RESUMO
Adenovirus vectors are widely used in gene therapy clinical trials, and preclinical studies with these vectors are often conducted in mice. It is therefore critical to understand whether mouse studies adequately predict the behavior of adenovirus vectors in humans. The most commonly-used adenovirus vectors are derived from adenovirus serotype 5 (Ad5). The Ad5 hexon protein can bind coagulation factor X (FX), and binding of FX has a major impact on vector interactions with other blood proteins. In mouse serum, FX protects Ad5 vectors from neutralization by natural antibodies and complement. In the current study, we similarly find that human FX inhibits neutralization of Ad5 vectors by human serum, and this finding is consistent among individual human sera. We show that human IgM and human IgG can each induce complement-mediated neutralization when Ad5 vectors are not protected by FX. Although mouse and human serum had similar effects on Ad5 vectors, we found that this was not true for a chimeric Ad5 vector that incorporated hexon regions from adenovirus serotype 48. Interestingly, this hexon-chimeric vector was neutralized by human serum, but not by mouse serum. These findings indicate that studies in mouse serum accurately predict the behavior of Ad5 vectors in human serum, but mouse serum is not an accurate model system for all adenovirus vectors.
Assuntos
Adenoviridae/imunologia , Proteínas do Capsídeo/imunologia , Vetores Genéticos , Testes de Neutralização , Adenoviridae/genética , Animais , Humanos , Imunoglobulina M/imunologia , Camundongos , Ligação ProteicaRESUMO
Natural IgM antibodies have an innate ability to recognize many viruses and viral-based gene therapy vectors. Naive mice have natural IgM antibodies that bind to adenoviruses, and these antibodies can profoundly affect the biodistribution and efficiency of gene delivery by adenovirus type 5 vectors. Here, we present protocols for isolating IgM from mouse serum, for assaying the concentration and adenoviral reactivity of mouse IgM, and for evaluating how natural antibodies and complement can synergize to neutralize adenovirus vectors.
Assuntos
Adenovírus Humanos/imunologia , Anticorpos Antivirais/imunologia , Vetores Genéticos/imunologia , Imunoglobulina M/imunologia , Adenovírus Humanos/classificação , Adenovírus Humanos/genética , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Proteínas do Sistema Complemento/imunologia , Ensaio de Imunoadsorção Enzimática , Técnicas de Transferência de Genes , Terapia Genética/efeitos adversos , Vetores Genéticos/genética , Humanos , Imunoglobulina M/sangue , CamundongosRESUMO
RATIONALE: During embryogenesis, hematopoietic cells appear in the myocardium prior to the initiation of coronary formation. However, their role is unknown. OBJECTIVE: Here we investigate whether pre-existing hematopoietic cells are required for the formation of coronary vasculature. METHODS AND RESULTS: As a model of for hematopoietic cell deficient animals, we used Runx1 knockout embryos and Vav1-cre; R26-DTA embryos, latter of which genetically ablates 2/3 of CD45(+) hematopoietic cells. Both Runx1 knockout embryos and Vav1-cre; R26-DTA embryos revealed disorganized, hypoplastic microvasculature of coronary vessels on section and whole-mount stainings. Furthermore, coronary explant experiments showed that the mouse heart explants from Runx1 and Vav1-cre; R26-DTA embryos exhibited impaired coronary formation ex vivo. Interestingly, in both models it appears that epicardial to mesenchymal transition is adversely affected in the absence of hematopoietic progenitors. CONCLUSION: Hematopoietic cells are not merely passively transported via coronary vessel, but substantially involved in the induction of the coronary growth. Our findings suggest a novel mechanism of coronary growth.
Assuntos
Diferenciação Celular/genética , Vasos Coronários/crescimento & desenvolvimento , Desenvolvimento Embrionário/genética , Coração/crescimento & desenvolvimento , Animais , Linhagem da Célula/genética , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Vasos Coronários/embriologia , Vasos Coronários/metabolismo , Embrião de Mamíferos , Transição Epitelial-Mesenquimal/genética , Camundongos , Camundongos KnockoutAssuntos
Aorta/citologia , Aorta/embriologia , Coração/embriologia , Proteínas de Homeodomínio/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/embriologia , Fatores de Transcrição/metabolismo , Animais , Vasos Coronários/citologia , Desenvolvimento Embrionário , Feminino , Genes Reporter , Proteína Homeobox Nkx-2.5 , Proteínas de Homeodomínio/genética , Camundongos , Músculo Liso Vascular/fisiologia , Miocárdio/metabolismo , Crista Neural/citologia , Crista Neural/embriologia , Fatores de Transcrição/genéticaRESUMO
Haematopoietic cells arise from spatiotemporally restricted domains in the developing embryo. Although studies of non-mammalian animal and in vitro embryonic stem cell models suggest a close relationship among cardiac, endocardial and haematopoietic lineages, it remains unknown whether the mammalian heart tube serves as a haemogenic organ akin to the dorsal aorta. Here we examine the haemogenic activity of the developing endocardium. Mouse heart explants generate myeloid and erythroid colonies in the absence of circulation. Haemogenic activity arises from a subset of endocardial cells in the outflow cushion and atria earlier than in the aorta-gonad-mesonephros region, and is transient and definitive in nature. Interestingly, key cardiac transcription factors, Nkx2-5 and Isl1, are expressed in and required for the haemogenic population of the endocardium. Together, these data suggest that a subset of endocardial/endothelial cells serve as a de novo source for transient definitive haematopoietic progenitors.
