Tripeptidyl Peptidase Research Articles

Post-translational Modification of Therapeutic Peptides By NisB, the Dehydratase of the Lantibiotic Nisin

Post-translationally introduced dehydroamino acids often play an important role in the activity and receptor specificity of biologically active peptides. In addition, a dehydroamino acid can be coupled to a cysteine to yield a cyclized peptide with increased biostability and resistance against proteolytic degradation and/or modified specificity. The lantibiotic nisin is an antimicrobial peptide produced by Lactococcus lactis. Its post-translational enzymatic modification involves NisB-mediated dehydration of serines and threonines and NisC-catalyzed coupling of cysteines to dehydroresidues, followed by NisT-mediated secretion. Here, we demonstrate that a L. lactis strain containing the nisBTC genes effectively dehydrates and secretes a wide range of medically relevant nonlantibiotic peptides among which variants of adrenocorticotropic hormone, vasopressin, an inhibitor of tripeptidyl peptidase II, enkephalin, luteinizing hormone-releasing hormone, angiotensin, and erythropoietin. For most of these peptides, ring formation was demonstrated. These data show that lantibiotic enzymes can be applied for the modification of peptides, thereby enabling the biotechnological production of dehydroresidue-containing and/or thioether-bridged therapeutic peptides with enhanced stability and/or modulated activities.

Catalytic Residues and Substrate Specificity of Recombinant Human Tripeptidyl Peptidase I (CLN2)

Tripeptidyl peptidase I (TTP-I), also known as CLN2, a member of the family of serine-carboxyl proteinases (S53), plays a crucial role in lysosomal protein degradation and a deficiency in this enzyme leads to fatal neurodegenerative disease. Recombinant human TPP-I and its mutants were analyzed in order to clarify the biochemical role of TPP-I and its mechanism of activity. Ser280, Glu77, and Asp81 were identified as the catalytic residues based on mutational analyses, inhibition studies, and sequence similarities with other family members. TPP-I hydrolyzed most effectively the peptide Ala-Arg-Phe*Nph-Arg-Leu (*, cleavage site) (k(cat)/K(m) = 2.94 microM(-1).s(-1)). The k(cat)/K(m) value for this substrate was 40 times higher than that for Ala-Ala-Phe-MCA. Coupled with other data, these results strongly suggest that the substrate-binding cleft of TPP-I is composed of only six subsites (S(3)-S(3)'). TPP-I prefers bulky and hydrophobic amino acid residues at the P(1) position and Ala, Arg, or Asp at the P(2) position. Hydrophilic interactions at the S(2) subsite are necessary for TPP-I, and this feature is unique among serine-carboxyl proteinases. TPP-I might have evolved from an ancestral gene in order to cleave, in cooperation with cathepsins, useless proteins in the lysosomal compartment.

AAV2-mediated CLN2 gene transfer to rodent and non-human primate brain results in long-term TPP-I expression compatible with therapy for LINCL

Late infantile neuronal ceroid lipofuscinosis (LINCL) is a fatal, autosomal recessive disease resulting from mutations in the CLN2 gene with consequent deficiency in its product tripeptidyl peptidase I (TPP-I). In the central nervous system (CNS), the deficiency of TPP-I results in the accumulation of proteins in lysosomes leading to a loss of neurons causing progressive neurological decline, and death by ages 10-12 years. To establish the feasibility of treating the CNS manifestations of LINCL by gene transfer, an adeno-associated virus 2 (AAV2) vector encoding the human CLN2 cDNA (AAV2CUhCLN2) was assessed for its ability to establish therapeutic levels of TPP-I in the brain. In vitro studies demonstrated that AAV2CUhCLN2 expressed CLN2 and produced biologically active TPP-I protein of which a fraction was secreted as the pro-TPP-I precursor and was taken up by nontransduced cells (ie, cross-correction). Following AAV2-mediated CLN2 delivery to the rat striatum, enzymatically active TPP-I protein was detected. By immunohistochemistry TPP-I protein was detected in striatal neurons (encompassing nearly half of the target structure) for up to 18 months. At the longer time points following striatal administration, TPP-I-positive cell bodies were also observed in the substantia nigra, frontal cerebral cortex and thalamus of the injected hemisphere, and the frontal cerebral cortex of the noninjected hemisphere. These areas of the brain contain neurons that extend axons into the striatum, suggesting that CNS circuitry may aid the distribution of the gene product. To assess the feasibility of human CNS delivery, a total of 3.6 x 10(11) particle units of AAV2CUhCLN2 was administered to the CNS of African green monkeys in 12 distributed doses. Assessment at 5 and 13 weeks demonstrated widespread detection of TPP-I in neurons, but not glial cells, at all regions of injection. The distribution of TPP-I-positive cells was similar between the two time points at all injection sites. Together, these data support the development of direct CNS gene transfer using an AAV2 vector expressing the CLN2 cDNA for the CNS manifestations of LINCL.

