A mouse mutant deficient in both neuronal ceroid lipofuscinosis-associated proteins CLN3 and TPP1.

Late-infantile neuronal ceroid lipofuscinosis (LINCL) and juvenile neuronal ceroid lipofuscinosis (JNCL) are inherited neurodegenerative diseases caused by mutations in the genes encoding lysosomal proteins tripeptidyl peptidase 1 (TPP1) and CLN3 protein, respectively. TPP1 is well-understood and, aided by animal models that accurately recapitulate the human disease, enzyme replacement therapy has been approved and other promising therapies are emerging. In contrast, there are no effective treatments for JNCL, partly because the function of the CLN3 protein remains unknown but also because animal models have attenuated disease and lack robust survival phenotypes. Mouse models for LINCL and JNCL, with mutations in Tpp1 and Cln3, respectively, have been thoroughly characterized but the phenotype of a double Cln3/Tpp1 mutant remains unknown. We created this double mutant and find that its phenotype is essentially indistinguishable from the single Tpp1-/- mutant in terms of survival and brain pathology. Analysis of brain proteomic changes in the single Tpp1-/- and double Cln3-/- ;Tpp1-/- mutants indicates largely overlapping sets of altered proteins and reinforces earlier studies that highlight GPNMB, LYZ2, and SERPINA3 as promising biomarker candidates in LINCL while several lysosomal proteins including SMPD1 and NPC1 appear to be altered in the Cln3-/- animals. An unexpected finding was that Tpp1 heterozygosity significantly decreased lifespan of the Cln3-/- mouse. The truncated survival of this mouse model makes it potentially useful in developing therapies for JNCL using survival as an endpoint. In addition, this model may also provide insights into CLN3 protein function and its potential functional interactions with TPP1.

Tralesinidase Alfa Enzyme Replacement Therapy Prevents Disease Manifestations in a Canine Model of Mucopolysaccharidosis Type IIIB.

Mucopolysaccharidosis type IIIB (MPS IIIB; Sanfilippo syndrome B; OMIM #252920) is a lethal, pediatric, neuropathic, autosomal recessive, and lysosomal storage disease with no approved therapy. Patients are deficient in the activity of N-acetyl-alpha-glucosaminidase (NAGLU; EC 3.2.150), necessary for normal lysosomal degradation of the glycosaminoglycan heparan sulfate (HS). Tralesinidase alfa (TA), a fusion protein comprised of recombinant human NAGLU and a modified human insulin-like growth factor 2, is in development as an enzyme replacement therapy that is administered via intracerebroventricular (ICV) infusion, thus circumventing the blood brain barrier. Previous studies have confirmed ICV infusion results in widespread distribution of TA throughout the brains of mice and nonhuman primates. We assessed the long-term tolerability, pharmacology, and clinical efficacy of TA in a canine model of MPS IIIB over a 20-month study. Long-term administration of TA was well tolerated as compared with administration of vehicle. TA was widely distributed across brain regions, which was confirmed in a follow-up 8-week pharmacokinetic/pharmacodynamic study. MPS IIIB dogs treated for up to 20 months had near-normal levels of HS and nonreducing ends of HS in cerebrospinal fluid and central nervous system (CNS) tissues. TA-treated MPS IIIB dogs performed better on cognitive tests and had improved CNS pathology and decreased cerebellar volume loss relative to vehicle-treated MPS IIIB dogs. These findings demonstrate the ability of TA to prevent or limit the biochemical, pathologic, and cognitive manifestations of canine MPS IIIB disease, thus providing support of its potential long-term tolerability and efficacy in MPS IIIB subjects. SIGNIFICANCE STATEMENT: This work illustrates the efficacy and tolerability of tralesinidase alfa as a potential therapeutic for patients with mucopolysaccharidosis type IIIB (MPS IIIB) by documenting that administration to the central nervous system of MPS IIIB dogs prevents the accumulation of disease-associated glycosaminoglycans in lysosomes, hepatomegaly, cerebellar atrophy, and cognitive decline.

Neuropathology of murine Sanfilippo D syndrome.

