DSpace Collection:https://hdl.handle.net/10171/50382024-03-28T22:30:39Z2024-03-28T22:30:39ZPreclinical models for prediction of immunotherapy outcomes and immune evasion mechanisms in genetically heterogeneous multiple myelomahttps://hdl.handle.net/10171/691852024-03-04T06:06:35Z2024-02-28T00:00:00ZTitle: Preclinical models for prediction of immunotherapy outcomes and immune evasion mechanisms in genetically heterogeneous multiple myeloma
Abstract: La falta de modelos experimentales que reflejen la heterogeneidad genética del mieloma
múltiple (MM) ha obstaculizado históricamente el avance de los descubrimientos
terapéuticos preclínicos. Para superar esta limitación, examinamos ratones diseñados
para expresar lesiones genéticas comunes del MM encontradas en pacientes, incluyendo
NF-kB, BCL2, MYC, TP53, KRAS, ciclina D1, MMSET/NSD2 y c-MAF. Estas lesiones fueron
activadas en células B maduras del centro germinal mediante el alelo cγ1-cre, lugar
donde se cree que se origina la enfermedad. Después de nuestro estudio, encontramos
dos modelos llamados BIcγ1 y MIcγ1, los cuales desarrollaron tumores de médula ósea
(MO) que cumplían con los elementos clave de la patogénesis del MM. Además, los
ratones mostraron respuesta in vivo a lenalidomida y combinaciones de lenalidomida,
las cuales actualmente son parte de la terapia estándar en pacientes con MM.
La caracterización de ambos modelos a través de ensayos celulares, moleculares e
inmunológicos reveló que la adquisición de la expresión de MYC condicionó el tiempo
de progresión y, a su vez, dictó mecanismos de evasión inmunológica que remodelaron
de manera diferente el microambiente de la MO. Los ratones MIcγ1 con progresión
rápida impulsada por la sobreexpresión inicial de MYC, exhibieron un alto número de
células T CD8+
activadas y una disminución de las células T reguladoras (Treg)
inmunosupresoras en la MO, mientras que los ratones BIcγ1, con adquisición tardía de
la expresión de MYC, mostraron una menor infiltración de células T CD8+
en la MO y una
infiltración superior de células Treg. De manera inesperada, los ratones MIcγ1 con
progresión rápida respondieron a la terapia de bloqueo de puntos de control inmunitario
(ICB), mientras que el modelo de progresión lenta BIcγ1 fue completamente refractario
a esta misma terapia. El estudio de las diferencias en la respuesta entre ambos modelos
murinos nos llevó a la conclusión de que una alta proporción de células T CD8+
frente a
células Treg en la MO predijo la respuesta a la terapia ICB.
A partir de estos nuevos modelos murinos, intentamos establecer líneas celulares de MM
derivadas de muestras de tumor primario, con el objetivo de utilizar un panel de líneas
celulares genéticamente diversas para probar las inmunoterapias. Entre ellas, las líneas
celulares MM5080 y MM8273 pudieron ser injertadas en ratones singénicos
inmunocompetentes, lo cual permitió probar la inmunoterapia in vivo. En estas líneas
celulares, evaluamos la hipótesis de si modular la proporción de células CD8+
/Treg podría
revertir la resistencia a los ICB. Nuestros hallazgos experimentales indicaron que al
aumentar la citotoxicidad de las células T CD8+ mediante la combinación de anticuerpos
monoclonales (moAbs) anti-PD-1 y anti-TIGIT, o al eliminar las células T reguladoras
(Treg) con un moAb anti-CD25, se revirtió la resistencia a la terapia PD-1/PD-L1, lo cual
resultó en un control prolongado del MM.
En conclusión, hemos generado modelos de MM similares a los encontrados en
pacientes, lo cual nos permite correlacionar los rasgos genéticos e inmunológicos del
MM con las respuestas a la terapia preclínica. Esto resulta fundamental para los ensayos
clínicos de nuevas inmunoterapias.2024-02-28T00:00:00ZEvaluation of Modified Vaccinia virus Ankara based locoregional immunotherapy in peritoneal carcinomatosis modelshttps://hdl.handle.net/10171/691842024-03-04T06:06:33Z2024-02-28T00:00:00ZTitle: Evaluation of Modified Vaccinia virus Ankara based locoregional immunotherapy in peritoneal carcinomatosis models
Abstract: The peritoneum is a serous membrane of mesodermal origin that coats the abdominal wall
and forms a lining on most abdominal organs. It consists of a thin layer of mesothelial cells
over a basal lamina and is divided into the parietal peritoneum, which covers the abdominal
and pelvic walls and the visceral peritoneum which surrounds the visceral organs (stomach,
spleen, liver and some parts of the intestine) (1–3). The space found between the parietal and
visceral peritoneum is called the peritoneal cavity and, in physiological conditions, contains
between 50-100 mL of peritoneal fluid that serves as lubricant reducing friction among
intraperitoneal organs during peristalsis and is provided with nutrients, growth factors,
cytokines and chemokines as well as immune cells (1, 2). Thus, the peritoneum plays a
crucial role in the maintenance of homeostasis in the peritoneal cavity mediating antigen
presentation, inflammatory responses, fibrosis and tissue repair (1).2024-02-28T00:00:00ZAdvances in precision and safety of CRISPR-based gene targeting for Primary Hyperoxaluria Type 1https://hdl.handle.net/10171/691652024-02-26T06:06:18Z2024-02-22T00:00:00ZTitle: Advances in precision and safety of CRISPR-based gene targeting for Primary Hyperoxaluria Type 12024-02-22T00:00:00ZModelling, design, fabrication and characterization of engineered human myocardium made with melt electrowriting and cardiac cells derived from hiPSCshttps://hdl.handle.net/10171/691522024-02-26T06:06:05Z2024-02-22T00:00:00ZTitle: Modelling, design, fabrication and characterization of engineered human myocardium made with melt electrowriting and cardiac cells derived from hiPSCs
Abstract: The adult human heart has evolved to become a highly specialized organ, whose continuous pumping
of blood is critical for survival. However, its ability to regenerate or self-repair following injury is very
limited, so consequently any event or disease resulting in damage to the heart poses a serious threat
to the patient. Moreover, cardiovascular diseases represent one of the most pressing healthcare
concerns nowadays, as they are the leading cause of death worldwide, and the number of cases is
only expected to increase in the following years. Despite great progress made over the years to treat
cardiovascular diseases, to date there is no therapy able to fully cure a heart that has been damaged.