Molecular architecture and assembly mechanism of Drosophila tripeptidyl peptidase II

In eukaryotes, tripeptidyl peptidase II (TPPII) is a crucial component of the proteolytic cascade acting downstream of the 26S proteasome in the ubiquitin-proteasome pathway. It is an amino peptidase belonging to the subtilase family removing tripeptides from the free N terminus of oligopeptides. The 150-kDa subunits of Drosophila TPPII assemble into a giant proteolytic complex of 6 MDa with a remarkable architecture consisting of two segmented and twisted strands that form a spindle-shaped structure. A refined 3D model has been obtained by cryoelectron microscopy, which reveals details of the molecular architecture and, in conjunction with biochemical data, provides insight into the assembly mechanism. The building blocks of this complex are apparently dimers, within which the 150-kDa monomers are oriented head to head. Stacking of these dimers leads to the formation of twisted single strands, two of which comprise the fully assembled spindle. This spindle also forms when TPPII is heterologously expressed in Escherichia coli, demonstrating that no scaffolding protein is required for complex formation and length determination. Reciprocal interactions of the N-terminal part of subunits from neighboring strands are probably involved in the formation of the native quaternary structure, lending the TPPII spindle a stability higher than that of single strands.

Tripeptidyl Peptidase II. An Oligomeric Protease Complex from Arabidopsis

The breakdown of most nuclear and cytoplasmic proteins involves their partial cleavage by the 26S proteasome followed by further disassembly to free amino acids by the combined action of endo- and exopeptidases. In animals, one important intermediate exopeptidase is tripeptidyl peptidase (TPP)II, which digests peptide products of the 26S proteasome and other endopeptidases into tripeptides. Here, we describe the purification and characterization of TPPII from Arabidopsis (Arabidopsis thaliana). Like its animal counterparts, Arabidopsis TPPII exists as a soluble, approximately 5- to 9-MD complex. Two related species of 153 and 142 kD are present in the purified preparations that are derived from a single TPP2 gene. Sequencing by Edman degradation of the intact polypeptides and mass spectrometry of proteolytic fragments demonstrated that the 142-kD form mainly differs from the 153-kD form by a truncation at the C-terminal end. This serine protease is a member of the subtilisin superfamily and is sensitive to the inhibitors alanine-alanine-phenylalanine-chloromethylketone and butabindide, which are diagnostic for the TPPII subfamily. The Arabidopsis TPP2 gene is widely expressed in many tissue types with related genes evident in other plant genomes. Whereas the 26S proteasome is essential, TPPII appears not as important for plant physiology. An Arabidopsis T-DNA mutant defective in TPP2 expression displays no phenotypic abnormalities and is not hypersensitive to either amino acid analogs or the 26S proteasome inhibitor MG132. As a consequence, plants likely contain other intermediate exopeptidases that assist in amino acid recycling.