Sanfilippo D syndrome (mucopolysaccharidosis type IIID) is a lysosomal storage disorder caused by the deficiency of N-acetylglucosamine-6-sulfatase (GNS). A mouse model was generated by constitutive knockout of the Gns gene. We studied affected mice and controls at 12, 24, 36, and 48 weeks of age for neuropathological markers of disease in the somatosensory cortex, primary motor cortex, ventral posterior nuclei of the thalamus, striatum, hippocampus, and lateral and medial entorhinal cortex. We found significantly increased immunostaining for glial fibrillary associated protein (GFAP), CD68 (a marker of activated microglia), and lysosomal-associated membrane protein-1 (LAMP-1) in Sanfilippo D mice compared to controls at 12 weeks of age in all brain regions. Intergroup differences were marked for GFAP and CD68 staining, with levels in Sanfilippo D mice consistently above controls at all age groups. Intergroup differences in LAMP-1 staining were more pronounced in 12- and 24-week age groups compared to 36- and 48-week groups, as control animals showed some LAMP-1 staining at later timepoints in some brain regions. We also evaluated the somatosensory cortex, medial entorhinal cortex, reticular nucleus of the thalamus, medial amygdala, and hippocampal hilus for subunit c of mitochondrial ATP synthase (SCMAS). We found a progressive accumulation of SCMAS in most brain regions of Sanfilippo D mice compared to controls by 24 weeks of age. Cataloging the regional neuropathology of Sanfilippo D mice may aid in understanding the disease pathogenesis and designing preclinical studies to test brain-directed treatments.

Biochemical evaluation of intracerebroventricular rhNAGLU-IGF2 enzyme replacement therapy in neonatal mice with Sanfilippo B syndrome.

Mucopolysaccharidosis IIIB (MPS IIIB, Sanfilippo syndrome type B) is caused by a deficiency in α-N-acetylglucosaminidase (NAGLU) activity, which leads to the accumulation of heparan sulfate (HS). MPS IIIB causes progressive neurological decline, with affected patients having an expected lifespan of approximately 20 years. No effective treatment is available. Recent pre-clinical studies have shown that intracerebroventricular (ICV) ERT with a fusion protein of rhNAGLU-IGF2 is a feasible treatment for MPS IIIB in both canine and mouse models. In this study, we evaluated the biochemical efficacy of a single dose of rhNAGLU-IGF2 via ICV-ERT in brain and liver tissue from Naglu-/- neonatal mice. Twelve weeks after treatment, NAGLU activity levels in brain were 0.75-fold those of controls. HS and ß-hexosaminidase activity, which are elevated in MPS IIIB, decreased to normal levels. This effect persisted for at least 4 weeks after treatment. Elevated NAGLU and reduced ß-hexosaminidase activity levels were detected in liver; these effects persisted for up to 4 weeks after treatment. The overall therapeutic effects of single dose ICV-ERT with rhNAGLU-IGF2 in Naglu-/- neonatal mice were long-lasting. These results suggest a potential benefit of early treatment, followed by less-frequent ICV-ERT dosing, in patients diagnosed with MPS IIIB.

Intravenous administration of scAAV9-Hexb normalizes lifespan and prevents pathology in Sandhoff disease mice.

Sandhoff disease (SD) is a rare inherited disorder caused by a deficiency of ß-hexosaminidase activity which is fatal because no effective treatment is available. A mouse model of Hexb deficiency reproduces the key pathognomonic features of SD patients with severe ubiquitous lysosomal dysfunction, GM2 accumulation, neuroinflammation and neurodegeneration, culminating in death at 4 months. Here, we show that a single intravenous neonatal administration of a self-complementary adeno-associated virus 9 vector (scAAV9) expressing the Hexb cDNA in SD mice is safe and sufficient to prevent disease development. Importantly, we demonstrate for the first time that this treatment results in a normal lifespan (over 700 days) and normalizes motor function assessed by a battery of behavioral tests, with scAAV9-treated SD mice being indistinguishable from wild-type littermates. Biochemical analyses in multiple tissues showed a significant increase in hexosaminidase A activity, which reached 10-15% of normal levels. AAV9 treatment was sufficient to prevent GM2 and GA2 storage almost completely in the cerebrum (less so in the cerebellum), as well as thalamic reactive gliosis and thalamocortical neuron loss in treated Hexb-/- mice. In summary, this study demonstrated a widespread protective effect throughout the entire CNS after a single intravenous administration of the scAAV9-Hexb vector to neonatal SD mice.

Synergistic effects of treating the spinal cord and brain in CLN1 disease.

