Inozyme Pharma : ENPP1-Fc expressing AAV vector prevents ectopic tissue calcification and restores bone parameters in ENPP1 deficient mice (presented at ASBMR 2022)

ENPP1-Fc expressing AAV vector prevents ectopic tissue calcification

and restores bone parameters in ENPP1 deficient mice

1. O'Brien1, L. Flaman 1, C. Sullivan 1, J. Howe 1, S. Jungles 1, A. Lynch 1, D. Schrier 1, Z. Cheng 1, Y. Sabbagh 1

1 Inozyme Pharma, Boston MA

INTRODUCTION

AAV-ENPP1RESTORES PLASMA PPi LEVELS

AAV-ENPP1 PREVENTS VERTEBRAL CALCIFICATION

AAV-ENPP1 RESTORES CRITICAL BONE PARAMETERS

ENPP1 Deficiency

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is the major enzyme that generates extracellular pyrophosphate (PPi), an inorganic metabolite with potent anti-calcificationactivity.1-3 Loss-of-function mutations lead to a state of ENPP1 Deficiency and hypopyrophosphatemia, which is associated with extensive calcification of the arteries, organs and joints. Infants with ENPP1 Deficiency present with severe cardiovascular complications and over 50% mortality in the first 6 months of life.5-6 Those who survive into childhood-adulthood typically develop hypophosphatemic rickets, characterized by growth plate abnormalities, bowed legs, short stature, and/or calcification of the joints and ligaments.4-6

AAV-ENPP1 Gene Therapy

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asj-2J,vehicle asj-2J AAV (5.0e12 vg/kg) asj-2J, AAV (2.5e13 vg/kg)

µ-Ct Cervical Spine

Fig 3. Representative examples of spine animations from the three groups analyzed. Red arrows highlight calcifications in the vehicle-treated asj-2Jgroup which are more prominent compared to the ENPP1-Fc-AAVtreated group

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asj-2J, vehicle

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An adeno-associated viral vector that expresses a modified human ENPP1-Fc under the control of a liver-specific promoter (AAV-ENPP1) was developed as a one-dose gene therapy to treat ENPP1 Deficiency.

Fig 1. Plasma ENPP1 activity levels (A) and Plasma PPi levels (B), at day 70 following

1. single IV dose on day 1 of ENPP1-Fc-AAV or vehicle. * p ≤ 0.05, **** p ≤ 0.0001

2D µ-Ct

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We previously reported that a single intravenous injection (2.5x1013 vg/kg) of

Spine

MAR

BFR

Ob. S/BS

AAV-ENPP1 in 2-week-oldEnpp1asj-2J/asj-2J mice (a murine model of ENPP1 Deficiency) resulted in sustained elevation of plasma ENPP1 activity during the 10-week study.7 It also rescued plasma PPi levels, prevented calcification

AAV-ENPP1 PREVENTS SOFT TISSUE CALCIFICATION

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/yr

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300

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in all organs analyzed, and inhibited the development of bone abnormalities.

OBJECTIVES

1. To analyze the impact of AAV-ENPP1 on prevention of bone and spinal

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of
(nmol/mg	2

X-ray skull and spine

Fig 4. Representative examples of 2D µ-CTimages showing head and spines of the three groups analyzed. Red arrows highlight calcifications in the vehicle-treated asj-2Jgroup which are more prominent compared to the ENPP1-Fc-AAVtreated group.

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ligament defects in Enpp1asj-2J/asj-2J mice

2. To evaluate the impact of lower dose AAV-ENPP1 therapy on PPi levels

and soft tissue calcification in Enpp1asj-2J/asj-2J mice

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AAV-ENPP1 PREVENTS DEVELOPMENT OF GROWTH PLATE ABNORMALITIES

asj/2J

WT

Vehicle

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5.0x1012 vg/kg

1.5x1013 vg/kg

asj-2J

Fig 6. Static and dynamic histomorphometry from day 70 following a single intravenous injection of AAV-ENPP1at 2.5e13 vg/kg. * p ≤ 0.05, ** p ≤ 0.01 *** p ≤ 0.001, **** p ≤ 0.0001

Cortical Thickness (Ct. Th.), Cortical Bone Area/Total Area (Ct. BA/TA), Trabecular Bone volume/total volume (Tb. BV/TV), Mineral apposition rate (MAR), Bone formation rate (BFR), Osteoblast surface/bone surface (Ob. S/BS).