Characterization of endoplasmic reticulum-associated degradation of a protein S mutant identified in a family of quantitative protein S deficiency

Introduction Misfolded and unassembled glycoproteins are eliminated from the endoplasmic reticulum (ER) lumen by the ER-associated degradation (ERAD).We previously identified a Tyr595Cys (Y595C) mutation of protein S (PS) in a family of a quantitative PS deficiency. The mutation causes intracellular degradation and decreased secretion of the Y595C mutant PS. The aim of the present study was to further characterize the molecular basis of the intracellular degradation of the mutant. Materials and methods We stably expressed the mutant in mammalian cells, and analyzed the intracellular localization of the protein. The intracellular degradation pathway was determined by pulse-chase analyses in the presence of various inhibitors of ERAD. Results and conclusions Endoglycosidase H digestion and immunofluorescence staining revealed the mutant being retained in the ER. Epoxomicin, a potent and specific proteasome inhibitor, and Ala-Ala-Phe-CH 2Cl (AAF), an inhibitor of tripeptidyl peptidase II (TPPII), suppressed the intracellular degradation of the mutant by about 65% and 50%, respectively. When epoxomicin was combined with AAF, the inhibitory effect was substantially enhanced. Although castanospermine, an inhibitor of glucosidases I and II, did not affect the degradation, kifunensine, an inhibitor of ER mannosidase I, suppressed it. Thus, it appears that the Y595C mutant is degraded through more than one pathway of ERAD, including the proteasome-dependent pathway and an alternate proteasome-independent pathway where proteases such as TPPII may be involved. Production of the critical B isoform of Man 8GlcNAc 2 targets the mutant for ERAD, however, the interaction with calnexin/calreticulin through monoglucosylated oligosaccharides may not be required for the degradation of the mutant.

Degradation of glyceraldehyde-3-phosphate dehydrogenase triggered by 4-hydroxy-2-nonenal and 4-hydroxy-2-hexenal

Lipid peroxidation products such as 4-hydroxy-2-nonenal (HNE) may be responsible for various pathophysiological events under oxidative stress, since they injure cellular components such as proteins and DNA. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is a key enzyme of glycolysis and has been reported to be a multifunctional enzyme, is one of the enzymes inhibited by HNE. Previous studies showed that GAPDH is degraded when incubated with acetylleucine chloromethyl ketone (ALCK), resulting in the liberation of a 23-kDa fragment. In this study, we examined whether GAPDH incubated with HNE or other aldehydes of lipid peroxidation products are degraded similarly to that with ALCK. The U937 cell extract was incubated with these aldehydes at 37 °C and analyzed by Western blotting using anti-GAPDH antibodies. Incubation with HNE or 4-hydroxy-2-hexenal (HHE) decreased GAPDH activity and GAPDH protein level, and increased the 23-kDa fragment, in time- and dose-dependent manners, but that with other aldehydes did not. Gel filtration using the Superose 6 showed that the GAPDH-degrading activity was eluted in higher molecular fractions than proteasome activity. The enzyme activity was detected at the basic range of pH and inhibited by serine protease inhibitors, diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride, but not by other protease inhibitors including a proteasome inhibitor, MG-132, and a tripeptidyl peptidase II (TPP II) inhibitor, AAF-CMK. These results suggest that GAPDH modified by HNE and HHE is degraded by a giant serine protease, releasing the 23-kDa fragment, not by proteasome or TPP II.

660. Quantitative Comparison of AAV Serotypes AAV2, AAV5, and AAVrh.10 Efficiency for CNS Gene Therapy Following Intracranial Gene Delivery

