Lack of germline transmission in male mice following a single intravenous administration of AAV5-hFVIII-SQ gene therapy.

Valoctocogene roxaparvovec (AAV5-hFVIII-SQ) is an adeno-associated virus serotype five gene therapy under investigation for the treatment of hemophilia A. Herein, we assessed the potential for germline transmission of AAV5-hFVIII-SQ in mice. Male B6.129S6-Rag2tm1Fwa N12 mice received a single intravenous dose of vehicle or 6 × 1013 vg/kg AAV5-hFVIII-SQ. Vehicle and AAV5-hFVIII-SQ-treated mice were mated with naïve females 4 days after dosing, when the concentration of vector genomes was expected to be at its peak in semen, and 37 days after dosing, when a full spermatogenesis cycle was estimated to be complete. Quantitative PCR was used to evaluate the presence of transgene DNA in liver and testes from F0 males dosed with AAV5-hFVIII-SQ and liver tissue of F1 offspring. Transgene DNA was detected in liver and testes of all F0 males dosed with AAV5-hFVIII-SQ, confirming successful transduction. Importantly, no transgene DNA was detected in any tested F1 offspring derived from F0 males dosed with AAV5-hFVIII-SQ. Using a novel 2-stage statistical model that takes into account the number of males dosed with AAV5-hFVIII-SQ and the number of offspring sired by these males, we estimate that the risk of germline transmission is <5% with a 99.2% confidence level.

Safety Findings of Dosing Gene Therapy Vectors in NHP With Pre-existing or Treatment-Emergent Anti-capsid Antibodies.

Replication-incompetent adeno-associated virus (AAV)-based vectors are nonpathogenic viral particles used to deliver therapeutic genes to treat multiple monogenic disorders. AAVs can elicit immune responses; thus, one challenge in AAV-based gene therapy is the presence of neutralizing antibodies against vector capsids that may prevent transduction of target cells or elicit adverse findings. We present safety findings from two 12-week studies in nonhuman primates (NHPs) with pre-existing or treatment-emergent antibodies. In the first study, NHPs with varying levels of naturally acquired anti-AAV5 antibodies were dosed with an AAV5-based vector encoding human factor VIII (hFVIII). In the second study, NHPs with no pre-existing anti-AAV antibodies were dosed with an AAV5-based vector carrying the beta subunit of choriogonadotropic hormone (bCG); this led to the induction of high-titer antibodies against the AAV5 capsid. Four weeks later, the same NHPs received an equivalent dose of an AAV5-based vector carrying human factor IX (hFIX). In both of these studies, the administration of vectors carrying hFVIII, bCG, and hFIX was well-tolerated in NHPs with no adverse clinical pathology or microscopic findings. These two studies demonstrate the safety of AAV-based vector administration in NHPs with either low-titer pre-existing anti-AAV5 antibodies or re-administration, even in the presence of high-titer antibodies.

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.

Intravitreal enzyme replacement inhibits progression of retinal degeneration in canine CLN2 neuronal ceroid lipofuscinosis.

CLN2 neuronal ceroid lipofuscinosis is a rare recessive hereditary retinal and neurodegenerative disease resulting from deleterious sequence variants in TPP1 that encodes the soluble lysosomal enzyme tripeptidyl peptidase-1 (TPP1). Children with this disorder develop normally, but starting at 2-4 years of age begin to exhibit neurological signs and visual deficits. Vision loss that progresses to blindness is associated with progressive retinal degeneration and impairment of retinal function. Similar progressive loss of retinal function and retinal degeneration occur in a dog CLN2 disease model with a TPP1 null sequence variant. Studies using the dog model were conducted to determine whether intravitreal injection of recombinant human TPP1 (rhTPP1) administered starting after onset of retinal functional impairment could slow or halt the progression of retinal functional decline and degeneration. TPP1-null dogs received intravitreal injections of rhTPP1 in one eye and vehicle in the other eye beginning at 23.5-25 weeks of age followed by second injections at 34-40 weeks in 3 out of 4 dogs. Ophthalmic exams, in vivo ocular imaging, and electroretinography (ERG) were repeated regularly to monitor retinal structure and function. Retinal histology was evaluated in eyes collected from these dogs when they were euthanized at end-stage neurological disease (40-45 weeks of age). Intravitreal rhTPP1 injections were effective in preserving retinal function (as measured with the electroretinogram) and retinal morphology for as long as 4 months after a single treatment. These findings indicate that intravitreal injection of rhTPP1 administered after partial loss of retinal function is an effective treatment for preserving retinal structure and function in canine CLN2 disease.