In consequence, there is a dire need to generate new strategies to repair the heart damage and
restore the lost cardiac function, as well as to develop accurate modelling platforms to advance in
the understanding of disease progression and assess the effectiveness of new drugs.
Since its advent, cardiac tissue engineering and regenerative medicine has been regarded as a
promising candidate to realise this enormous challenge. Given its interdisciplinary nature, scientific
breakthroughs in different areas such as cellular reprogramming, polymer chemistry, and additive
manufacturing technologies have resulted in the advancement of cardiac tissue engineering and
regenerative medicine over the years. One of such cornerstone discoveries was the generation of
induced pluripotent stem cells and subsequent differentiation to different cardiac phenotypes, and
the present Thesis revolves around their application to generate patient-specific cardiac disease
models and humanised engineered functional cardiac minitissues.
Firstly, we reprogrammed peripheral blood mononuclear cells from a transthyretin amyloid
cardiomyopathy patient, resulting in the generation of a new cell line carrying a c.128G>A
(p.Ser43Asn) mutation in the transthyretin gene. Experiments demonstrated the efficacy and safety
of the approach, confirming the pluripotency of the cells, the presence of the disease-causing
mutation, and the removal of reprogramming vectors. This cell line, which is now available in a
repository, can be used to investigate disease biology, molecular mechanisms and progression; as
well as an advanced cellular model to test novel therapeutic strategies.
Secondly, we aimed to generate functional human minitissues by combining human cardiomyocytes
derived from induced pluripotent stem cells and tridimensional fibrillar scaffolds generated with the
technology of melt electrowriting. Compared to conventional two-dimensional cell culture, the
cardiac minitissues demonstrated enhanced maturation, with a significant increase in conduction
velocity, presence of connexin 43 and expression of cardiac-associated genes such as MYL2, GJA5
and SCN5A, and isoform ratios MYH7/MYH6 and MYL2/MYL7 after 28 days in culture. When
investigating the effect of the scaffold fibres on the cells, we found that cardiomyocytes placed close
to the fibre were arranged parallel to it, but that alignment was progressively lost towards the centre
of the scaffold pore. We then used these data to develop simulations capable of accurately
reproducing the experimental performance. In-depth gauging of the structural disposition and
intercellular connectivity allowed us to develop an improved computational model able to predict
the relationship between cardiac cell alignment and functional performance. This study lays down
the path for advancing in the development of in silico tools to predict cardiac biofabricated tissue
evolution after generation, and maps the route towards more accurate and biomimetic tissue
manufacture.
We next aimed at increasing the biological representativity of the cardiac minitissues, by
implementing a few changes in cellular (addition of induced pluripotent stem cell-derived cardiac
fibroblasts) and hydrogel (substitution of Matrigel for fibrin) composition. We also sought to control
cardiomyocyte behaviour based on melt electrowritten scaffold geometry. For this, we hypothesized
that diamond-based scaffolds would induce cardiomyocyte contraction in the direction of least
mechanical resistance, i.e., the small diagonal of the diamonds. The characterization of the new
cardiac minitissues demonstrated functional maturation consistent with the previous work in terms
of gene expression and conduction velocity, although the observed low initial cell retention within
the scaffold highlighted the need of new strategies to improve cell seeding efficiency. When
comparing contractile dynamics between melt electrowritten scaffolds made with square,
rectangular, and diamond-shaped pores, we found that the latter resulted in significantly faster,
stronger and aligned contraction in the direction that we had anticipated. The potential use of the
cardiac minitissues as therapy agents was tested by implanting the constructs in a murine model of
chronic myocardial infarction. Compared to controls, implanted animals showed significant
improvement, including higher left ventricular ejection fraction and greater wall thickness.
Finally, in another attempt to enhance the biological representativity of the constructs, a proof of
concept was made to generate melt electrowritten ellipsoidal scaffolds with controlled pore
architecture. In summary, the present Thesis revolves around human induced pluripotent stem cells
and melt electrowriting as cornerstone tools for cardiac tissue engineering and regenerative
medicine efforts. By combining both and iteratively optimising the design and experimental
conditions, we were able to generate human functional cardiac minitissues of increased biological
relevance.2024-02-22T00:00:00Z