Top of pageAbstract Late infantile neuronal ceroid lipofuscinosis (Batten disease) is a fatal pediatric neurodegenerative disease resulting from mutation of the CLN2 gene encoding for a tripeptidyl peptidase (TPP-I). Deficiency of TPP-I results in aberrant degradation of membrane proteins in the lysosome with development of inclusion bodies which lead to neuronal cell death throughout the brain. We have previously shown that AAV2 based vectors expressing human CLN2 cDNA driven by the CMV-enhanced chicken-b-actin hybrid promoter can mediate long term expression of TPP-I in rat brain. In the present study, we have compared the ability of an AAV2-based vector to mediate distribution of TPP-I production in the rat CNS to that of recombinant AAV2-based vectors pseudotyped with capsids derived from human AAV5 and rh.10, a rhesus macaque derived AAV. Vectors (2.5|[times]|109 genome copies) were injected into the striatum of 8 wk old male F344 rats. Fluorometric enzyme assay for TPP-I in the injected region after 4 wk revealed that while AAV2- and AAV5-mediated gene delivery produced TPP-I enzyme levels that were 1.2 and 1.8 fold of endogenous (p<0.01 compared to naive) respectively, AAVrh.10-mediated delivery provided TPP-I levels that approached 17-fold above endogenous levels (p<0.01 compared to either naive or AAV5-treated animals). Immunohistochemical staining showed vector-derived TPP-I in the striatum of rats injected with each of the three vectors, with AAV5 and AAVrh.10 producing the greatest striatal filling. Volumetric analysis showed that while infection by AAV2-derived vector lead to TPP-I detection in 5.1|[plusmn]|0.4% of the striatum, AAV5 and AAVrh.10 resulted in TPP-I expression in 46.9|[plusmn]|2.9% and 51.2|[plusmn]|6.1% of the striatum respectively. Co-immunofluorescence studies demonstrated that virtually all neurons in the region of AAVrh.10 injection were TPP-I positive but no glia stained TPP-I positive. When the whole brain was examined, AAVrh.10 mediated more extensive TPP-I expression, with TPP-I being detected in the globus pallidus and basal forebrain. There was also prominent substantia nigra staining at 4 wk with AAVrh.10 delivery, suggesting retrograde distribution. In addition TPP-I positive cells were readily detected in the frontal cortex and the thalamus, both of which project to the location of the injection in the striatum. When non-striatal staining was quantitated, AAV2 administration resulted in TPP-I positive cells in 0.4|[plusmn]|0.1% of the hemisphere compared to 4.8|[plusmn]|0.6% for AAV5 and 8.6|[plusmn]|1.4% for AAVrh.10. The data show that capsids from other AAV serotypes give more widespread and higher levels of gene transfer than AAV2 vectors and suggests that high transgene expression levels may correlate with a wider distribution of transgene product. In particular, AAV vectors pseudotyped with the capsid from AAVrh.10 may be applicable to gene therapy for diffuse neurological diseases.

427. AAV2- and AAV5-Mediated CNS Delivery of Human CLN2 Reduces Lysosomal Storage in a Mouse Model of Late Infantile Neuronal Ceroid Lipofuscinosis

Late infantile neuronal ceroid lipofuscinosis (LINCL) is a neurometabolic disorder caused by mutations in CLN2, which encodes lysosomal tripeptidyl peptidase (TPP-1). Lack of TPP-1 results in intracellular accumulation of autofluorescent storage material within the CNS, neurodegeneration and death typically in childhood. In this study, we injected clinical-grade AAV vectors containing the human CLN2 cDNA into the LINCL mouse brain to determine therapeutic efficacy. A total of 3.6 |[times]| 109 genome copies of AAV2CUhCLN2 (n=5) or AAV5CUhCLN2 (n=6) were stereotaxically injected into the motor cortex, thalamus, and cerebellum of both hemispheres at 6 weeks of age. At 13 weeks post-injection (19 weeks of age), mice were sacrificed and brains hemispheres were separated from each other. The left hemisphere was analyzed for TPP-1 enzyme activity, and the right hemisphere was processed for immunohistochemistry and detection of autoflourescent storage material. Brains treated with AAV2CUhCLN2 contained TPP-1 activity at each of three injection tracts equivalent to that of levels in the brains of heterozygote which are phenotypically normal. In contrast, AAV5CUhCLN2-injected brains had approximately two fold wild-type levels of enzyme activity within these tracts. Immunohistochemistry and confocal microscopy of the right hemispheres showed the presence of TPP-1 protein in the lysosomes and neuronal processes of cells at the injection sites. Compared to control animals (AAV2CUNull, sham, uninjected), there was a marked reduction of autofluorescent storage material with AAV2CUCLN2 and AAV5CUCLN2 in the injected regions which include motor cortex, thalamus, cerebellum, as well as other regions such as the striatum and the hippocampus. There was no statistical difference in the amount of autofluorescent reduction mediated by the two serotype vectors despite the higher levels of enzyme activity with AAV5CUCLN2. Behavioral testing on the rotorod showed improvement of cerebellar motor function at 8 and 10 weeks post-injection with both viral vectors. This study demonstrates that the LINCL brain is responsive to TPP-1 replacement with different AAV serotype vectors, and that heterozygote levels of enzyme activity is sufficient to reverse lysosomal pathology and partially restore behavioral function.