Infantile neuronal ceroid lipofuscinosis (INCL, or CLN1 disease) is an inherited neurodegenerative storage disorder caused by a deficiency of the lysosomal enzyme palmitoyl protein thioesterase 1 (PPT1). It was widely believed that the pathology associated with INCL was limited to the brain, but we have now found unexpectedly profound pathology in the human INCL spinal cord. Similar pathological changes also occur at every level of the spinal cord of PPT1-deficient (Ppt1-/- ) mice before the onset of neuropathology in the brain. Various forebrain-directed gene therapy approaches have only had limited success in Ppt1-/- mice. Targeting the spinal cord via intrathecal administration of an adeno-associated virus (AAV) gene transfer vector significantly prevented pathology and produced significant improvements in life span and motor function in Ppt1-/- mice. Surprisingly, forebrain-directed gene therapy resulted in essentially no PPT1 activity in the spinal cord, and vice versa. This leads to a reciprocal pattern of histological correction in the respective tissues when comparing intracranial with intrathecal injections. However, the characteristic pathological features of INCL were almost completely absent in both the brain and spinal cord when intracranial and intrathecal injections of the same AAV vector were combined. Targeting both the brain and spinal cord also produced dramatic and synergistic improvements in motor function with an unprecedented increase in life span. These data show that spinal cord pathology significantly contributes to the clinical progression of INCL and can be effectively targeted therapeutically. This has important implications for the delivery of therapies in INCL, and potentially in other similar disorders.

Partial rescue of neuropathology in the murine model of PKU following administration of recombinant phenylalanine ammonia lyase (pegvaliase).

Pegylated recombinant phenylalanine ammonia lyase (pegvaliase) is an enzyme substitution therapy being evaluated for the treatment of phenylketonuria (PKU). PKU is characterized by elevated plasma phenylalanine, which is thought to lead to a deficiency in monoamine neurotransmitters and ultimately, neurocognitive dysfunction. A natural history evaluation in a mouse model of PKU demonstrated a profound decrease in tyrosine hydroxylase (TH) immunoreactivity in several brain regions, beginning at 4weeks of age. Following treatment with pegvaliase, the number of TH positive neurons was increased in several brain regions compared to placebo treated ENU2 mice.

Efficacy of phosphodiesterase-4 inhibitors in juvenile Batten disease (CLN3).

OBJECTIVE: Juvenile neuronal ceroid lipofuscinosis (JNCL), or juvenile Batten disease, is a pediatric lysosomal storage disease caused by autosomal recessive mutations in CLN3, typified by blindness, seizures, progressive cognitive and motor decline, and premature death. Currently, there is no treatment for JNCL that slows disease progression, which highlights the need to explore novel strategies to extend the survival and quality of life of afflicted children. Cyclic adenosine monophosphate (cAMP) is a second messenger with pleiotropic effects, including regulating neuroinflammation and neuronal survival. Here we investigated whether 3 phosphodiesterase-4 (PDE4) inhibitors (rolipram, roflumilast, and PF-06266047) could mitigate behavioral deficits and cell-specific pathology in the Cln3Δex7/8 mouse model of JNCL. METHODS: In a randomized, blinded study, wild-type (WT) and Cln3Δex7/8 mice received PDE4 inhibitors daily beginning at 1 or 3 months of age and continuing for 6 to 9 months, with motor deficits assessed by accelerating rotarod testing. The effect of PDE4 inhibitors on cAMP levels, astrocyte and microglial activation (glial fibrillary acidic protein and CD68, respectively), lysosomal pathology (lysosomal-associated membrane protein 1), and astrocyte glutamate transporter expression (glutamate/aspartate transporter) were also examined in WT and Cln3Δex7/8 animals. RESULTS: cAMP levels were significantly reduced in the Cln3Δex7/8 brain, and were restored by PF-06266047. PDE4 inhibitors significantly improved motor function in Cln3Δex7/8 mice, attenuated glial activation and lysosomal pathology, and restored glutamate transporter expression to levels observed in WT animals, with no evidence of toxicity as revealed by blood chemistry analysis. INTERPRETATION: These studies reveal neuroprotective effects for PDE4 inhibitors in Cln3Δex7/8 mice and support their therapeutic potential in JNCL patients. Ann Neurol 2016;80:909-923.

Evolution of structural abnormalities in the rat brain following in utero exposure to maternal immune activation: A longitudinal in vivo MRI study.