Intravitreal enzyme replacement preserves retinal structure and function in canine CLN2 neuronal ceroid lipofuscinosis.

CLN2 neuronal ceroid lipofuscinosis is a hereditary neurodegenerative disorder characterized by progressive vision loss, neurological decline, and seizures. CLN2 disease results from mutations in TPP1 that encodes the lysosomal enzyme tripeptidyl peptidase-1 (TPP1). Children with CLN2 neuronal ceroid lipofuscinosis experience ocular disease, characterized by progressive retinal degeneration associated with impaired retinal function and gradual vision loss culminating in total blindness. A similar progressive loss of retinal function is also observed in a dog CLN2 model with a TPP1 null mutation. A study was conducted to evaluate the efficacy of periodic intravitreal injections of recombinant human (rh) TPP1 in inhibiting retinal degeneration and preserving retinal function in the canine model. TPP1 null dogs received periodic intravitreal injections of rhTPP1 in one eye and vehicle in the other eye beginning at approximately 12 weeks of age. Ophthalmic exams, in vivo ocular imaging, and electroretinography (ERG) were repeated regularly to monitor retinal structure and function. Retinal histology was evaluated in eyes collected from these dogs when they were euthanized at end-stage neurological disease (43-46 weeks of age). Intravitreal rhTPP1 dosing prevented disease-related declines in ERG amplitudes in the TPP1-treated eyes. At end-stage neurologic disease, TPP1-treated eyes retained normal morphology while the contralateral vehicle-treated eyes exhibited loss of inner retinal neurons and photoreceptor disorganization typical of CLN2 disease. The treatment also prevented the development of disease-related focal retinal detachments observed in the control eyes. Uveitis occurred secondary to the administration of the rhTPP1 but did not hinder the therapeutic benefits. These findings demonstrate that periodic intravitreal injection of rhTPP1 preserves retinal structure and function in canine CLN2 disease.

Ex Vivo Gene Therapy: Graft-versus-host Disease (GVHD) in NSG™ (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) Mice Transplanted with CD34+ Human Hematopoietic Stem Cells.

A therapeutic option for monogenic disorders is gene therapy with ex vivo-transduced autologous hematopoietic stem cells (HSCs). Safety or efficacy studies of ex vivo-modified HSCs are conducted in humanized mouse models after ablation of the murine bone marrow and transfer of human CD34+ HSCs. Engrafted human CD34+ cells migrate to bone marrow and differentiate into various human hematopoietic lineages. A 12-week study was conducted in NSG™ mice to evaluate engraftment, differentiation, and safety of human CD34+ cells that were transduced (ex vivo) with a proprietary lentiviral vector encoding a human gene (BMRN-1) or a mock (green fluorescent protein) vector. Several mice intravenously injected with naive CD34+ cells or transduced CD34+ cells had variable lymphohistiocytic inflammatory cell infiltrates and microgranulomas in the liver and lungs consistent with graft-versus-host disease (GVHD). Spleen, bone marrow, stomach, reproductive tract, but not the skin had similar inflammatory changes. Ex vivo viral transduction of CD34+ cells did not impact engraftment or predispose to xenogeneic GVHD.

Gene Therapy with BMN 270 Results in Therapeutic Levels of FVIII in Mice and Primates and Normalization of Bleeding in Hemophilic Mice.

Hemophilia A is an X-linked bleeding disorder caused by mutations in the gene encoding the factor VIII (FVIII) coagulation protein. Bleeding episodes in patients are reduced by prophylactic therapy or treated acutely using recombinant or plasma-derived FVIII. We have made an adeno-associated virus 5 vector containing a B domain-deleted (BDD) FVIII gene (BMN 270) with a liver-specific promoter. BMN 270 injected into hemophilic mice resulted in a dose-dependent expression of BDD FVIII protein and a corresponding correction of bleeding time and blood loss. At the highest dose tested, complete correction was achieved. Similar corrections in bleeding were observed at approximately the same plasma levels of FVIII protein produced either endogenously by BMN 270 or following exogenous administration of recombinant BDD FVIII. No evidence of liver dysfunction or hepatocyte endoplasmic reticulum stress was observed. Comparable doses in primates produced similar levels of circulating FVIII. These preclinical data support evaluation of BMN 270 in hemophilia A patients.