969. Development of Non-Invasive Imaging Strategies to Assess the Impact of Gene Therapy: Phenotypic Assessment of the Neurodegenerative CNS Disorder Late Infantile Neuronal Ceroid Lipofuscinosis Using Magnetic Resonance Spectroscopy

Several animal models of neurodegenerative lysosomal storage disease have been successfully treated by intracranial gene transfer. Among the most challenging aspects of moving these strategies to the clinic is to identify objective outcome parameters to measure the success of gene therapy. Classical phenotyping with measurement of neurological functional parameters and/or survival will take years of observation, it is not ethical to obtain brain biopsies following treatment for histological analysis, and there are limited numbers of children with these disorders to develop comparable treated and control groups. We have commenced a program of AAV-mediated gene therapy for the CNS manifestations of late infantile neuronal ceroid lipofuscinosis (LINCL), a fatal childhood neurodegenerative lysosomal storage disease with no known therapy resulting from mutations in the CLN2 gene and a deficiency of the lysosomal protease tripeptidyl peptidase I. As part of this program, we have assessed whether magnetic resonance spectroscopy (MRS) is sufficiently sensitive and reproducible to assess the impact of intracranial gene transfer in humans with neurodegenerative diseases. To date, 8 children with LINCL (ages 6.25 to 13.75 yr old) have been successfully scanned and four of these children were scanned twice at intervals from 17 to 126 days. MRS scans were performed in a 3.0 Tesla GE Signa LX scanner generating 1H images of 4 brain sections of 15 mm thickness separated by 3.5 mm. Strong MRS signals from pericranial lipid resonances and water were suppressed. The raw data were processed to give the concentrations of four metabolites, lactate, choline, creatine (Cr) and N-acetylacetate (NAA) in multiple voxels (8 |[times]| 8 |[times]| 15 mm). Analysis was confined to those regions of the cortex that were accessible for surgical administration of gene transfer vectors. Initially we assessed the NAA/Cr ratio, an index of neuronal function. The NAA/Cr ratio in the target area was 1.60 |[plusmn]| 0.16 in the youngest LINCL patient and 0.58 |[plusmn]|0.18 in the oldest. The NAA/Cr ratio decreased with a slope of |[minus]|0.10/yr (r2=0.43; 95% confidence intervals |[minus]|0.186 to |[minus]|0.019/yr). The difference in NAA/Cr ratio between the first and second scans from the same subjects was |[minus]|0.52|[plusmn]|0.53/yr suggesting that the NAA/Cr ratio is poorly reproducible in duplicate scans of similar brains. Assuming a low estimate of the overall coefficient of variation in measurement of NAA/Cr ratio of 20% (from spatial variability and variability among scans), we calculated that if the NAA/Cr ratio was stabilized by gene therapy in one third of the voxels in the target area, this would be evident by statistical analysis at 18 months post-surgery. We conclude that MRS may provide objective evidence of local changes in metabolite concentration following gene transfer, but to accomplish this will require repeat scans over greater than 1 yr following gene transfer.

Functional consequence and clinical importance of distinct mutations in CLN2, the gene associated with classical late infantile ceroid lipofuscinosis

The neuronal ceroid lipofuscinoses (NCL) are lysosomal diseases characterized by progressive neurodegeneration resulting in loss of vision, seizures, mental regression, behavioral changes, movement disorders and premature death. One of the most frequent of these diseases is classical late infantile NCL caused by mutations in the CLN2 gene and is related to defects of lysosomal tripeptidyl peptidase I (TPP-I).

Assembly/disassembly and enzymatic properties of the Tripeptidyl peptidase II supercomplex from Drosophila melanogaster

Assembly/disassembly and enzymatic properties of the Tripeptidyl peptidase II supercomplex from Drosophila melanogaster

Inhibitor-based validation of a homology model of the active-site of tripeptidyl peptidase II

A homology model of the active site region of tripeptidyl peptidase II (TPP II) was constructed based on the crystal structures of four subtilisin-like templates. The resulting model was subsequently validated by judging expectations of the model versus observed activities for a broad set of prepared TPP II inhibitors. The structure–activity relationships observed for the prepared TPP II inhibitors correlated nicely with the structural details of the TPP II active site model, supporting the validity of this model and its usefulness for structure-based drug design and pharmacophore searching experiments.

