Cristiana Ulpiano

Class 2019

PhD grant: PD/BD/150336/2019

PhD thesis

Mesenchymal Stromal Cells (MSC)-derived extracellular vesicles for the delivery of RNAi to modulate angiogenesis in malignancy

Supervisors

• Gabriel A. Monteiro, Professor @ IST/ULisboa, PT

• Cláudia L. da Silva, Professor @ IST/ULisboa, PT

PhD Degree complete: Discussion on June 5, 2024

THESIS ABSTRACT

Besides participating in physiological angiogenesis, vascular endothelial growth factor (VEGF) contributes to excessive angiogenesis-related disorders. RNA interference (RNAi)-based strategies have shown promising results in modulating angiogenesis. Expression vectors encoding short-hairpin RNAs (shRNAs) are used as alternative RNAi effectors, and minicircles (MC) are a favourable expressing system. Extracellular vesicles (EVs) emerge as delivery systems for nucleic acids due to their innate potential to deliver functional cargo and cross biological barriers. Due to their intrinsic therapeutic benefits and efficient ex vivo expansion capacity, mesenchymal stromal cells (MSCs) are widely explored as EV producers.
This work aimed to develop an anti-angiogenic gene-based therapy using shRNA-expressing MCs targeting VEGF regulatory players and MSC-EVs as a delivery system, being divided into three stages.
In the first stage, the shRNA-expressing MCs targeting VEGF-A and its receptor (VEGFR2) were developed. After large-scale production and purification of the supercoiled MCs, transfection experiments showed that 4 days after microporation MC-shVEGF induced a knockdown of ~78% and MC-shVEGFR2 a knockdown of ~56% in human breast cancer cells and umbilical vein endothelial cells, respectively.
In the second stage, we established a platform for manufacturing MSC-EVs. Combining serum-/xeno(geneic)-free exosome-depleted human platelet lysate (hPL)-supplemented medium and a stirred-tank reactor, the system sustained a 3-day continuous EV production. When combined with scalable EV isolation, this platform yielded MSC-EVs with accepted biochemical/biophysical characteristics, at clinically relevant numbers.
In the third stage, MSC-EVs were engineered by directly loading the MCs. GFP-targeting MC loading was tested by passive incubation, microporation, sonication and using a transfection reagent. The results showed that low loading efficiencies (<7%) were obtained and no significant decrease in fluorescent intensity after EV-MC delivery to GFP+cells was observed.
Overall, this work provided important insights regarding MSC-EVs as delivery systems. Moreover, it demonstrated the potential of MC-derived RNAi systems as a non-viral therapeutic approach for excessive angiogenesis. Lastly, the MSC-EV manufacturing platform established herein constitutes an important step towards making MSC-EV-based therapies widely available in clinical settings.

RESUMO DA TESE

Além de participar na angiogénese fisiológica, o fator de crescimento endotelial vascular (VEGF) contribui para condições relacionados com angiogénese excessiva. Estratégias com RNA de interferência (RNAi) demonstram resultados promissores na modulação angiogénica. Vetores de expressão que codificam short hairpin RNAs (shRNAs) são usados como efetores de RNAi, e os minicírculos (MC) são vetores vantajosos. Vesículas extracelulares (EVs) surgem como sistemas de entrega de ácidos nucleicos devido ao seu potencial inato para entregar carga funcional e ultrapassar barreiras biológicas, e as células mesenquimais estromais (MSCs) têm sido exploradas como fonte produtora de EVs.
Este trabalho visou desenvolver uma terapia génica anti-angiogénica à base de MCs que expressam shRNA (MC-shRNA) que silenciam reguladores do VEGF, utilizando MSC-EVs como sistema de entrega, estando dividido em três etapas.
Na primeira etapa, foram desenvolvidos MC-shRNA que inibem o VEGF-A e o seu recetor (VEGFR2). Após produção e purificação dos MCs superenrolados, experiências de transfeção demonstraram que MC-shVEGF induziu uma supressão máxima de ~78%, e MC-shVEGFR2 de ~56% em células de cancro da mama e células endoteliais, respetivamente.
Na segunda etapa, estabelecemos uma plataforma para produção de MSC-EVs. A combinação de meio cultura sem soro e componentes xenogénicos suplementado com lisado plaquetário humano (hPL) desprovido de EVs e reatores de tanque agitado, permitiu a produção contínua de EVs. Após o isolamento, esta plataforma proporcionou MSC-EVs com características bioquímicas/biofísicas aceites em números clinicamente relevantes.
Na terceira etapa, as MSC-EVs foram modificadas pelo carregamento direto dos MCs. MSC-EVs foram carregadas com MC anti-GFP por incubação passiva, microporação, sonicação e utilizando um reagente de transfeção. Os resultados demonstraram eficiências de carregamento baixas (<7%), não sendo observada uma diminuição na intensidade de fluorescência após a entrega das EV-MC a células GFP+.
Globalmente, este trabalho fornece perspetivas relevantes sobre MSC-EVs como sistema de entrega, demonstra o potencial dos MC-shRNA como abordagem terapêutica não viral para angiogénese excessiva e a plataforma de produção de MSC-EVs estabelecida representa um passo importante no estudo generalizado de terapias com EVs.

PUBLICATIONS

Papers

Bandarra-Tavares, H.; Franchi-Mendes, T.; Ulpiano, C.; Morini, S.; Kaur, N.; Harris-Becker, A.; Vemuri, M.C.; Cabral, J.M.S.; Fernandes-Platzgummer, A.; da Silva, C.L. Dual Production of Human Mesenchymal Stromal Cells and Derived Extracellular Vesicles in a Dissolvable Microcarrier-Based Stirred Culture System, Cytotherapy, 2024, pp.1 – 8. doi:10.1016/j.jcyt.2024.03.001.