Genetic and environmental risk factors for psychiatric disorders are suggested to disrupt the trajectory of brain maturation during adolescence, leading to the development of psychopathology in adulthood. Rodent models are powerful tools to dissect the specific effects of such risk factors on brain maturational profiles, particularly when combined with Magnetic Resonance Imaging (MRI; clinically comparable technology). We therefore investigated the effect of maternal immune activation (MIA), an epidemiological risk factor for adult-onset psychiatric disorders, on rat brain maturation using atlas and tensor-based morphometry analysis of longitudinal in vivo MR images. Exposure to MIA resulted in decreases in the volume of several cortical regions, the hippocampus, amygdala, striatum, nucleus accumbens and unexpectedly, the lateral ventricles, relative to controls. In contrast, the volumes of the thalamus, ventral mesencephalon, brain stem and major white matter tracts were larger, relative to controls. These volumetric changes were maximal between post-natal day 50 and 100 with no differences between the groups thereafter. These data are consistent with and extend prior studies of brain structure in MIA-exposed rodents. Apart from the ventricular findings, these data have robust face validity to clinical imaging findings reported in studies of individuals at high clinical risk for a psychiatric disorder. Further work is now required to address the relationship of these MRI changes to behavioral dysfunction and to establish thier cellular correlates.

Towards a new understanding of NCL pathogenesis.

The Neuronal Ceroid Lipofuscinoses (NCLs, Batten disease) are a group of inherited neurodegenerative disorders that have been traditionally grouped together on the basis of certain shared clinical and pathological features. However, as the number of genes that appear to cause new forms of NCL continues to grow, it is timely to reassess our understanding of the pathogenesis of these disorders and what groups them together. The various NCL subtypes do indeed share features of a build-up of autofluorescent storage material, progressive neuron loss and activation of the innate immune system. The characterisation of animal models has highlighted the selective nature of neuron loss and its intimate relationship with glial activation, rather than the generalised build-up of storage material. More recent data provide evidence for the pathway-dependent nature of pathology, the contribution of glial dysfunction, and the involvement of new brain regions previously thought to be unaffected, and it is becoming apparent that pathology extends beyond the brain. These data have important implications, not just for therapy, but also for our understanding of these disorders. However, looking beneath these broadly similar pathological themes evidence emerges for marked differences in the nature and extent of these events in different forms of NCL. Indeed, given the widely different nature of the mutated gene products it is perhaps more surprising that these disorders resemble each other as much as they do. Such data raise the question whether we should rethink the collective grouping of these gene deficiencies together, or whether it would be better to consider them as separate entities. This article is part of a Special Issue entitled: Current Research on the Neuronal Ceroid Lipofuscinoses (Batten Disease).

Guidelines for incorporating scientific knowledge and practice on rare diseases into higher education: neuronal ceroid lipofuscinoses as a model disorder.

This article addresses the educational issues associated with rare diseases (RD) and in particular the Neuronal Ceroid Lipofuscinoses (NCLs, or CLN diseases) in the curricula of Health Sciences and Professional's Training Programs. Our aim is to develop guidelines for improving scientific knowledge and practice in higher education and continuous learning programs. Rare diseases (RD) are collectively common in the general population with 1 in 17 people affected by a RD in their lifetime. Inherited defects in genes involved in metabolism are the commonest group of RD with over 8000 known inborn errors of metabolism. The majority of these diseases are neurodegenerative including the NCLs. Any professional training program on NCL must take into account the medical, social and economic burdens related to RDs. To address these challenges and find solutions to them it is necessary that individuals in the government and administrative authorities, academia, teaching hospitals and medical schools, the pharmaceutical industry, investment community and patient advocacy groups all work together to achieve these goals. The logistical issues of including RD lectures in university curricula and in continuing medical education should reflect its complex nature. To evaluate the state of education in the RD field, a summary should be periodically up dated in order to assess the progress achieved in each country that signed up to the international conventions addressing RD issues in society. It is anticipated that auditing current practice will lead to higher standards and provide a framework for those educators involved in establishing RD teaching programs world-wide.

Diagnosis of neuronal ceroid lipofuscinosis type 2 (CLN2 disease): Expert recommendations for early detection and laboratory diagnosis.