A voyage to the inner space of cells.

The title I have chosen for my personal recollections describes, in a nutshell, the direction my scientific endeavors from the time of my Ph.D. thesis, which I began in 1970, to the present day. Over the years, I have changed fields a number of times. There were periods when I was preoccupied with the development of methods; at other times, the focus was on biological problems. Science can be advanced by new hypotheses about how things work, which can be tested and proven right or wrong, and by new methods which enable us to tackle questions that we were unable to address with the existing methods. Or, as Richard Feynman put it, “Science means, sometimes, a special method of finding things out. Sometimes it means the body of knowledge arising from the things found out. It may also mean the new things you can do when you have found something out, or the actual doing of new things. This last field is usually called technology. . . .” (R.P. Feynman in the John Danz Lectures, 1963 [Feynman 1998]).

RECOMBINANT INTERFERON-γ SECRETED BY CHINESE HAMSTER OVARY-320 CELLS CULTIVATED IN SUSPENSION IN PROTEIN-FREE MEDIA IS PROTECTED AGAINST EXTRACELLULAR PROTEOLYSIS BY THE EXPRESSION OF NATURAL PROTEASE INHIBITORS AND BY THE ADDITION OF PLANT PROTEIN HYDROLYSATES TO THE CULTURE MEDIUM

Biosafety requirements increasingly restrict the cultivation of mammalian cells producing therapeutic glycoproteins to conditions that are devoid of any compound of animal origin. On cultivation in serum-free media, the proteases inhibitors, usually found in serum, cannot protect secreted recombinant proteins against unwanted endogenous proteolysis. Chinese hamster ovary (CHO) cells, secreting recombinant human interferon-gamma (CHO-320 cell line) and cultivated in suspension in an original protein-free medium, expressed at least two members of the matrix metalloproteinases (MMP), either at the cell surface (proMMP-14 and MMP-14) or secreted (proMMP-9). In addition, tissue- and urinary-type plasminogen activators were also secreted in such culture conditions. At the cell surface, dipeptidyl peptidase IV and tripeptidyl peptidase II (TPPII) activities were also detected, and their activities decreased during time course of batch cultures. The proteolytic activities of these proteins were counterbalanced by (1) their expression as zymogens (proMMP-9, proMMP-14), (2) the expression of their natural inhibitors, tissue inhibitors of metalloproteinases-1 and -2 and plasminogen activator inhibitor-1 (PAI-1), or (3) the addition of plant protein hydrolysates to the culture medium, acting as a nonspecific source of TPPII inhibitors. This study points out that, even in protein-free media, recombinant proteins secreted by CHO cells are actively protected against physiological and unwanted extracellular proteolysis either by endogenous or by exogenous inhibitors.

Mannose 6-Phosphorylated Proteins Are Required for Tumor Necrosis Factor-induced Apoptosis: DEFECTIVE RESPONSE IN I-CELL DISEASE FIBROBLASTS

Whereas caspases are essential components in apoptosis, other proteases seem to be involved in programmed cell death. This study investigated the role of lysosomal mannose 6-phosphorylated proteins in tumor necrosis factor (TNF)-induced apoptosis. We report that fibroblasts isolated from patients affected with inclusion-cell disease (ICD), having a deficient activity of almost all lysosomal hydrolases, are resistant to the toxic effect of TNF. These mutant cells exhibited a defect in TNF-induced caspase activation, Bid cleavage, and release of cytochrome c. In contrast, TNF-induced p42/p44 MAPK activation and CD54 expression remained unaltered. Human ICD lymphoblasts and fibroblasts derived from mice nullizygous for Igf2 and the two mannose 6-phosphate (M6P) receptors, Mpr300 and Mpr46, which develop an ICD-like phenotype, were also resistant to CD95 ligand and TNF, respectively. Moreover, correction of the lysosomal enzyme defect of ICD fibroblasts, using a medium enriched in M6P-containing proteins, enabled restoration of sensitivity to TNF. This effect was blocked by exogenous M6P but not by cathepsin B or L inhibitors. Altogether, these findings suggest that some M6P-bearing glycoproteins modulate the susceptibility to TNF-induced apoptosis. As a matter of fact, exogenous tripeptidyl peptidase 1, a lysosomal carboxypeptidase, could sensitize ICD fibroblasts to TNF. These observations highlight the hitherto unrecognized role of some mannose 6-phosphorylated proteins such as tripeptidyl peptidase 1 in the apoptotic cascade triggered by TNF.