Ulpiano, C., da Silva, C.L., & Monteiro, G.A. Bioengineered Mesenchymal-Stromal-Cell-Derived Extracellular Vesicles as an Improved Drug Delivery System: Methods and Applications. Biomedicines, 2023, 11(4), 1231. DOI: 10.3390/biomedicines11041231

Ulpiano, C., da Silva, C.L., Monteiro, G.A. Mesenchymal Stromal Cells (MSCs): A Promising Tool for Cell-Based Angiogenic Therapy, Current Gene Therapy, 2021, 21(5), pp. 382 – 405. doi:10.2174/1566523221666210917114353.

Book chapter

Silva, M., Ulpiano, C., Bernardes, N., Monteiro, G.A., da Silva, C.L. Gene delivery as a tool to improve the therapeutic features of mesenchymal stromal cells: methods and applications, in Gene Delivery: Methods and Applications, Zimmer, V. (ed.), Nova Science Publishers, ISBN 9781536162684 (2019)

Oral Communications

Ulpiano, C., and da Silva, C.L, Exosome-depleted hPL Supplements Can Support Robust Cell-derived EV Production, at Aventacell’s Global Showcase Presentation in ISCT 2023, Paris, France (2023).

Tavares, H.T., Franchi-Mendes, T., Ulpiano, C., Morini, S., Becker, A.H., Cabral, J.M.S., Vemuri, M.C., Fernandes-Platzgummer, A., da Silva, C.L., Microcarrier-based xeno(geneic)-free culture system for the scalable production of Extracellular vesicles derived from Wharton’s jelly-Mesenchymal stromal cells. In 2nd PNEV meeting, Lisbon, Portugal (2023).

Ulpiano, C., Silva, R.M., Rosa, S.S., Fuzeta, M.A., Fernandes-Platzgummer, A., da Silva, C.L., Monteiro, G.A., Extracellular Vesicles from Mesenchymal Stromal Cells as potential angiogenic modulators through the delivery of RNAi-expressing Minicircles. In Student Network on EVs (SNEV), online virtual format (2023).

Ulpiano, C., Fernandes-Platzgummer, A., da Silva, C.L., Monteiro, G.A., Extracellular Vesicles from Mesenchymal Stromal Cells as potential angiogenic modulators through the delivery of RNAi-expressing Minicircles. In ESACT-UK 33rd Annual Conference, Nottingham, UK (2023).

Ulpiano, C., da Silva, C.L., Monteiro, G.A., Minicircle-derived RNA interference as a novel gene-based anti-angiogenic therapy. In MicroBiotec’21 – Congress of Microbiology and Biotechnology, online virtual format (2021).

Poster Communications

Ulpiano,C., Franchi-Mendes, T., Salvador, W., Tavares, H., Rosa, S.S., Fernandes-Platzgummer, A., da Silva, C.L., Monteiro, G.A., Extracellular Vesicles from Mesenchymal Stromal Cells as potential angiogenic modulators through the delivery of RNAi-expressing Minicircles. In ESGCT 30th Annual congress, Brussels, Belgium (2023).

Ulpiano, C., Fernandes-Platzgummer, A., da Silva, C.L., Monteiro, G.A., Engineering Extracellular Vesicles from Mesenchymal Stromal Cells using Minicircle-derived RNAi Technology. In 27th ESACT meeting, Lisbon, Portugal (2022).

Ulpiano, C., da Silva, C.L., Monteiro, G.A., Minicircle-derived RNA interference as a novel gene-based anti-angiogenic therapy. In IST PhD Open Days, Lisboa, Portugal (2021).

Ulpiano, C., da Silva, C.L., Monteiro, G.A.,“RNAi as a tool to inhibit the angiogenic potential of human Mesenchymal Stem/Stromal Cells in malignancy”, MicroBiotec’19 Congress, December 5-7, 2019, Coimbra, Portugal

DOCTORAL PROGRAM (36 ECTS)

Curricular units:

• General Doctoral Training (6 ECTS)

• Advanced Experimental Techniques and methodologies (6 ECTS)

• Bioentrepreneurship (6 ECTS)

• Nanobiotechnology (6 ECTS)

• Outreach and Teaching Skills – Department of Bioengineering (6 ECTS)

• Advanced Topics in Bioengineering and Biosciences (6 ECTS)

Advanced courses:

• Advanced Therapies: Scientific Basis and Clinical Use, organized by Universidad Internacional del Mar – Universidad de Murcia (Spain), July 11 – Jul 15, 2022, online format.

• Basic of Extracellular Vesicles Course, organized in collaboration between the International Society for Extracellular Vesicles (ISEV), University of California Irvine (USA), University of Gothenburg (Sweden) and Pohang University of Science and Technology (South Korea), January 4 – February 8, 2022, online format.

• Workshop in Imaging in Biosciences, organized by TIMB3 twinning project – ITQB NOVA, February 7, 2022, Oeiras, Portugal.

• Champalimaud Research Symposium 2019 – Tissue Environment in Health and Disease, sponsored by the International Union of Biochemistry and Molecular Biology (IUBMB), October 8-10, 2019, Lisbon, Portugal

• 10th Course on Animal Cell Technology organized by the European Society of Animal Cell Technology, performed in virtual mode, from 21st to 25th September 2020.