Immunogenicity to cerliponase alfa intracerebroventricular enzyme replacement therapy for CLN2 disease: Results from a Phase 1/2 study.

Treatment with intracerebroventricular (ICV)-delivered cerliponase alfa enzyme replacement therapy (ERT) in a Phase 1/2 study of 24 subjects with CLN2 disease resulted in a meaningful preservation of motor and language (ML) function and was well tolerated. Treatment was associated with anti-drug antibody (ADA) production in the cerebrospinal fluid (CSF) of 6/24 (25%) and in the serum of 19/24 (79%) of clinical trial subjects, respectively, over a mean exposure of 96.4 weeks (range 0.1-129 weeks). Neutralizing antibodies (NAb) were not detected in the CSF of any of the subjects. No events of anaphylaxis were reported. Neither the presence of serum ADA nor drug-specific immunoglobulin E was associated with the incidence or severity of hypersensitivity adverse events. Serum and CSF ADA titers did not correlate with change in ML score. Therefore, the development of an ADA response to cerliponase alfa is not predictive of an adverse safety profile or poor treatment outcome.

Maternal phenylketonuria syndrome: studies in mice suggest a potential approach to a continuing problem.

BackgroundUntreated phenylketonuria (PKU), one of the most common human genetic disorders, usually results in mental retardation. Although a protein-restricted artificial diet can prevent retardation, dietary compliance in adults is often poor. In pregnant PKU women, noncompliance can result in maternal PKU syndrome, where high phenylalanine (Phe) levels cause severe fetal complications. Enzyme substitution therapy using Phe ammonia lyase (PAL) corrects PKU in BTBR Phe hydroxylase (Pahenu2) mutant mice, suggesting a potential for maternal PKU syndrome treatment in humans.MethodsWe reviewed clinical data to assess maternal PKU syndrome incidence in pregnant PKU women. We treated female PKU mice (on normal diet) with PAL, stabilizing Phe at physiological levels, and mated them to assess pregnancy outcomes.ResultsPatient records show that, unfortunately, the efficacy of diet to prevent maternal PKU syndrome has not significantly improved since the problem was first noted 40 years ago. PAL treatment of pregnant PKU mice shows that offspring of PAL-treated dams survive to adulthood, in contrast to the complete lethality seen in untreated mice, or limited survival seen in mice on a PKU diet.ConclusionPAL treatment reduced maternal PKU syndrome severity in mice and may have potential for human PKU therapy.

Reveglucosidase alfa (BMN 701), an IGF2-Tagged rhAcid α-Glucosidase, Improves Respiratory Functional Parameters in a Murine Model of Pompe Disease.

Pompe disease is a rare neuromuscular disorder caused by an acid α-glucosidase (GAA) deficiency resulting in glycogen accumulation in muscle, leading to myopathy and respiratory weakness. Reveglucosidase alfa (BMN 701) is an insulin-like growth factor 2-tagged recombinant human acid GAA (rhGAA) that enhances rhGAA cellular uptake via a glycosylation-independent insulin-like growth factor 2 binding region of the cation-independent mannose-6-phosphate receptor (CI-MPR). The studies presented here evaluated the effects of Reveglucosidase alfa treatment on glycogen clearance in muscle relative to rhGAA, as well as changes in respiratory function and glycogen clearance in respiratory-related tissue in a Pompe mouse model (GAAtm1Rabn/J). In a comparison of glycogen clearance in muscle with Reveglucosidase alfa and rhGAA, Reveglucosidase alfa was more effective than rhGAA with 2.8-4.7 lower EC50 values, probably owing to increased cellular uptake. The effect of weekly intravenous administration of Reveglucosidase alfa on respiratory function was monitored in Pompe and wild-type mice using whole body plethysmography. Over 12 weeks of 20-mg/kg Reveglucosidase alfa treatment in Pompe mice, peak inspiratory flow (PIF) and peak expiratory flow (PEF) stabilized with no compensation in respiratory rate and inspiratory time during hypercapnic and recovery conditions compared with vehicle-treated Pompe mice. Dose-related decreases in glycogen levels in both ambulatory and respiratory muscles generally correlated to changes in respiratory function. Improvement of murine PIF and PEF were similar in magnitude to increases in maximal inspiratory and expiratory pressure observed clinically in late onset Pompe patients treated with Reveglucosidase alfa (Byrne et al., manuscript in preparation).

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.

