Ex vivo transfer of the Hoxc-8-interacting domain of Smad1 by a tropism-modified adenoviral vector results in efficient bone formation in a rabbit model of spinal fusion.
METHODS
Ex vivo gene transfer for spinal fusion.


OBJECTIVE
This study aimed to evaluate ex vivo transfer of the nuclear-localized Hoxc-8-interacting domain of Smad1 (termed Smad1C) to rabbit bone marrow stromal cells (BMSCs) by a tropism-modified human adenovirus serotype 5 (Ad5) vector as a novel therapeutic approach for spinal fusion.


BACKGROUND
Novel approaches are needed to improve the success of bone union after spinal fusion. One such approach is the ex vivo transfer of a gene encoding an osteoinductive factor to BMSCs which are subsequently reimplanted into the host. We have previously shown that heterologous expression of the Hoxc-8-interacting domain of Smad1 in the nuclei of osteoblast precursor cells is able to stimulate the expression of genes related to osteoblast differentiation and induce osteogenesis in vivo. Gene delivery vehicles based on human Ad5 are well suited for gene transfer for spinal fusion because they can mediate high-level, short-term gene expression. However, Ad5-based vectors with native tropism poorly transduce BMSCs, necessitating the use of vectors with modified tropism to achieve efficient gene transfer.


METHODS
The gene encoding Smad1C was transferred to rabbit BMSCs by an Ad5 vector with native tropism or a vector retargeted to alphav integrins, which are abundantly expressed on rabbit BMSCs. Transduced BMSCs were maintained in osteoblastic differentiation medium for 30 days. Alkaline phosphatase activity was determined and cells stained for calcium deposition. As positive controls for osteogenesis, we used Ad5 vectors expressing bone morphogenetic protein 2. As negative controls, BMSCs were mock-transduced or transduced with an Ad5 vector expressing beta-galactosidase. In an immunocompetent rabbit model of spinal fusion, transduced BMSCs were coated onto absorbable gelatin sponge and implanted between decorticated transverse processes L6 and L7 of 8-week-old female New Zealand white rabbits. Animals were killed 4 weeks after implantation of the sponges, the fusion masses harvested and the area of new bone quantified using image analysis software.


RESULTS
The Smad1C-expressing tropism-modified Ad5 vector mediated a significantly higher level of alkaline phosphatase activity and calcium deposition in transduced rabbit BMSCs than all other vectors. The rabbit BMSCs transduced ex vivo with the Smad1C-expressing tropism-modified Ad5 vector mediated a greater amount of new bone formation than BMSCs transduced with any other vector.


CONCLUSIONS
Delivery of the Smad1C gene construct to BMSCs by an alphav integrin-targeted Ad5 vector shows promise for spinal fusion and other applications requiring the formation of new bone in vivo.
Douglas
Joanne T
JT
Division of Human Gene Therapy, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
Rivera
Angel A
AA
Lyons
Gray R
GR
Lott
Patricia F
PF
Wang
Dezhi
D
Zayzafoon
Majd
M
Siegal
Gene P
GP
Cao
Xu
X
Theiss
Steven M
SM
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D016026:Bone Marrow Transplantation / Q000379:methods*; D001861:Bone Regeneration / Q000235:genetics; D002478:Cells, Cultured; D004195:Disease Models, Animal; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D018398:Homeodomain Proteins / Q000235:genetics; D039564:Integrin alphaV / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D011817:Rabbits; D051898:Smad1 Protein / Q000235:genetics*; D013123:Spinal Fusion / Q000379:methods*; D017154:Stromal Cells / Q000166:cytology / Q000378:metabolism / Q000637:transplantation*; D016896:Treatment Outcome
D018398:Homeodomain Proteins; C489816:Hoxc8 protein, mouse; D039564:Integrin alphaV; D051898:Smad1 Protein