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Generation of a Mammalian Achondroplasia Model in Micropigs Jennifer S.C. Peng 1a, Eran Rom 2a, Michael Malka 2, Felicia Ang 1, Tammy P.J. Wu 1, Alvin Goh 1, Jerry W. Lee 1, Lisa Soyeon Park 1, Avner Yayon 2 1 PWG

2 ProCore

Genetics Pte Ltd, 15 Tech Park Crescent, Singapore 638117

ABSTRACT

Photography and Micro-CT were Taken in the Tibia bones after Animal Termination

There were no group differences throughout the whole experimental period

Achondroplasia (ACH) is an autosomal dominant syndrome, characterized by marked short-limbs, rhizomelic short stature, macrocephaly, low nasal bridge, frontal bossing, narrowing of spinal cord column and thorax. ACH patients suffer also from delayed motor development, persistent or recurrent middle-ear dysfunction and bowing of lower legs. Most ACH cases share a gain-of-function mutation of Gly380Arg aminoacid substitution in the transmembrane domain of FGFR3. It is believed that the malfunction of FGFR3 persistent overactivation contributes to ACH etiology and pathology. Physiologically, FGF18 activates FGFR2 and FGFR3 in the epiphyseal growth plate and the adjacent subchondal bone in vivo and in vitro. It was recently, found (Yayon, unpublished observation) that direct injection of FGF18 into the knee joint space resulted in premature growth plate closure in mice, which suggested that intraarticular administration of FGF18 may mimic ACH symptoms by reaching and activating FGFR3 in the growth plate. The purpose of the present study was to develop a first of its kind, large animal model for ACH by targeting FGFR3 in micropigs with either wild-type FGF18 or a FGFR3selective variant of FGF18 (FGF18v1) as a novel platform for the development of specific and safe therapeutics for ACH. Following several intraarticular administrations of FGF18 and FGF18v1, the micropigs were sacrificed, and their femur and tibia were harvested for further evaluation. The bone length of femur and tibia were measured, and the growth plate morphology was analyzed by imaging with C-arm-X-ray and Micro-CT and by histopathological examinations. Consistent with the preliminary results in mice, we found that these treatments dramatically induced bone shortening and growth plate closure in both the femur and the tibia of these micropigs. Further studies are now underway to optimize and validate the ACH micropig model. Until now, there are still no known cures or treatments for ACH. Following the establishment of this newly established ACH micropig model, a spectrum of FGF18 activation antagonists can be examined as potential treatments for ACH in the future.

Bone Photography

Growth Plate Morphology Showed Mild to Medium Disorganization and Degeneration in the Proliferati ve Zone (P) in the FGF18v1 Treatment Group

Micro-CT

Femur Right

Left

Tibia

Femur

a Equal

Ltd., 7 Golda Meir St., Weizmann Science Park, Nes-Ziona, 7400, Israel

Tibia Right

Left

contribution

A Schematic Model for Achondroplasia Induction in the Growth plate of PWG Micropigs Growth Plate (Epiphyseal Pate)

FGF18 Right

Left

FGFR3

Femur Right FGF18

FGF18

High Dose

High Dose

1M001

1M001

Zone of Resting Cartilage

Left

R

BW (g)

FGF18v1 Low Dose 2M001

FGF18 High Dose FGF18v1 Low Dose FGF18v1 High Dose

7000 6000

Right

R P

P FGF18v1 Low Dose 2M001

P

Zone of Proliferation

H

H

5000

P

Left

Tibia C

C

4000

R

3000

H

H

C

C R

R

R

Zone of

Right

Hypertrophy

FGF18v1 High Dose 3M001

2000

FGF18v1 High Dose 3M001

1000

P

P

P

P

Bone of Diaphysis

Left

0 -28

0

6

14 20 28 Experimental Day

35

42

H

H

H

H

48 C

C

C

C

Key Words: Achondroplasia, FGF18, FGFR3, Micropig, Porcine Disease model

Photo of Hind Limbs and C-Arm X Ray were Taken in the baseline Phase Experimental Design

Photo of Hind Limbs

C-Arm X Ray

Bone Length of Femur and Tibia Bones was Reduc ed by FGF18v1 Treatment

A. Measurement in Growth Plate Thickness of Femur Bones

A. Bone Length Measurement in Femur Bones Treatment

cm 12

 4 weeks old of PWG Micropigs were selected  7 intraarticular injections into right hind limbs in weekly base

FGF18

Group 2:

Group 3.

