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| Author(s): |
Franciele de Lima
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | Campinas, SP. |
| Institution: | Universidade Estadual de Campinas (UNICAMP). Faculdade de Ciências Médicas |
| Defense date: | 2023-03-14 |
| Examining board members: |
Erich Vinicius De Paula;
Margareth Castro Ozelo;
Marilda de Souza Gonçalves;
Fabíola Traina;
Bidossessi Wilfried Hounkpe
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| Advisor: | Erich Vinicius De Paula |
| Abstract | |
Sickle cell anemia (SCA) is the most frequent hemoglobinopathy in the world, one of its main pathological mechanisms being chronic intravascular hemolysis. Heme is a fundamental molecule for several biological processes, but in diseases where there is a lot of hemolysis there is an increase of free heme in the circulation that can be toxic to cells and tissues. Hemopexin (HPX) is a circulating protein responsible for removing free heme from circulation, whose levels may be depleted in conditions of chronic hemolysis, such as in SCA. Thus, increasing HPX levels represents an attractive strategy to mitigate the deleterious effects of heme in these conditions. Gene therapy with adeno-associated viral vectors (AAV) is a treatment strategy that in recent years has accumulated several successes in humans. Our objective was to use a vector based on an adeno-associated virus serotype 8 (AAV8) that expresses human HPX (hHPX) or murine HPX (mHPX) protein in animal models of hemolysis, evaluating its efficacy and safety. C57Bl/6j mice were injected with vector doses and expression was evaluated by ELISA, Western Blot and qPCR. Furthermore, the biological activity of the transgenic hHPX was confirmed using two different models of heme challenge consisting of serial injections of heme or phenylhydrazine-induced hemolysis. We also used an animal model of FA, the Townes, to evaluate safety and efficacy profiles of this vector. In C57Bl/6j mice sustained hHPX expression was confirmed for up to 26 weeks in plasma. Expression was dose-dependent and not associated with clinical signs of toxicity. hHPX levels were significantly reduced by heme infusions and phenylhydrazine-induced hemolysis. No clinical toxicity or laboratory signs of liver damage were observed in the short-term heme challenge studies. In the Townes model, characterized by sustained hemolysis, hHPX expression was confirmed in the liver, but circulating levels did not reach the expected levels. Furthermore, using the mHPX vector, we saw that it was able to increase murine HPX expression in C57Bl/6j mice in a sustained and dose-dependent manner. In Townes mice, we observed a significant increase in mHPX gene expression, but circulating levels remained low. However, we noticed a tendency towards weight gain in animals injected with the mHPX vector, which suggests its effectiveness. Our results confirm the feasibility and safety of HPX gene transfer even in heme overload models. Furthermore, the use of a vector that expresses murine HPX has become an interesting strategy with greater therapeutic potential, suggesting that the use of mHPX can modulate parameters that were not able to be evaluated with hHPX (AU) | |
| FAPESP's process: | 18/23484-7 - Evaluation of the efficacy and safety of a strategy based on gene therapy with a viral vector for expression of hemopexin in animal models of Sickle Cell Disease |
| Grantee: | Franciele de Lima |
| Support Opportunities: | Scholarships in Brazil - Doctorate |