studying high titer viral vector producer cells. Gene Ther, May 4. 
  9. Castro R, Fernandes P, Laske T, Sousa MFQ, Genzel Y, Scharfenberg K, Alves PM, Coroadinha AS. 2015. Production of canine adenovirus type 2 in serum-free suspension cultures of MDCK cells. Appl Microbiol Biotechnol Sep;99(17):7059-68
  10. Nestola P, Peixoto C, Silva RJS, Alves PM, Mota JPB, Carrondo MJT 2015. Improved virus purification processes for vaccines and gene therapy. Biotechnology and Bioengineering, 112(5):843-57. 
  11. Fernandes P, Almeida AI, Kremer EJ, Alves PM & Coroadinha AS. 2015. Canine helper-dependent vectors production: implications of Cre activity and co-infection on adenovirus propagation. Scientific Reports 5: 9135
  12. Fernandes P, Simão D, Guerreiro MR, Kremer EJ, Coroadinha AS, Alves PM. 2015. Impact of adenovirus life cycle on the generation of canine helper-dependent vectors. Gen Ther 22, 40-49.
  13. Nestola P, Villain L, Peixoto C, Martins DL, Alves PM, Carrondo MJT, Mota JPB. 2014. Impact of grafting on the design of new membrane adsorbers for adenovirus purification. Journal of Biotechnology, 181: 1–11. 
  14. Nestola P, Silva RJS, Peixoto C, Alves PM, Carrondo MJT, Mota JPB. 2014. Adenovirus purification by two-column, size-exclusion, simulated countercurrent chromatography. Journal of Chromatography A,1347: 111–121.   
  15. Rodrigues AF, Formas-Oliveira AS, Bandeira, VS, Alves PM, Hu WS, Coroadinha AS. 2013. Metabolic pathways recruited in the production of a recombinant enveloped virus: mining targets for process and cell engineering. Metab. Eng., 20, 131-137. 
  16. Fernandes P, Peixoto C, Santiago VM, Kremer EJ, Coroadinha AS, Alves PM, 2013. Bioprocess development for canine adenovirus type 2 vectors. Gene Ther. Apr;20(4):353-60. 
  17. Fernandes P, Santiago VM, Rodrigues AF, Tomás H, Kremer EJ, Alves PM, Coroadinha AS. 2013. Impact of E1 and Cre on Adenovirus Vector Amplification: Developing MDCK CAV-2-E1 and E1-Cre Transcomplementing Cell Lines. PLoS One. 2013;8(4):e60342. 
  18. Rodrigues AF, Amaral AI, Veríssimo V, Alves PM, Coroadinha AS, 2012. Adaptation of retrovirus producer cells to serum deprivation: Implications in lipid biosynthesis and vector production. Biotechnol Bioeng, 109 (5): 1269-79. 
  19. Carrondo MJ, Panet A, Wirth D, Coroadinha AS, Cruz P, Falk H, Schucht R, Dupont F, Geny-Fiamma C, Merten OW, Hauser H, 2011. Integrated strategy for the production of therapeutic retroviral vectors. Hum Gene Ther, 22(3): 370-79. 
  20. Gama-Norton L, Botezatu L, Herrmann S, Schweizer M, Alves PM, Hauser H, Wirth D, 2011. Lentivirus production is influenced by SV40 large T-antigen and chromosomal integration of the vector in HEK293 cells. Hum Gene Ther, 22(10):1269-79. 
  21. Real G, Monteiro F, Burger C, Alves PM, 2011. Improvement of lentiviral transfer vectors using cis-acting regulatory elements for increased gene expression. Appl Microbiol Biotechnol, 91:1581-91. 
  22. Cruz PE, Rodrigues T, Carmo M, Wirth D, Amaral AI, Alves PM, Coroadinha AS, 2011. Manufacturing of retroviruses. Methods Mol Biol, 737:157-82. 
  23. Silva AC, Peixoto C, Lucas T, Kuppers C, Cruz PE, Alves PM, Kochanek S, 2010. Adenovirus vector production and purification. Curr Gene Ther, 10(6):437-55.