Administration of a Replication-Deficient Adeno-Associated Virus Gene Transfer Vector Expressing the HumanCLN2cDNA to the Brain of Children with Late Infantile Neuronal Ceroid Lipofuscinosis

Late infantile neuronal ceroid lipofuscinosis (LINCL) is a fatal childhood neurodegenerative lysosomal storage disease with no known therapy. There are estimated to be 200 to 300 children in the United States at any one time with the disease. LINCL is a genetic disease resulting from a deficiency of tripeptidyl peptidase I (TPP-I), a proteolytic enzyme encoded by CLN2, the gene that is mutated in individuals with LINCL. The subjects are chronically ill, with a progressive CNS disorder that invariably results in death, typically by age 8 to 12 years. The strategy of this clinical study is based on the concept that persistent expression in the CNS of the normal CLN2 cDNA with production of sufficient amounts of TPP-I should prevent further loss of neurons, and hence limit disease progression. To assess this concept, an adeno-associated virus vector (AAV2CUh-CLN2) will be used to transfer to and express the human CLN2 cDNA in the brain of children with LINCL. The vector consists of the AAV2 capsid enclosing the 4278-base single-stranded genome consisting of the two inverted terminal repeats of AAV serotype 2 and an expression cassette composed of the human cytomegalovirus (CMV) enhancer, the chicken beta-actin promoter/splice donor and 5' end of the intron, the 3' end of the rabbit P-globin intron and splice acceptor, the human CLN2 cDNA with an optimized Kozak translation initiation signal, and the polyadenylation/transcription stop codon from rabbit 3-globin. The proposed study will include 10 individuals and will be divided into two parts. Group A, to be studied first, will include four individuals with the severe form of the disease. Group B of the trial will include six individuals with a moderate form of the disease. After direct intracranial administration of the vector, there will be neurological assessment based on the LINCL clinical rating scale and magnetic resonance imaging/magnetic resonance spectroscopy assessment of the brain in regions of vector administration. The data generated will help evaluate two hypotheses: (1) that it is safe to carry out direct intracranial administration of the AAV2cuhCLN2 vector to the CNS of individuals with LINCL, and (2) that administration of the AAV2cuhCLN2 vector will slow down or halt the progression of the disease in the central nervous system.

Novel Prolyl Tri/Tetra-Peptidyl Aminopeptidase from Streptomyces mobaraensis: Substrate Specificity and Enzyme Gene Cloning

The prolyl peptidase that removes the tetra-peptide of pro-transglutaminase was purified from Streptomyces mobaraensis mycelia. The substrate specificity of the enzyme using synthetic peptide substrates showed proline-specific activity with not only tripeptidyl peptidase activity, but also tetrapeptidyl peptidase activity. However, the enzyme had no other exo- and endo-activities. This substrate specificity is different from proline specific peptidases so far reported. The enzyme gene was cloned, based on the direct N-terminal amino acid sequence of the purified enzyme, and the entire nucleotide sequence of the coding region was determined. The deduced amino acid sequence revealed an N-terminal signal peptide sequence (33 amino acids) followed by the mature protein comprising 444 amino acid residues. This enzyme shows no remarkable homology with enzymes belonging to the prolyl oligopeptidase family, but has about 65% identity with three tripeptidyl peptidases from Streptomyces lividans, Streptomyces coelicolor, and Streptomyces avermitilis. Based on its substrate specificity, a new name, "prolyl tri/tetra-peptidyl aminopeptidase," is proposed for the enzyme.

