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. 2015 Nov 11;7(313):313ra180.
doi: 10.1126/scitranslmed.aac6191.

AAV gene transfer delays disease onset in a TPP1-deficient canine model of the late infantile form of Batten disease

Martin L Katz  1 , Luis Tecedor  2 , Yonghong Chen  2 , Baye G Williamson  3 , Elena Lysenko  2 , Fred A Wininger  3 , Whitney M Young  1 , Gayle C Johnson  4 , Rebecca E H Whiting  3 , Joan R Coates  3 , Beverly L Davidson  5
Affiliations

AAV gene transfer delays disease onset in a TPP1-deficient canine model of the late infantile form of Batten disease

Martin L Katz et al. Sci Transl Med. .

Abstract

The most common form of the childhood neurodegenerative disease late infantile neuronal ceroid lipofuscinosis (also called Batten disease) is caused by deficiency of the soluble lysosomal enzyme tripeptidyl peptidase 1 (TPP1) resulting from mutations in the TPP1 gene. We tested whether TPP1 gene transfer to the ependyma, the epithelial lining of the brain ventricular system, in TPP1-deficient dogs would be therapeutically beneficial. A one-time administration of recombinant adeno-associated virus (rAAV) expressing canine TPP1 (rAAV.caTPP1) resulted in high expression of TPP1 predominantly in ependymal cells and secretion of the enzyme into the cerebrospinal fluid leading to clinical benefit. Diseased dogs treated with rAAV.caTPP1 showed delays in onset of clinical signs and disease progression, protection from cognitive decline, and extension of life span. By immunostaining and enzyme assay, recombinant protein was evident throughout the brain and spinal cord, with correction of the neuropathology characteristic of the disease. This study in a naturally occurring canine model of TPP1 deficiency highlights the utility of AAV transduction of ventricular lining cells to accomplish stable secretion of recombinant protein for broad distribution in the central nervous system and therapeutic benefit.

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Conflict of interest statement

Competing interests: B.L.D. is a cofounder of Spark Therapeutics Inc., a gene therapy company. B.L.D. is on the scientific advisory board of Sarepta Therapeutics and Marina Biotech and consults for Wave Life Biosciences.