Neuronal ceroid lipofuscinoses (NCLs) are a heterogeneous group of lysosomal storage disorders. NCLs include the rare autosomal recessive neurodegenerative disorder neuronal ceroid lipofuscinosis type 2 (CLN2) disease, caused by mutations in the tripeptidyl peptidase 1 (TPP1)/CLN2 gene and the resulting TPP1 enzyme deficiency. CLN2 disease most commonly presents with seizures and/or ataxia in the late-infantile period (ages 2-4), often in combination with a history of language delay, followed by progressive childhood dementia, motor and visual deterioration, and early death. Atypical phenotypes are characterized by later onset and, in some instances, longer life expectancies. Early diagnosis is important to optimize clinical care and improve outcomes; however, currently, delays in diagnosis are common due to low disease awareness, nonspecific clinical presentation, and limited access to diagnostic testing in some regions. In May 2015, international experts met to recommend best laboratory practices for early diagnosis of CLN2 disease. When clinical signs suggest an NCL, TPP1 enzyme activity should be among the first tests performed (together with the palmitoyl-protein thioesterase enzyme activity assay to rule out CLN1 disease). However, reaching an initial suspicion of an NCL or CLN2 disease can be challenging; thus, use of an epilepsy gene panel for investigation of unexplained seizures in the late-infantile/childhood ages is encouraged. To confirm clinical suspicion of CLN2 disease, the recommended gold standard for laboratory diagnosis is demonstration of deficient TPP1 enzyme activity (in leukocytes, fibroblasts, or dried blood spots) and the identification of causative mutations in each allele of the TPP1/CLN2 gene. When it is not possible to perform both analyses, either demonstration of a) deficient TPP1 enzyme activity in leukocytes or fibroblasts, or b) detection of two pathogenic mutations in trans is diagnostic for CLN2 disease.

Origin of terminal voltage variations due to self-mixing in terahertz frequency quantum cascade lasers.

We explain the origin of voltage variations due to self-mixing in a terahertz (THz) frequency quantum cascade laser (QCL) using an extended density matrix (DM) approach. Our DM model allows calculation of both the current-voltage (I-V) and optical power characteristics of the QCL under optical feedback by changing the cavity loss, to which the gain of the active region is clamped. The variation of intra-cavity field strength necessary to achieve gain clamping, and the corresponding change in bias required to maintain a constant current density through the heterostructure is then calculated. Strong enhancement of the self-mixing voltage signal due to non-linearity of the (I-V) characteristics is predicted and confirmed experimentally in an exemplar 2.6 THz bound-to-continuum QCL.

Systemic gene delivery following intravenous administration of AAV9 to fetal and neonatal mice and late-gestation nonhuman primates.

Several acute monogenic diseases affect multiple body systems, causing death in childhood. The development of novel therapies for such conditions is challenging. However, improvements in gene delivery technology mean that gene therapy has the potential to treat such disorders. We evaluated the ability of the AAV9 vector to mediate systemic gene delivery after intravenous administration to perinatal mice and late-gestation nonhuman primates (NHPs). Titer-matched single-stranded (ss) and self-complementary (sc) AAV9 carrying the green fluorescent protein (GFP) reporter gene were intravenously administered to fetal and neonatal mice, with noninjected age-matched mice used as the control. Extensive GFP expression was observed in organs throughout the body, with the epithelial and muscle cells being particularly well transduced. ssAAV9 carrying the WPRE sequence mediated significantly more gene expression than its sc counterpart, which lacked the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) sequence. To examine a realistic scale-up to larger models or potentially patients for such an approach, AAV9 was intravenously administered to late-gestation NHPs by using a clinically relevant protocol. Widespread systemic gene expression was measured throughout the body, with cellular tropisms similar to those observed in the mouse studies and no observable adverse events. This study confirms that AAV9 can safely mediate systemic gene delivery in small and large animal models and supports its potential use in clinical systemic gene therapy protocols.

An anti-neuroinflammatory that targets dysregulated glia enhances the efficacy of CNS-directed gene therapy in murine infantile neuronal ceroid lipofuscinosis.

