Targeting Adeno-Associated Virus Vectors for Local Delivery to Fractures and Systemic Delivery to the Skeleton

Lucinda R Lee; Lauren Peacock; Leszek Lisowski; David G Little; Craig F Munns; Aaron Schindeler

doi:10.1016/j.omtm.2019.08.010

Targeting Adeno-Associated Virus Vectors for Local Delivery to Fractures and Systemic Delivery to the Skeleton

Mol Ther Methods Clin Dev. 2019 Sep 5:15:101-111. doi: 10.1016/j.omtm.2019.08.010. eCollection 2019 Dec 13.

Authors

Lucinda R Lee^{1

2}, Lauren Peacock¹, Leszek Lisowski^{3

4}, David G Little^{1

2}, Craig F Munns^{2

5}, Aaron Schindeler^{1

2

3}

Affiliations

¹ Orthopaedic Research and Biotechnology Unit, The Children's Hospital at Westmead, Westmead, NSW, Australia.
² The University of Sydney, Discipline of Child and Adolescent Health, Faculty of Health and Medicine, Sydney, NSW, Australia.
³ Translational Vectorology Group, Children's Medical Research Institute, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW 2006, Australia.
⁴ Military Institute of Hygiene and Epidemiology, The Biological Threats Identification and Countermeasure Centre, 24-100 Puławy, Poland.
⁵ Institute of Endocrinology and Diabetes, The Children's Hospital at Westmead, Westmead, NSW, Australia.

Abstract

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.

Keywords: AAV; adeno-associated virus; bone; capsid variants; osteoblasts.