Figures

Fig. 1
Fig. 1. TPP1 and TPP1-NAbs in CSF after rAAV.caTPP1 infusion
(A, C, E, and E′) An increase in TPP1 in CSF was measured with an enzyme activity assay (see also table S2). (B, D, F, and F′) NAbs in CSF were determined by a neutralization assay: TPP1-deficient mouse embryonic fibroblasts were exposed to a mixture of canine TPP1 plus dog CSF collected before and after treatment. Data were normalized to baseline (pretreatment) values. (A and B) TPP1 activity and NAbs in two dogs (DC013 and DC014) given mycophenolate mofetil treatment starting at day 44 relative to vector infusion (see table S1 for information on vector dose and drug treatment for each dog). (C and D) TPP1 activity and NAbs in two dogs (DC015 and DC016) given mycophenolate mofetil starting at day 33 relative to vector infusion. (E, E′, F, and F′) TPP1 activity and NAbs in three dogs (DC018, DC019, and DC020) given mycophenolate mofetil starting at day −5 relative to vector infusion. Vector infusions were at about 90 days of age in all cases. *TPP1 in CSF from DC019 was 0.3 pmol/mg protein or 100% of normal. **CSF from DC020 showed no detectable TPP1 uptake at this time point, indicating high levels of NAbs.
Fig. 2
Fig. 2. rAAV.caTPP1 delivery delays disease onset in TPP1-deficient dogs
(A) Onset of proprioceptive response deficits in treated versus untreated TPP1-deficient dogs. (B and C) Onset of pathological nystagmus and menace response deficits in treated versus untreated TPP1-deficient dogs. (D) Onset of pupillary light reflex abnormalities in rAAV. caTPP1-treated TPP1-deficient dogs versus untreated dogs. (E) Onset of cerebellar ataxia in rAAV.caTPP1-treated TPP1-deficient dogs versus untreated dogs. (F) Onset of proprioceptive response deficits in the forelimbs of treated versus untreated TPP1-deficient dogs. (G) Intention tremor onset in rAAV.caTPP1-treated dogs compared to untreated TPP1-deficient dogs. *P < 0.05, nonparametric Mann-Whitney test (see also table S2).
Fig. 3
Fig. 3. rAAV.caTPP1 treatment delays onset of cognitive deficits and increases life span of TPP1-deficient dogs
(A) Cognitive deficits were assessed by the T-maze (18) in rAAV.caTPP1-treated dogs (DC019 and DC020) and untreated dogs (DC024 and DC025) from 4 months (1 month after injection) onward (see also table S2). The asterisk denotes the last time point that dogs were capable of completing the T-maze because of motor impairment or behavioral problems. (B) Survival plot of untreated TPP1-deficient dogs (solid line) and rAAV.caTPP1-treated TPP1-deficient dogs (dashed and dotted lines). Mycophenolate mofetil treatment was initiated 33 days after vector delivery (dashed line) or 5 days before vector delivery (dotted line).
Fig. 4
Fig. 4. TPP1 activity in brain parenchyma of TPP1-deficient dogs
TPP1 activity at the experimental end points taken from punches from the indicated regions (see also table S2). (A) TPP1 activity in ependyma. wt, wild type. (B) TPP1 activity in periventricular or meningeal regions. TPP1 activity is relative to that found in normal dogs (see fig. S2). Open bars, dogs DC013 and DC014, which received mycophenolate mofetil treatment starting at day 44 relative to vector infusion; gray bars, dogs DC015 and DC016, which received mycophenolate mofetil treatment starting at day 33 relative to vector infusion; dark gray bars, dogs DC018, DC019, and DC020, which received mycophenolate mofetil treatment starting at day −5 relative to vector infusion; black bars, normal dogs.
Fig. 5
Fig. 5. Biodistribution of TPP1 in rAAV.caTPP1-treated TPP1-deficient dogs
Representative immunohistochemical staining for canine TPP1 in treated versus untreated TPP1-deficient dogs. (A) TPP1-immunopositive cells in the septum (Spt) and caudate nucleus (Caud) in the forebrain, hypothalamus (HT) at the level of the third ventricle, and the vestibular area (VA) near the fourth ventricle. Scale bar, 100 μm. (B) TPP1-immunopositive cells near the central canal and neurons along the spinal cord: cervical 3 (C3), thoracic 5 (T5), and lumbar 7 (L7). Scale bar, 400 μm. (C) TPP1-immunopositive cells spanning caudal to rostral regions. pfCx, prefrontal cortex; cgCX, cingulate cortex; tmpCx, temporal cortex; pirCx, piriform cortex; occCx, occipital cortex; CbCx, cerebellar cortex. Scale bar, 100 μm. (Insets) High-magnification images of TPP1-positive cells demonstrate cellular morphology and the punctate staining typical of lysosomal localization.
Fig. 6
Fig. 6. rAAV.caTPP1 treatment reduces astrocytic activation
Glial astrocytosis measured by immunoreactivity for GFAP in treated versus untreated TPP1-deficient dogs. Representative photomicrographs from the caudate, septum, motor cortex (mCx), and cingulate cortex (cgCx) of untreated (n = 3) and rAAV.caTPP1-treated dogs (n = 5). GFAP staining of an untreated wt dog is shown for reference (n = 1). Scale bars, 500 μm for caudate and septum and 1 mm for mCx and cgCx photomicrographs.
Fig. 7
Fig. 7. rAAV.caTPP1 treatment reduces storage burden
(A) Representative images of autofluorescent material in layer II/III of the occipital cortex (top panels) or spinal cord (layer C3, bottom panels) observed by confocal microscopy. Scale bar, 20 μm. (B) Immunolabeling for subunit C of mitochondrial ATP synthase (SCMAS) accumulation in rAAV.caTPP1-treated versus untreated TPP1-deficient dogs. Scale bar, 100 μm. (C) p62 and SCMAS quantified by Western blot in tissue lysates harvested from thalamus, cerebellum, or occipital cortex. *P < 0.05; **P < 0.01 by nonparametric Mann-Whitney test of treated (n = 6) versus untreated (n = 6) TPP1-deficient dogs.
Fig. 8
Fig. 8. TPP1 activity in peripheral organs
TPP1 activity in homogenized samples of peripheral tissues (spleen, heart, liver, and kidney) of untreated or rAAV.caTPP1-treated TPP1-deficient dogs or normal healthy dogs (see table S2). Nonparametric Kruskal-Wallis (P < 0.01) test with Dunn’s post hoc test (*P < 0.05).
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