  24. Coroadinha AS, Gama-Norton L, Amaral AI, Hauser H, Alves PM, Cruz PE, 2010. Production of retroviral vectors. Curr Gene Ther, 10(6):456-73.
  25. Bernal V, Carinhas N, Yokomizo A, Carrondo MJ, Alves PM, 2009. Cell density effect in the Baculovirus-Insect cells system: a quantitative analysis of energetic metabolism. Biotechnol Bioeng, 104(1):162-80. 
  26. Carinhas N, Bernal V, Yokomizo AY, Carrondo MJ, Oliveira R, Alves PM, 2009. Baculovirus production for gene therapy: the role of cell density, multiplicity of infection and medium exchange. Appl Microbiol Biotechnol, 81(6):1041-49. 
  27. Rodrigues AF, Carmo M, Alves PM, Coroadinha AS, 2009. Retroviral vector production under serum deprivation: the role of lipids. Biotechnol Bioeng, 104(6):1171-81. 
  28. Vicente T, Peixoto C, Carrondo MJ, Alves PM, 2009. Virus production for clinical gene therapy. Methods Mol Biol, 542:447-70. 
  29. Vicente T, Peixoto C, Carrondo MJ, Alves PM, 2009. Purification of recombinant baculoviruses for gene therapy using membrane processes. Gene Ther, 16:766-775. 
  30. Carmo M, Alves A, Rodrigues AF, Coroadinha AS, Carrondo MJ, Alves PM, Cruz PE, 2009 Stabilization of gammaretroviral and lentiviral vectors: from production to gene transfer. J Gene Med, 11:670-78. 
  31. Ferreira TB, Perdigão R, Silva AC, Zhang C, Aunins JG, Carrondo MJ, Alves PM, 2009. 293 cell cycle synchronisation in adenovirus vector production. Biotechnol Prog, 25(1):235-43. 
  32. Carmo M, Dias JD, Panet A, Coroadinha AS, Carrondo MJ, Alves PM, Cruz PE, 2009. Thermosensitivity of the reverse transcription process as an inactivation mechanism of lentiviral vectors. Hum Gene Ther, 20(10):1168-76. 
  33. Amaral AI, Coroadinha AS, Merten O-W, Alves PM, 2008. Improving retroviral vectors production: role of carbon sources in lipid biosynthesis. J Biotechnol, 138: 57-66. 
  34. Carmo M, Panet A, Carrondo MJ, Alves PM, Cruz PE, 2008. From retroviral vector production to gene transfer: spontaneous inactivation is caused by loss of reverse transcription capacity. J Gene Med, 10:383-91. 
  35. Rodrigues T, Alves A, Lopes A, Carrondo MJ, Alves PM, Cruz PE, 2008. Removal of envelope protein-free retroviral vectors by anion exchange chromatography to improve product quality. J Sep Sci, 31:3509-18. 
  36. Carrondo MJ, Merten OW, Haury M, Alves PM, Coroadinha AS, 2008. Impact of retroviral vector components stoichiometry on packaging cell lines: effects on productivity and vector quality. Hum Gene Ther, 19:199-210. 
  37. Ferreira TB, Carrondo MJ, Alves PM, 2007. Effect of ammonia production on intracellular pH consequent effect on adenovirus vector production. J Biotechnol, 129(3):433-38. 
  38. Rodrigues T, Carvalho A, Carmo M, Carrondo MJ, Alves PM, Cruz PE, 2007. Scaleable purification process for gene therapy retroviral vectors. J Gene Med, 9(4):233-43. 
  39. Rodrigues T, Carrondo MJ, Alves PM, Cruz PE, 2007. Purification of retroviral vectors for clinical application: biological implication and technological challenges. J Biotechnol, 127:520-41. 
  40. Wirth D, Gama-Norton L, Riemer P, Sandhu U, Schucht R, Hauser H, 2007. Road to precision: recombinase-based targeting technologies for genome engineering. Curr Opin Biotechnol, 18:411–19. 