STUDY DESIGN AND METHODS

Descending Aorta

Lung

CONCLUSIONS

2-week-oldEnpp1asj-2J/asj-2J mice were given a single 2.5x1013 vg/kg dose of AAV-ENPP1 or vehicle, and bone microarchitecture and dynamic histomorphometry were evaluated 10 weeks after injection. Fixed cervical spines were scanned and evaluated using Scanco µCT35 (Scanco Medical, AG, Switzerland) by UMass Chan Medical School Bone Core with an X-ray energy intensity of 55kV with a current of 145mA and 400ms integration time. Quantitative analyses were carried out using IPL software (Scanco Medical, AG, Switerland). The cervical areas were scanned with 12µm voxels (1024 x 1024 pixels) and 2D images were generated using IRW Ver 4.2 software (Siemens, USA). Decalcified tibia were embedded using paraffin wax, sectioned at 6µm, and stained using Safranin O by UMass Chan Medical School Bone Core.

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Fig 5. Representative images of Safranin O and Fast Green-stainedimages from the tibia from all three AAV dosed groups and controls. Red asterisk denotes hypertrophic chondrocytes.

REFERENCES

6. Rutsch F, et al. Circ Cardiovasc Genet. 2008;1(2):133-

A single 1.5e12 vg/kg intravenous dose of AAV-ENPP1 is sufficient to elevate plasma pyrophosphate to levels seen in WT animals, prevent ectopic calcification in soft tissues, and prevent the development of rickets in an ENPP1-Deficient mouse model.

A single intravenous dose of AAV-ENPP1 as low as 5.0e1012 vg/kg is sufficient to prevent vertebral calcification in ENPP1-Deficient mice. Based on these results AAV-ENPP1 could prove an effective gene therapy for disorders of vertebral calcification such as ossification of the posterior longitudinal ligament (OPLL).

ACKNOWLEDGEMENTS & DISCLOSURES

Prior experiments demonstrating efficacy of AAV-ENPP1 on plasma PPi and ectopic calcification 10 weeks after injection were repeated in mice treated with three lower (single) doses of AAV-ENPP1 (1.5x1012, 5.0x1012, and 1.5x1013 vg/kg).

asj-2J

Figure 2. Terminal calcium levels of various tissues. WT and Asj-2J,vehicle treated are shown in red and blue, respectively. AAV-ENPP1-Fctreated Asj-2Jgroups are shown in green (1.5e12 vg/kg), purple (5.0e12 vg/kg) and orange (1.5e13 vg/kg). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001,

**** p ≤ 0.0001

1.	Mackenzie NC et al. Bone. 2012;51(5):961-968.	140.
2.	Johnson K et al. J Bone Miner Res. 2003;18(6):994-1004.7.	O'Brien K, et al. Treatment with an AAV vector
3. Orriss IR et al. Curr Opin Pharmacol. 2016;28:57-68.	expressing ENPP1-Fc prevents ectopic tissue
4. Ferreira CR et al. Genet Med. 2021;23(2):396-407	calcification and restores bone parameters in ENPP1
5.	Ferreira CR et al. J Bone Miner Res. 2021;36(11):2193-2202	deficient mice. ASBMR Annual Meeting; October 2021.
		San Diego, CA.

All study authors are current or former employees and stockholders in Inozyme Pharma

The authors would like to thank: Biomere Research Models, Worcester MA. (Kristin Sapp, Sue Champagne, and Amy Descarreaux); UMASS Chan Medical School Bone Analysis Core (Jae- Hyuck Shim PhD and Yeon-Suk Yang); UMASS Chan Medical School Image Processing and Analysis Core: (Mohammad Shazeeb PhD)