Targeting Adeno-Associated Virus Vectors for Local Delivery to Fractures and Systemic Delivery to the Skeleton
| Autor: | David G. Little, Aaron Schindeler, Leszek Lisowski, Lauren Peacock, Craig F Munns, Lucinda R. Lee |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
lcsh:QH426-470 Transgene viruses Cre recombinase adeno-associated virus Gene delivery Biology medicine.disease_cause capsid variants bone Article 03 medical and health sciences Transduction (genetics) 0302 clinical medicine Genetics medicine lcsh:QH573-671 Enhancer Molecular Biology Adeno-associated virus lcsh:Cytology osteoblasts Promoter AAV Molecular biology lcsh:Genetics 030104 developmental biology 030220 oncology & carcinogenesis Molecular Medicine Alkaline phosphatase |
| Zdroj: | Molecular Therapy: Methods & Clinical Development, Vol 15, Iss, Pp 101-111 (2019) Molecular Therapy. Methods & Clinical Development |
| ISSN: | 2329-0501 |
| Popis: | A panel of 18 recombinant adeno-associated virus (rAAV) variants, both natural and engineered, constitutively expressing Cre recombinase under the cytomegalovirus early enhancer/chicken β actin (CAG) promoter, were screened for their ability to transduce bone in Ai9 fluorescent reporter mice. Transgenic Cre-induced tdTomato expression served as a measure of transduction efficiency and alkaline phosphatase (AP) activity as an osteoblastic marker. Single injections of AAV8, AAV9, and AAV-DJ into midshaft tibial fractures yielded robust tdTomato expression in the callus. Next, the bone cell-specific promoters Sp7 and Col2.3 were tested to restrict Cre expression in an alternate model of systemic delivery by intravenous injection. Although CAG promoter constructs packaged into AAV8 produced high levels of tdTomato in the bone, liver, heart, spleen, and kidney, bone-specific promoter constructs restricted Cre expression to osseous tissues. AAV variants were further tested in vitro in a human osteoblast cell line (hFOB1.19), measuring GFP reporter expression by flow cytometry after 72 h. AAV2, AAV5, and AAV-DJ showed the highest transduction efficiency. In summary, we produced AAV vectors for selective and high-efficiency in vivo gene delivery to murine bone. The AAV8-Sp7-Cre vector has significant practical applications for inducing gene deletion postnatally in floxed mouse models. Graphical Abstract |
| Databáze: | OpenAIRE |
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