Pharmacodynamics, pharmacokinetics and biodistribution of recombinant human N-acetylgalactosamine 4-sulfatase after 6months of therapy in cats using different IV infusion durations.

BACKGROUND: Mucopolysaccharidosis VI (MPS VI) is a lysosomal storage disease characterized by an absence or marked reduction of lysosomal N-acetylgalactosamine-4-sulfatase activity. Affected individuals have widespread accumulation of unmetabolized glycosaminoglycan substrates leading to detrimental effects. Recombinant human N-acetylgalactosamine 4-sulfatase (rhASB) is an approved enzyme replacement therapy for patients with MPS VI. Despite the known efficacy of weekly 4-h rhASB infusions, some clinicians wish to treat patients using reduced infusion times. This study compared the pharmacodynamics, pharmacokinetics, and tissue biodistribution of rhASB when administered as 2- and 4-h intravenous infusions using a feline model of MPS VI. METHODS: Study animals were MPS VI-affected cats that demonstrate clinical signs and biochemical derangements similar to human MPS VI patients. Beginning at age 4weeks, animals received weekly 2-h (N=6) or 4-h (N=6) IV infusions of rhASB for 26weeks (Naglazyme® [galsulfase] Solution for Intravenous Infusion; BioMarin Pharmaceutical, Inc.). The control group consisted of untreated MPS VI-affected cats (N=6). The pharmacokinetic parameters of plasma rhASB and urinary glycosaminoglycan were determined at weeks 13 and 26. Animals were euthanized 48h after the last infusion and tissue concentration of ASB, GAG and ß-glucuronidase were measured in the liver, spleen, aorta, and kidney. Skeletal and ophthalmological evaluations were performed within 2weeks of euthanasia. RESULTS: At week 13, the mean AUC0-t in animals treated with 4-h infusions was similar to 2-h infusions while the Cmax of the 4-h infusion was 50% of the 2-h infusion. By week 26, the mean AUC0-t of the 4-h infusion was 1.3-fold higher than the 2-h infusion (p<0.05) while Cmax of the 4-h infusion was 70% of the 2-h infusion (p<0.05). Among animals treated with 2- and 4-h infusions, there was no difference in urinary GAG excretion, tissue GAG storage, tissue galsulfase activity, and ß-glucuronidase but all were significantly different than control animals (for each, p<0.001). Radiographic skeletal abnormality scores for animals were also similar for both treatment groups and significantly higher than control animals (p<0.001). There was no significant difference in corneal clouding scores among treated and untreated animals. CONCLUSIONS: There was no significant difference in clinical outcomes when rhASB was administered to MPS VI affected cats as 2- and 4-h infusions over 26weeks. Additional studies may determine if shorter infusion times are appropriate for MPS VI patients without significant infusion-associated reactions.

Workshop Proceedings: Streamlined Development of Safety Assessment Programs Supporting Orphan/Rare Diseases-Are We There Yet?

A workshop entitled "Streamlined Development of Safety Assessment Programs Supporting Orphan/Rare Diseases-Are We There Yet?" was held at the 36th Annual Meeting of the American College of Toxicology in Summerlin, Nevada. The workshop was sponsored by Shire and Ultragenyx and was designed to present the nonclinical considerations for the development of various products for rare diseases. A panel of experts from industry and government highlighted the nonclinical considerations in developing toxicology programs supporting rare disease therapeutics, challenges in preclinical safety assessment, reviewed the current guidance, and presented the progress that has been made to date. The main learning from the workshop was that nonclinical testing of therapeutics targeting rare disease warrants special considerations, and early collaboration between sponsors and health authorities may help optimize the scope and timing of the supportive studies. Specific examples for nonclinical development programs for enzyme replacement therapy (ERT) were presented. Although the symposium focused on ERTs, the concepts are broadly applicable.

Evaluation of the therapeutic potential of a CNP analog in a Fgfr3 mouse model recapitulating achondroplasia.