FGF18_High Dose

FGF18v1_Low Dose

FGF18v1_High Dose

Control

400 350

10

300

8

250

6

200 150

4

100 50 0

0 1M003

1M004

1F002

Interim Sacrifice

Group 1:

Treatment

Control

2

High Dose

Growth Plate Thickness in Femur and Tibia Bones was Reduced by FGF18v1 Treatment

1M001

1M001

1M002

Terminal Sacrifice

1F001

2M003

Recovery Sacrifice

2M004

2F002

Interim Sacrifice

FGF18: High Dose

2M001

2M002

Terminal Sacrifice

2F001

3M003

Recovery Sacrifice

3M004

3F002

Interim Sacrifice

FGF18v1: Low Dose

3M001

3M002

Terminal Sacrifice

3F001

1M003

B7-319

Recovery Sacrifice

FGF18v1: High Dose

1M004

1F002

Interim Sacrifice

Recovery Sacrifice

1M001

1M002

Terminal Sacrifice

1F001

2M003

Recovery Sacrifice

2M004

FGF18: High Dose

No Treatment Control

2F002

Interim Sacrifice

2M001

2M002

Terminal Sacrifice

2F001

3M003

Recovery Sacrifice

3M004

3F002

Interim Sacrifice

FGF18v1: Low Dose

3M001

3M002

Terminal Sacrifice

3F001

B7-319

Recovery Sacrifice

Recovery Sacrifice

FGF18v1: High Dose

No Treatment Control

B. Measurement in Growth Plate Thickness of Tibia Bones B. Bone Length Measurement in Tibia Bones

Treatment

Control

350 Treatment

cm 12

1.

As the dosing periods prolonged, the ACH phenotypes could be ob served more obviously.

2. Limping, shaking, and tremors were observed when walking

Control

300 250

10

FGF18v1 Low Dose 2M001

200

8

150

6

100 50

4

0

2

1M003

1M004

1F002

1M001

Interim Sacrifice

0 1M003

1M004

1F002

Interim Sacrifice

1M001

1M002

Terminal Sacrifice

1F001

2M003

Recovery Sacrifice

2M004

FGF18: High Dose

3. The treated right hind limbs showed somehow shorter than the non-i njected left hind limbs, especially in the FGF18v1_High Dose Group.

2F002

Interim Sacrifice

2M001

2M002

Terminal Sacrifice

2F001

3M003

Recovery Sacrifice

3M004

3F002

Interim Sacrifice

FGF18v1: Low Dose

3M001

3M002

Terminal Sacrifice

3F001

B7-319

Recovery Sacrifice

Recovery Sacrifice

FGF18v1: High Dose

1M002

Terminal Sacrifice

1F001

2M003

Recovery Sacrifice

2M004

2F002

2M001

Interim Sacrifice

FGF18: High Dose

2M002

Terminal Sacrifice

2F001

3M003

Recovery Sacrifice

3M004

3F002

3M001

Interim Sacrifice

FGF18v1: Low Dose

3M002

Terminal Sacrifice

3F001

B7-319

Recovery Sacrifice

Recovery Sacrifice

FGF18v1: High Dose

No Treatment Control

No Treatment Control

C. Changes in Growth Plate Thickness in Femur and Tibia Bones

4. The changes in growth plate thickness are observed both in the fem ur and the tibia in all the three groups .

C. Changes in Bone Length Measurement in Femur and Tibia Bones

Femur Femur

FGF18v1

8% 6% 4%

5. The disorganization and the degeneration of epiphyseal hyaline carti lage matrix in the proliferative zone showed both in the groups of F GF18v1_Low Dose and FGF18v1_High Dose

High Dose

2% 0% -2%

3M001

-4%

1M003

-6% 1M003

1M004 Interim Sacrifice

1F002

1M001

1M002

Terminal Sacrifice FGF18: High Dose

1F001 Recovery Sacrifice

2M003

2M004 Interim Sacrifice

2F002

2M001

2M002

Terminal Sacrifice FGF18v1: Low Dose

2F001 Recovery Sacrifice

3M003

3M004 Interim Sacrifice

3F002

3M001

3M002

Terminal Sacrifice FGF18v1: High Dose

Tibia

50% 40% 30% 20% 10% 0% -10% -20% -30% -40%

Tibia

3F001

B7-319

Recovery Sacrifice

Recovery Sacrifice No Treatment Control

1M004 Interim Sacrifice

1F002

1M001

1M002

Terminal Sacrifice FGF18: High Dose

1F001 Recovery Sacrifice

2M003

2M004 Interim Sacrifice

2F002

2M001

2M002

Terminal Sacrifice FGF18v1: Low Dose

2F001 Recovery Sacrifice

3M003

3M004 Interim Sacrifice

3F002

3M001

3M002

Terminal Sacrifice FGF18v1: High Dose

3F001

B7-319

Recovery Sacrifice

Recovery Sacrifice No Treatment Control

Acknowledgemenet 

PWG Genetics Pte Ltd

Procore Ltd

Singapore Israel Inductrial Research and Development Foundation(SIIRD)

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