A Pilot-Scale Expressed Sequence Tag Analysis of Beauveria Bassiana Gene Expression Reveals a Tripeptidyl Peptidase that is Differentially Expressed in vivo

The entomopathogen Beauveria bassiana is a dimorphic fungus that displays an in vivo-specific, yeast-like parasitic phase. In order to study the transcriptome of B. bassiana during this unique developmental phase, we developed a method to harvest in vivo B. bassiana cells from infected Manduca sexta larvae. The infected hemolymph was collected just prior to insect death and subjected to gradient centrifugation, which allowed for separation of the B. bassiana in vivo-produced cells from remaining insect hemocytes. Total RNA was extracted from the harvested fungal cells and used to construct a cDNA library that is representative of B. bassiana gene expression in vivo. Expressed Sequence Tags (ESTs) were generated and led to the cloning of two protease genes. One of these proteases was identified as a tripeptidyl peptidase (Bb TPP). The Bb TPP protease was shown to be up-regulated during infection, and identification of a signal peptide suggested that the enzyme is secreted in the host hemolymph. Although its activity and role have yet to be characterized, the Bb TPP protease appears as a likely candidate for being involved in B. bassiana pathogenesis. The identification of this novel, up-regulated protease also suggests that random sequencing from our in vivo cDNA library may be a valuable step towards identifying biologically active metabolites produced in vivo by B. bassiana.

433. Micro-Utrophin as a Therapeutic Protein in rAAV Mediated Gene Therapy for Duchenne Muscular Dystrophy

Late infantile neuronal ceroid lipofuscinosis (LINCL), a pediatric neurodegenerative lysosomal storage disorder, results from mutations in the CLN2 gene that lead to a deficiency in tripeptidyl peptidase (TPP-I) and consequent progressive destruction of neurons. Studies in our laboratory have demonstrated long term TPP-I expression in the CNS following CNS gene transfer of AAV2CUhCLN2 (an AAV2-based vector expressing the human CLN2 cDNA) in the rat and non-human primate. To prepare for a possible clinical trial, the present study tests the hypothesis that direct CNS injection of AAV2CUhCLN2 to the CNS of rats and non-human primates at doses scalable to humans has an acceptable long term safety profile. Fischer 344 rats were injected bilaterally in the striatum with 2 × 1010 particle units (pu) of AAV2CUhCLN2, using PBS as a control. At 13 wk, vector and PBS-injected rats were sacrificed (n=6 /group), and blood was collected and the brain and distant organs were assessed by histopathology. The data showed no significant differences between control and vector groups for complete blood count, blood chemistry, and neutralizing anti-AAV2 antibody levels. CNS administration of AAV2CUhCLN2 did not result in any pathological changes in the brain that were attributable to the vector, although microscopic changes were observed along the tract consistent with needle trauma. Changes observed in distant organs were of the type commonly observed in rats and occurred at similar frequencies in AAV2CUhCLN2 and PBS-injected animals. A total dose of 3.6 × 1010 or 3.6 × 1011 pu of AAV2CUhCLN2 was administered to the CNS of 18 African Green monkeys at 12 locations targeting the caudate nucleus, hippocampus and overlying cortices. Monkeys (n=3) at each dose were sacrificed at 1, 13, and 26 wk following injection. The controls consisted of sham-injected, PBS-injected, and AAV2CUNull-injected (3.6 × 1011 pu) monkeys. Using ANOVA analysis for repeated measurements, there were no significant differences among vector-injected and control groups in any parameter of the general assessment or comprehensive mammalian profile (blood chemistry panel, complete blood count) pre-administration, on the day of administration, and day 3, 7, 14, 28, 56, 91, 180 post-administration. There were no abnormal behaviors observed in any group in videotaped neurological assessment where behaviors was quantified prior to administration compared to day 7, 14, 28, 56, 91, 180 post-administration. Histopathological examination of the CNS demonstrated that injection of AAV2CUhCLN2 produced slight to mild and transient white matter edema (spongiosis) with reactive glial cells in the corona radiata of the cerebrum along the injection tract and in the surrounding white matter. This abmormality likely attributable to the vehicle, was not observed at 13 and 26 wk. Together with the long term gene expression following gene transfer, these findings support the initiation of clinical trials to assess the safety of AAV2CUhCLN2 administration to individuals with LINCL.