Achondroplasia (ACH), the most common form of dwarfism, is an inherited autosomal-dominant chondrodysplasia caused by a gain-of-function mutation in fibroblast-growth-factor-receptor 3 (FGFR3). C-type natriuretic peptide (CNP) antagonizes FGFR3 downstream signaling by inhibiting the pathway of mitogen-activated protein kinase (MAPK). Here, we report the pharmacological activity of a 39 amino acid CNP analog (BMN 111) with an extended plasma half-life due to its resistance to neutral-endopeptidase (NEP) digestion. In ACH human growth-plate chondrocytes, we demonstrated a decrease in the phosphorylation of extracellular-signal-regulated kinases 1 and 2, confirming that this CNP analog inhibits fibroblast-growth-factor-mediated MAPK activation. Concomitantly, we analyzed the phenotype of Fgfr3(Y367C/+) mice and showed the presence of ACH-related clinical features in this mouse model. We found that in Fgfr3(Y367C/+) mice, treatment with this CNP analog led to a significant recovery of bone growth. We observed an increase in the axial and appendicular skeleton lengths, and improvements in dwarfism-related clinical features included flattening of the skull, reduced crossbite, straightening of the tibias and femurs, and correction of the growth-plate defect. Thus, our results provide the proof of concept that BMN 111, a NEP-resistant CNP analog, might benefit individuals with ACH and hypochondroplasia.

Neutral endopeptidase-resistant C-type natriuretic peptide variant represents a new therapeutic approach for treatment of fibroblast growth factor receptor 3-related dwarfism.

Achondroplasia (ACH), the most common form of human dwarfism, is caused by an activating autosomal dominant mutation in the fibroblast growth factor receptor-3 gene. Genetic overexpression of C-type natriuretic peptide (CNP), a positive regulator of endochondral bone growth, prevents dwarfism in mouse models of ACH. However, administration of exogenous CNP is compromised by its rapid clearance in vivo through receptor-mediated and proteolytic pathways. Using in vitro approaches, we developed modified variants of human CNP, resistant to proteolytic degradation by neutral endopeptidase, that retain the ability to stimulate signaling downstream of the CNP receptor, natriuretic peptide receptor B. The variants tested in vivo demonstrated significantly longer serum half-lives than native CNP. Subcutaneous administration of one of these CNP variants (BMN 111) resulted in correction of the dwarfism phenotype in a mouse model of ACH and overgrowth of the axial and appendicular skeletons in wild-type mice without observable changes in trabecular and cortical bone architecture. Moreover, significant growth plate widening that translated into accelerated bone growth, at hemodynamically tolerable doses, was observed in juvenile cynomolgus monkeys that had received daily subcutaneous administrations of BMN 111. BMN 111 was well tolerated and represents a promising new approach for treatment of patients with ACH.

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.

Enzyme replacement therapy attenuates disease progression in a canine model of late-infantile neuronal ceroid lipofuscinosis (CLN2 disease).

Using a canine model of classical late-infantile neuronal ceroid lipofuscinosis (CLN2 disease), a study was conducted to evaluate the potential pharmacological activity of recombinant human tripeptidyl peptidase-1 (rhTPP1) enzyme replacement therapy administered directly to the cerebrospinal fluid (CSF). CLN2 disease is a hereditary neurodegenerative disorder resulting from mutations in CLN2, which encodes the soluble lysosomal enzyme tripeptidyl peptidase-1 (TPP1). Infants with mutations in both CLN2 alleles develop normally but in the late-infantile/early-childhood period undergo progressive neurological decline accompanied by pronounced brain atrophy. The disorder, a form of Batten disease, is uniformly fatal, with clinical signs starting between 2 and 4 years of age and death usually occurring by the early teenage years. Dachshunds homozygous for a null mutation in the canine ortholog of CLN2 (TPP1) exhibit a similar disorder that progresses to end stage at 10.5-11 months of age. Administration of rhTPP1 via infusion into the CSF every other week, starting at approximately 2.5 months of age, resulted in dose-dependent significant delays in disease progression, as measured by delayed onset of neurologic deficits, improved performance on a cognitive function test, reduced brain atrophy, and increased life span. Based on these findings, a clinical study evaluating the potential therapeutic value of rhTPP1 administration into the CSF of children with CLN2 disease has been initiated.

Recombinant human tripeptidyl peptidase-1 infusion to the monkey CNS: safety, pharmacokinetics, and distribution.