Infantile neuronal ceroid lipofuscinosis (INCL) is an inherited neurodegenerative lysosomal storage disease (LSD) caused by a deficiency in palmitoyl protein thioesterase-1 (PPT1). Studies in Ppt1(-/-) mice demonstrate that glial activation is central to the pathogenesis of INCL. Astrocyte activation precedes neuronal loss, while cytokine upregulation associated with microglial reactivity occurs before and concurrent with neurodegeneration. Therefore, we hypothesized that cytokine cascades associated with neuroinflammation are important therapeutic targets for the treatment of INCL. MW01-2-151SRM (MW151) is a blood-brain barrier penetrant, small-molecule anti-neuroinflammatory that attenuates glial cytokine upregulation in models of neuroinflammation such as traumatic brain injury, Alzheimer's disease, and kainic acid toxicity. Thus, we used MW151, alone and in combination with CNS-directed, AAV-mediated gene therapy, as a possible treatment for INCL. MW151 alone decreased seizure susceptibility. When combined with AAV-mediated gene therapy, treated INCL mice had increased life spans, improved motor performance, and eradication of seizures. Combination-treated INCL mice also had decreased brain atrophy, astrocytosis, and microglial activation, as well as intermediary effects on cytokine upregulation. These data suggest that MW151 can attenuate seizure susceptibility but is most effective when used in conjunction with a therapy that targets the primary genetic defect.

Post-assembly Modification of Tetrazine-Edged Fe(II)4L6 Tetrahedra.

Post-assembly modification (PAM) is a powerful tool for the modular functionalization of self-assembled structures. We report a new family of tetrazine-edged Fe(II)4L6 tetrahedral cages, prepared using different aniline subcomponents, which undergo rapid and efficient PAM by inverse electron-demand Diels-Alder (IEDDA) reactions. Remarkably, the electron-donating or -withdrawing ability of the para-substituent on the aniline moiety influences the IEDDA reactivity of the tetrazine ring 11 bonds away. This effect manifests as a linear free energy relationship, quantified using the Hammett equation, between σ(para) and the rate of the IEDDA reaction. The rate of PAM can thus be adjusted by varying the aniline subcomponent.

Cathepsin F mutations cause Type B Kufs disease, an adult-onset neuronal ceroid lipofuscinosis.

Kufs disease, an adult-onset neuronal ceroid lipofuscinosis, is challenging to diagnose and genetically heterogeneous. Mutations in CLN6 were recently identified in recessive Kufs disease presenting as progressive myoclonus epilepsy (Type A), whereas the molecular basis of cases presenting with dementia and motor features (Type B) is unknown. We performed genome-wide linkage mapping of two families with recessive Type B Kufs disease and identified a single region on chromosome 11 to which both families showed linkage. Exome sequencing of five samples from the two families identified homozygous and compound heterozygous missense mutations in CTSF within this linkage region. We subsequently sequenced CTSF in 22 unrelated individuals with suspected recessive Kufs disease, and identified an additional patient with compound heterozygous mutations. CTSF encodes cathepsin F, a lysosomal cysteine protease, dysfunction of which is a highly plausible candidate mechanism for a storage disorder like ceroid lipofuscinosis. In silico modeling suggested the missense mutations would alter protein structure and function. Moreover, re-examination of a previously published mouse knockout of Ctsf shows that it recapitulates the light and electron-microscopic pathological features of Kufs disease. Although CTSF mutations account for a minority of cases of type B Kufs, CTSF screening should be considered in cases with early-onset dementia and may avoid the need for invasive biopsies.

Intrathecal enzyme replacement therapy improves motor function and survival in a preclinical mouse model of infantile neuronal ceroid lipofuscinosis.

The neuronal ceroid lipofuscinoses (NCLs) are a group of related hereditary lysosomal storage disorders characterized by progressive loss of neurons in the central nervous system resulting in dementia, loss of motor skills, seizures and blindness. A characteristic intralysosomal accumulation of autofluorescent storage material occurs in the brain and other tissues. Three major forms and nearly a dozen minor forms of NCL are recognized. Infantile-onset NCL (CLN1 disease) is caused by severe deficiency in a soluble lysosomal enzyme, palmitoyl-protein thioesterase-1 (PPT1) and no therapy beyond supportive care is available. Homozygous Ppt1 knockout mice reproduce the known features of the disease, developing signs of motor dysfunction at 5 months of age and death around 8 months. Direct delivery of lysosomal enzymes to the cerebrospinal fluid is an approach that has gained traction in small and large animal models of several other neuropathic lysosomal storage diseases, and has advanced to clinical trials. In the current study, Ppt1 knockout mice were treated with purified recombinant human PPT1 enzyme delivered to the lumbar intrathecal space on each of three consecutive days at 6 weeks of age. Untreated PPT1 knockout mice and wild-type mice served as additional controls. Four enzyme concentration levels (0, 2.6, 5.3 and 10.6 mg/ml of specific activity 20 U/mg) were administered in a volume of 80 µl infused over 8 min. Each group consisted of 16-20 mice. The treatment was well tolerated. Disease-specific survival was 233, 267, 272, and 284days for each of the four treatment groups, respectively, and the effect of treatment was highly significant (p<0.0001). The timing of motor deterioration was also delayed. Neuropathology was improved as evidenced by decreased autofluorescent storage material in the spinal cord and a decrease in CD68 staining in the cortex and spinal cord. The improvements in motor function and survival are similar to results reported for preclinical studies involving other lysosomal storage disorders, such as CLN2/TPP1 deficiency, for which intraventricular ERT is being offered in clinical trials. If ERT delivery to the CSF proves to be efficacious in these disorders, PPT1 deficiency may also be amenable to this approach.

