Intrathecal enzyme replacement therapy in a preclinical model:

Infantile Batten Disease Jui-Yun Lu1,3, Hemanth R. Nelvagal4, Lingling Wang1,3, Shari G. Birnbaum2, Jonathan D. Cooper4 and Sandra L. Hofmann1,3

From the Departments of 1Internal Medicine, 2Psychiatry, and the 3Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, TX 75390-8593, USA, and the 4Pediatric Storage Disorders Laboratory, Department of Basic and Clinical Neuroscience, King’s Health Partners Centre for Neurodegeneration, James Black Centre, Institute of

Psychiatry, Psychology & Neuroscience, King’s College London, 125 Coldharbour Lane, London SE5 9NU, UK

INTRODUCTION

Infantile Batten disease is caused by deficiency in an enzyme, palmitoyl-protein thioesterase (PPT1). Lack of PPT1 activity causes accumulation of material in the lysosomal compartment of cells. Experience in other lysosomal disease shows that administration of the missing enzyme (enzyme replacement therapy, ERT) by direct delivery to the spinal fluid may be helpful in preventing further accumulation and arresting progression of the disease. In this study we tested a preparation of the enzyme in mice. Treatment was given on three consecutive days in PPT1 knockout mice at 6 weeks of age.

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Fig. 2. Effect of intrathecal (IT) ERT on latency to fall from a constant speed rotating rod (Rotarod). All tests were terminated after 60 seconds. (A) Two trials were conducted daily on each of three consecutive days at 3, 5, 6, 7, 8, and 9 months of age. Maximum latency to fall on the third testing day is reported. (B)Detail of trial results at age 7 months. Individual trials on each of the three testing days are shown.

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WHAT THIS MEANS FOR THERAPY

PPT1 injected into the cerebrospinal fluid at the lumbar area of mice (intrathecal injection) prevented decline in motor function and improved survival of mice with the CLN1 form of Batten Disease. The treatment was only given at one point in time but had a measurable effect. The effect was similar to the effect seen for CLN2 enzyme in the CLN2 mouse model. CLN2 enzyme (TPP1) is currently being administered to late infantile Batten children through the cerebrospinal fluid. If this approach is helpful in CLN2 disease, our results suggest that the same approach would be helpful in CLN1 disease.

Fig. 3. Effect of IT ERT on disease-specific (A) and overall (B) survival. Both disease-specific (p<0.001) and overall survival (p<0.001) were improved. Higher doses were more effective. Disease-specific mean survivals were 238, 233, 267, 272, and 284 days for control (uninjected), 0, 2.6, 5,3, 10.6 mg/ml PPT1 injected groups, respectively.

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Fig. 6. PPT1 can be found in the brain of the Ppt1 knockout mouse cerebellum at varying times after intrathecal injection. (A) Ppt1 knockout mouse, age 6 weeks, injected with aCSF (B-D) Ppt1 knockout mice, age 6 weeks, injected with 10 .6 mg/ml PPT1 and sacrificed at 1 day (B), 3 weeks (C) or 5 months (D) after the injection. (E) Wild-type control mouse brain. Brown staining indicates PPT1 immunostaining. Bar, 20 microns.

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Fig. 4 and 5. IT ERT improves brain and spinal cord pathology. Fig. 4 shows the microglial marker CD68 from three randomly selected 7 month-old mice from each group indicated and Fig. 5 shows autofluorescent storage material. Different areas of the brain and spinal cord are shown. (A) S1 barrel field cortex (B) VPM/VPL; (C) LGnD; (D) Dorsal cervical spinal cord (E) Ventral cervical spinal cord (F) Dorsal lumbar spinal cord (G) Ventral lumbar spinal cord.

Fig. 1. Experimental design. Four groups of 16-20 Ppt1 knockout mice at six weeks of age were randomized to receive three consecutive daily lumbar spinal infusions of human recombinant PPT1 in 80 µl over 8 minutes at concentrations of 0, 2.6, 5.3 or 10.6 mg/ml. Rotarod testing was performed by a blinded observer at 3, 5, 6, 7, 8 and 9 months of age. Three mice from each group were sacrificed at seven months for pathological examination. Unmanipulated groups of wild-type and Ppt1 knockout mice (n=15) were tested for comparison.

Acknowledgements: The authors wish to thank Drs. Peter Lobel and David Sleat for advice on the experimental design, Dr. James Richardson and John Shelton of the UT Southwestern Molecular Pathology Core for PPT1 immunostaining and microscopy, and Lauren Peca for performing the behavioral tests. This work was funded by Taylor’s Tale, the Batten Disease Support and Research Association, the Batten Disease Family Association, and a King’s College London Graduate School International Studentship award to HRN.