CLN2 disease is caused by deficiency in tripeptidyl peptidase-1 (TPP1), leading to neurodegeneration and death. The safety, pharmacokinetics (PK), and CNS distribution of recombinant human TPP1 (rhTPP1) were characterized following a single intracerebroventricular (ICV) or intrathecal-lumbar (IT-L) infusion to cynomolgus monkeys. Animals received 0, 5, 14, or 20mg rhTPP1, ICV, or 14 mg IT-L, in artificial cerebrospinal fluid (aCSF) vehicle. Plasma and CSF were collected for PK analysis. Necropsies occurred at 3, 7, and 14 days post-infusion. CNS tissues were sampled for rhTPP1 distribution. TPP1 infusion was well tolerated and without effect on clinical observations or ECG. A mild increase in CSF white blood cells (WBCs) was detected transiently after ICV infusion. Isolated histological changes related to catheter placement and infusion were observed in ICV treated animals, including vehicle controls. The CSF and plasma exposure profiles were equivalent between animals that received an ICV or IT-L infusion. TPP1 levels peaked at the end of infusion, at which point the enzyme was present in plasma at 0.3% to 0.5% of CSF levels. TPP1 was detected in brain tissues with half-lives of 3-14 days. CNS distribution between ICV and IT-L administration was similar, although ICV resulted in distribution to deep brain structures including the thalamus, midbrain, and striatum. Direct CNS infusion of rhTPP1 was well tolerated with no drug related safety findings. The favorable nonclinical profile of ICV rhTPP1 supports the treatment of CLN2 by direct administration to the CNS.

Enzyme replacement therapy delays pupillary light reflex deficits in a canine model of late infantile neuronal ceroid lipofuscinosis.

Late-infantile neuronal ceroid lipofuscinosis (CLN2 disease) is a hereditary neurological disorder characterized by progressive retinal degeneration and vision loss, cognitive and motor decline, seizures, and pronounced brain atrophy. This fatal pediatric disease is caused by mutations in the CLN2 gene which encodes the lysosomal enzyme tripeptidyl peptidase-1 (TPP1). Utilizing a TPP1-/- Dachshund model of CLN2 disease, studies were conducted to assess the effects of TPP1 enzyme replacement administered directly to the CNS on disease progression. Recombinant human TPP1 (rhTPP1) or artificial cerebrospinal fluid vehicle was administered to CLN2-affected dogs via infusion into the CSF. Untreated and vehicle treated affected dogs exhibited progressive declines in pupillary light reflexes (PLRs) and electroretinographic (ERG) responses to light stimuli. Studies were undertaken to determine whether CSF administration of rhTPP1 alters progression of the PLR and ERG deficits in the canine model. rhTPP1 administration did not inhibit the decline in ERG responses, as rhTPP1 treated, vehicle treated, and untreated dogs all exhibited similar progressive and profound declines in ERG amplitudes. However, in some of the dogs treated with rhTPP1 there were substantial delays in the appearance and progression of PLR deficits compared with untreated or vehicle treated affected dogs. These findings indicate that CSF administration of TPP1 can attenuate functional impairment of neural pathways involved in mediating the PLR but does not prevent loss of retinal responses detectable with ERG.

The role of inorganic pyrophosphate in aortic valve calcification.

BACKGROUND AND AIM OF THE STUDY: Aortic valve (AV) calcification is a major cause of morbidity and mortality, yet the molecular mechanisms involved are poorly understood. Hence, an ex vivo model of calcification in intact AVs was developed in order to test the role of orthophosphate and pyrophosphate (PPi), both of which factors are known to influence vascular calcification. METHODS: Porcine AV leaflets were cultured in serum-free medium under static conditions for eight days, over which time leaflet architecture and viability were preserved. Calcification was measured as the incorporation of 45Ca, with confirmation by Alizarin Red staining. RESULTS: Calcification required both a high phosphate concentration (3.8 mM) and removal of PPi with alkaline phosphatase or inorganic pyrophosphatase. Calcification occurred predominantly on the fibrosa and was arrested by the bisphosphonate etidronate, a non-hydrolyzable analog of PPi. Leaflets released PPi into the medium, and this was enhanced by MLS38949, a specific inhibitor of tissue non-specific alkaline phosphatase (TNAP). Furthermore, leaflets synthesized PPi from extracellular ATP, which was reduced by ß,γ-methylene-ATP, an inhibitor of ectonucleotide pyrophosphorylase phosphodiesterase (NPP1). CONCLUSION: The ex vivo AV calcification model developed in the present study showed that extracellular PPi, produced by valvular tissue, is a potent inhibitor of valvular calcification. In addition to synthesis, hydrolysis by TNAP also controls PPi levels and calcification. The results suggest that a decreased synthesis or increased hydrolysis of pyrophosphate may contribute to valvular calcification, and that bisphosphonates or inhibitors of TNAP are potential preventive strategies of the process. TNAP are potential preventive strategies.