Thesis etd-06242021-103945 |
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Thesis type
Tesi di laurea magistrale
Author
MACI, FRANCESCA
URN
etd-06242021-103945
Thesis title
Apolipoprotein A-I Milano, orally administered via genetically engineered rice plants, exerts anti-inflammatory effects in vivo: new insights and tools to study this innovative delivery strategy
Department
BIOLOGIA
Course of study
BIOTECNOLOGIE MOLECOLARI
Supervisors
relatore Prof. Giovannoni, Roberto
Keywords
- apolipoproteinA-I Milano
- atherosclerosis
- cardiovascular diseases
- HDL therapy
- high-density lipoproteins
- molecular farming
Graduation session start date
13/07/2021
Availability
Withheld
Release date
13/07/2024
Summary
According to the World Health Organization, atherosclerotic cardiovascular disease (ASCVD) is a major cause of disability and premature death throughout the world. Therefore, the research and development of therapeutic options to prevent or reverse the progression of this pathologic condition is an ongoing critical issue.
The inverse relationship between high-density lipoprotein cholesterol (HDL-C) and cardiovascular disease (CVD) risk has been well established for many years, making HDL particles an attractive therapeutic target. However, shortcomings concerning manufacturing issues and the drawbacks of the typical intravenous route of administration of straight forward HDL-based therapies have made it difficult to fully exploit their potential, which thus requires innovative strategies.
This thesis is part of a research project focused on the development of a novel production and delivery system of apolipoprotein A-I Milano (ApoA-IM), a genetic variant of the major structural and functional component of HDL, apolipoprotein A-I (apoA-I), which possesses pronounced anti-atherosclerotic and anti-inflammatory properties.
In a recent study, our group has developed genetically engineered rice plants expressing the ApoA-IM protein in their seeds, a strategy which overcome difficulties concerning the purification of the therapeutic protein and allows its safe oral administration via the seed’s extract, the so called “APO-rice”. The atheroprotective efficacy of the treatment with the APO-rice was also demonstrated both in vitro and in an atherosclerotic mouse model.
Here we present a next step in the examination of the APO-rice therapeutic potential. One of the main goals of this thesis is a deeper understanding of the performance of rice seeds as a bioreactor for the production of the ApoA-IM and its suitability as a delivery system.
Two other critical aspects we have tried to address consist in the biodistribution of the orally administered ApoA-IM and the complex molecular mechanisms underlying the anti-atherogenic effects observed in the treated mice.
In this view, the quantity of ApoA-IM protein among different protein subfractions of the transgenic rice flour has been characterized by means of enzyme-linked immunosorbent assay (ELISA) and Western Blot, thus providing a closer look on the expression of the recombinant protein into the transgenic seeds. The rice flour has also been processed to reduce the content of starch by enzymatic hydrolysis, in order to improve its degree of water solubility in the perspective of an optimization of the APO-rice oral administration.
We have also investigated, again using ELISA and Western Blot, the presence of the ApoA-IM in the protein extracts of tissues (liver, intestine, brain and cerebellum) harvested from animals treated with the APO-rice, in order to assess its biodistribution. However, this has proved to be one of the most challenging analyses. Indeed, our attempts to detect the recombinant protein in the animal organs have not succeeded for reasons which in part reside in the intrinsic ApoA-IM metabolic properties, especially the enhanced catabolic turnover the protein undergoes.
As for the molecular effects induced in the animals by the APO-rice treatment, evaluations of the endogenous ApoA-I levels have revealed an interesting trend of overexpression of hepatic mouse ApoA-I protein. Further analyses in this sense are needed, but this data certainly represents an important clue for the interpretation of the APO-rice effects.
Finally, in this thesis by constructing a bicistronic vector co-expressing the ApoA-IM and the EGFP reporter, along with a set of control plasmids, we have provided new fundamental tools for future in vitro studies concerning the molecular response induced by the ApoA-IM protein.
While further evaluations and improvements of the APO-rice system are certainly needed, the new insights and experimental results achieved in this work have hopefully provided an important step forward in the direction of an innovative and promising therapeutic agent against cardiovascular disease.
The inverse relationship between high-density lipoprotein cholesterol (HDL-C) and cardiovascular disease (CVD) risk has been well established for many years, making HDL particles an attractive therapeutic target. However, shortcomings concerning manufacturing issues and the drawbacks of the typical intravenous route of administration of straight forward HDL-based therapies have made it difficult to fully exploit their potential, which thus requires innovative strategies.
This thesis is part of a research project focused on the development of a novel production and delivery system of apolipoprotein A-I Milano (ApoA-IM), a genetic variant of the major structural and functional component of HDL, apolipoprotein A-I (apoA-I), which possesses pronounced anti-atherosclerotic and anti-inflammatory properties.
In a recent study, our group has developed genetically engineered rice plants expressing the ApoA-IM protein in their seeds, a strategy which overcome difficulties concerning the purification of the therapeutic protein and allows its safe oral administration via the seed’s extract, the so called “APO-rice”. The atheroprotective efficacy of the treatment with the APO-rice was also demonstrated both in vitro and in an atherosclerotic mouse model.
Here we present a next step in the examination of the APO-rice therapeutic potential. One of the main goals of this thesis is a deeper understanding of the performance of rice seeds as a bioreactor for the production of the ApoA-IM and its suitability as a delivery system.
Two other critical aspects we have tried to address consist in the biodistribution of the orally administered ApoA-IM and the complex molecular mechanisms underlying the anti-atherogenic effects observed in the treated mice.
In this view, the quantity of ApoA-IM protein among different protein subfractions of the transgenic rice flour has been characterized by means of enzyme-linked immunosorbent assay (ELISA) and Western Blot, thus providing a closer look on the expression of the recombinant protein into the transgenic seeds. The rice flour has also been processed to reduce the content of starch by enzymatic hydrolysis, in order to improve its degree of water solubility in the perspective of an optimization of the APO-rice oral administration.
We have also investigated, again using ELISA and Western Blot, the presence of the ApoA-IM in the protein extracts of tissues (liver, intestine, brain and cerebellum) harvested from animals treated with the APO-rice, in order to assess its biodistribution. However, this has proved to be one of the most challenging analyses. Indeed, our attempts to detect the recombinant protein in the animal organs have not succeeded for reasons which in part reside in the intrinsic ApoA-IM metabolic properties, especially the enhanced catabolic turnover the protein undergoes.
As for the molecular effects induced in the animals by the APO-rice treatment, evaluations of the endogenous ApoA-I levels have revealed an interesting trend of overexpression of hepatic mouse ApoA-I protein. Further analyses in this sense are needed, but this data certainly represents an important clue for the interpretation of the APO-rice effects.
Finally, in this thesis by constructing a bicistronic vector co-expressing the ApoA-IM and the EGFP reporter, along with a set of control plasmids, we have provided new fundamental tools for future in vitro studies concerning the molecular response induced by the ApoA-IM protein.
While further evaluations and improvements of the APO-rice system are certainly needed, the new insights and experimental results achieved in this work have hopefully provided an important step forward in the direction of an innovative and promising therapeutic agent against cardiovascular disease.
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