  41. Coroadinha AS, Silva AC, Pires E, Coelho A, Alves PM, Carrondo MJ, 2006. Effect of osmotic pressure on the production of retroviral vectors: enhancement in vector stability. Biotechnol Bioeng, 94(2):322-9. 
  42. Rodrigues T, Carvalho A, Roldao A, Carrondo MJ, Alves PM, Cruz PE, 2006. Screening anion-exchange chromatographic matrices for isolation of onco-retroviral vectors. J Chromatogr B, 837(1-2):59-68. 
  43. Schucht R, Coroadinha AS, Zanta-Boussif MA, Verhoeyen E, Carrondo MJ, Hauser H, Wirth D, 2006. A new generation of retroviral producer cells: predictable and stable virus production by Flp-mediated site-specific integration of retroviral vectors. Mol Ther, 14(2):285-92. 
  44. Carmo M, Faria TQ, Falk H, Coroadinha AS, Teixeira M, Merten OW, Geny-Fiamma C, Alves PM, Danos O, Panet A, Carrondo MJ, Cruz PE, 2006. Relationship between retroviral vector membrane and vector stability. J Gen Virol, 87(5):1349-56. 
  45. Cruz PE, Silva AC, Roldao A, Carmo M, Carrondo MJ, Alves PM, 2006. Screening of novel excipients for improving the stability of retroviral and adenoviral vectors. Biotechnol Prog, 22(2):568-76. 
  46. Coroadinha AS, Ribeiro J, Roldão A, Cruz PE, Alves PM, Merten O-W, Carrondo MJ, 2006. Effect of medium sugar source on the production of retroviral vectors for gene therapy. Biotechnol Bioeng, 94(1):24-36. 
  47. Coroadinha AS, Alves PM, Santos SS, Cruz PE, Merten, OW, Carrondo MJ, 2006. Retrovirus producer cell line metabolism: implications on viral productivity. Appl Microbiol Biotechnol. 72(6):1125-35. 
  48. Coroadinha AS, Schucht R, Gama-Norton L, Wirth D, Hauser H, Carrondo MJ, 2006. The use of recombinase mediated cassette exchange in retroviral vector producer cell lines: predictability and efficiency by transgene exchange. J Biotechnol, 124(2):457-68. 
  49. Ferreira TB, Alves PM, Aunins JG, Carrondo MJ, 2005. Use of adenoviral vectors as veterinary vaccines. Gene Ther, 12(S1):73-83. 
  50. Carmo M,  Peixoto CC, Coroadinha AS, Alves PM, Cruz PE, Carrondo MJ, 2004. Quantitation of MLV-based retroviral vectors using real-time RT-PCR. J Virol Methods, 119:115-9. 
  51. Cruz PE, L Maranga, MJ Carrondo, 2002. Integrated process optimization: lessons from retrovirus and virus-like particle production. J Biotechnol, 99:199-214. 
  52. Merten OW, Cruz PE, Rochette C, Geny-Fiamma C, Bouquet C, Goncalves D, Danos O, Carrondo MJ, 2001. Comparison of different bioreactor systems for the production of high titer retroviral vectors. Biotechnol Prog, 17:326-35. 
  53. Morais VA, Serpa J, Palma AS, Costa T, Maranga L, Costa J, Wu N, Ataai MM, 2001. Expression and characterization of recombinant human alpha-3/4-fucosyltransferase III from Spodoptera frugiperda (Sf9) and Trichoplusia ni (Tn) cells using the baculovirus expression system. Biochem J, 353:719-25. 
  54. Cruz PE, Almeida JS, Murphy PN, Moreira JL, Carrondo MJ, 2000. Modeling retrovirus production for gene therapy. 1. Determination of optimal bioreaction mode and harvest strategy. Biotechnol Prog, 16:213-21. 
  55. Cruz PE, Goncalves D, Almeida J, Moreira JL, Carrondo MJ, 2000. Modeling retrovirus production for gene therapy. 2. Integrated optimization of bioreaction and downstream processing. Biotechnol Prog, 16:350-57.