● NOGGIN
GENE THERAPY FOR THE PREVENTION OF BMP INDUCED HETEROTOPIC OSSIFICATION IN THE MOUSE:
IMPLICATIONS FOR THE TREATMENT OF FIBRODYSPLASIA OSSIFICANS PROGRESSIVA AND OTHER DISORDERS
OF ECTOPIC OSSIFICATION IN HUMANS.
+*Glaser M. D., D L.; *****Kaplan, M. D., F S.; **Wang, Ph.D. L.; ***Economides, Ph.D., A N. (D-Regeneron Pharmaceuticals); **Wilson, M.D.,Ph.D., J M. (DTargeted Genetics Inc./ GSK); ***Stahl, Ph.D., N. (D-Regeneron Pharmaceuticals); ****Shore, Ph. D., E M.
+*University of Pennsylvania Department of Orthopaedic Surgery, Philadelphia, Pennsylvania. 215-662-4091, Fax: 215-349-5928, [email protected]
INTRODUCTION: Disorders of heterotopic ossification occur in a wide
variety of disease processes, and prevention regimens using indomethacin and
radiation therapy have had varying success. However, for individuals with
more severe forms of heterotopic ossification such as fibrodysplasia ossificans
progressiva (FOP), conventional interventions have failed to halt progression
of the disease. Although the genetic causes of FOP and other disorders of
heterotopic ossification remain unknown, overexpression of bone
morphogenetic protein-4 (BMP4) and underexpression of the BMP antagonist
noggin may have important inductive roles in the pathogenesis of such
disorders.
The purpose of this study was to develop an effective gene therapy
approach for the prevention of BMP mediated heterotopic ossification that
would be applicable in principle to the treatment of patients with FOP. To
achieve this goal, we used an adenovirus-mediated gene transfer of noggin, a
potent secreted extracellular antagonist of the osteogenic morphogen, BMP4.
METHODS: A mouse model of induced heterotopic osteogenesis was
developed using an injectable osteoconductive material (carrier) impregnated
with BMP4. This model permits the definitive identification of the stages of
endochondral bone formation (Figure 1). The abdominal musculature of 32
C57/b6 mice (with IACUC review board approval) was injected on one side
of the midline with 250µl of carrier alone, and on the contra-lateral side with
250µl of carrier combined with recombinant human BMP4 at a final
concentration of either 50µg/ml or 12.5µg/ml. Both doses of BMP4 were
previously determined to consistently produce heterotopic ossification in a
dose-dependent manner. Half of the animals were pretreated for 4 days with
1x1011 viral particles/mouse of recombinant adenovirus carrying the human
noggin gene (AdhNGdeltaB2, a modified noggin gene engineered to increase
protein bioavailability). Implants were recovered at 7 and 14 days after
injection. Standard histological techniques were used to evaluate the
histologic stages of bone formation and to identify specific cell types present
in the tissue.
RESULTS: Experimental and control implants were recovered from all
animals. The implants containing BMP4-induced an aggressive,
fibroproliferative lesion with early cartilage formation at 7 days (Figure 2a).
and heterotopic ossification at 14 days. However, in animals treated with
noggin-containing adenovirus, the implants with BMP4 demonstrated a
minimal, mixed inflammatory cell infiltrate at 7 days (Figure 2b) and a thin
pseudocapsule several cell layers thick surrounding the unresorbed plug at 14
days, indistinguishable from carrier implants with no BMP.
DISCUSSION: This study demonstrates that the delivery of noggin through a
gene therapy approach successfully prevents BMP4 induced heterotopic
ossification in a mouse model. It provides proof-of-concept that a secreted
morphogen antagonist can be produced in vivo and act systemically to prevent
BMP4-mediated heterotopic ossification that is clinically relevant to
catastrophic disorders of progressive heterotopic ossification in humans.
Numerous obstacles need to be overcome before the application of noggin
gene therapy could be considered for the treatment of patients who have FOP
and other disorders of heterotopic ossification. These include the development
of safe and effective viral vectors and inducible promoters for the systemic
and durable delivery of genes in humans and ultimately the development of
improved animal models based upon knowledge of the molecular genetics of
FOP and other disorders of heterotopic ossification. Nevertheless, for patients
suffering from FOP, stable gene transfer of an inducible BMP4 antagonist
may offer a solution where all other modalities have failed.
A
M
M
I
F
I
F
I
B
F
C
C
F
B
B
C
H
H
Figure 1. BMP induced bone formation (200x, 400x). A) Day 14, implant
without BMP. B) Day 7, with BMP. Early stages of endochondral bone
formation with fibroproliferation and chondrogenesis. C) Day 14, with BMP,
immature bone with marrow cavities and hematopoietic elements.
F
A
C
I
I
B
I
M
C
S
M
I
M
Figure 2. Noggin therapy (100x, 200x). A) Day 7, BMP implant without
noggin treatment shows early stages of bone formation. B) Day 7, BMP
implant with noggin treatment. Note clear lack of induction with a distinct
muscle/implant border.
Key:
M=muscle;
S=skin;
I=implant;
B=bone;
C=cartilage;
F=fibroproliferation; H=Hematopoietic cells
**University of Pennsylvania Institute for Human Gene Therapy,
Philadelphia, Pennsylvania.
***Regeneron Pharmaceuticals, Tarrytown, New York.
****University of Pennsylvania Department of Orthoapedic Surgery and
Genetics, Philadelphia, Pennsylvania.
*****University of Pennslyvania Department of Orthopaedic Surgery and
Medicine, Philadelphia, Pennsylvania.
48th Annual Meeting of the Orthopaedic Research Society
Poster No: 0493