Nonclinical evaluation of CNS-administered TPP1 enzyme replacement in canine CLN2 neuronal ceroid lipofuscinosis.

The CLN2 form of neuronal ceroid lipofuscinosis, a type of Batten disease, is a lysosomal storage disorder caused by a deficiency of the enzyme tripeptidyl peptidase-1 (TPP1). Patients exhibit progressive neurodegeneration and loss of motor, cognitive, and visual functions, leading to death by the early teenage years. TPP1-null Dachshunds recapitulate human CLN2 disease. To characterize the safety and pharmacology of recombinant human (rh) TPP1 administration to the cerebrospinal fluid (CSF) as a potential enzyme replacement therapy (ERT) for CLN2 disease, TPP1-null and wild-type (WT) Dachshunds were given repeated intracerebroventricular (ICV) infusions and the pharmacokinetic (PK) profile, central nervous system (CNS) distribution, and safety were evaluated. TPP1-null animals and WT controls received 4 or 16mg of rhTPP1 or artificial cerebrospinal fluid (aCSF) vehicle every other week. Elevated CSF TPP1 concentrations were observed for 2-3 days after the first ICV infusion and were approximately 1000-fold higher than plasma levels at the same time points. Anti-rhTPP1 antibodies were detected in CSF and plasma after repeat rhTPP1 administration, with titers generally higher in TPP1-null than in WT animals. Widespread brain distribution of rhTPP1 was observed after chronic administration. Expected histological changes were present due to the CNS delivery catheters and were similar in rhTPP1 and vehicle-treated animals, regardless of genotype. Neuropathological evaluation demonstrated the clearance of lysosomal storage, preservation of neuronal morphology, and reduction in brain inflammation with treatment. This study demonstrates the favorable safety and pharmacology profile of rhTPP1 ERT administered directly to the CNS and supports clinical evaluation in patients with CLN2 disease.

Pathogenesis and therapies for infantile neuronal ceroid lipofuscinosis (infantile CLN1 disease).

The neuronal ceroid lipofuscinoses (NCL, Batten disease) are a group of inherited neurodegenerative diseases. Infantile neuronal ceroid lipofuscinosis (INCL, infantile Batten disease, or infantile CLN1 disease) is caused by a deficiency in the soluble lysosomal enzyme palmitoyl protein thioesterase-1 (PPT1) and has the earliest onset and fastest progression of all the NCLs. Several therapeutic strategies including enzyme replacement, gene therapy, stem cell-mediated therapy, and small molecule drugs have resulted in minimal to modest improvements in the murine model of PPT1-deficiency. However, more recent studies using various combinations of these approaches have shown more promising results; in some instances more than doubling the lifespan of PPT1-deficient mice. These combination therapies that target different pathogenic mechanisms may offer the hope of treating this profoundly neurodegenerative disorder. Similar approaches may be useful when treating other forms of NCL caused by deficiencies in soluble lysosomal proteins. Different therapeutic targets will need to be identified and novel strategies developed in order to effectively treat forms of NCL caused by deficiencies in integral membrane proteins such as juvenile neuronal ceroid lipofuscinosis. Finally, the challenge with all of the NCLs will lie in early diagnosis, improving the efficacy of the treatments, and effectively translating them into the clinic. This article is part of a Special Issue entitled: The Neuronal Ceroid Lipofuscinoses or Batten Disease.