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BMN 250 is being developed as enzyme replacement therapy for Sanfilippo type B, a primarily neurological rare disease, in which patients have deficient lysosomal alpha-N-acetylglucosaminidase (NAGLU) enzyme activity. BMN 250 is taken up in target cells by the cation-independent mannose 6-phosphate receptor (CI-MPR, insulin-like growth factor 2 receptor), which then facilitates transit to the lysosome. BMN 250 is dosed directly into the central nervous system via the intracerebroventricular (ICV) route, and the objective of this work was to compare systemic intravenous (IV) and ICV delivery of BMN 250 to confirm the value of ICV dosing. We first assess the ability of enzyme to cross a potentially compromised blood–brain barrier in the Naglu−/−mouse model and then assess the potential for CI-MPR to be employed for receptor-mediated transport across the blood–brain barrier. In wild-type and Naglu−/−mice, CI-MPR expression in brain vasculature is high during the neonatal period but virtually absent by adolescence. In contrast, CI-MPR remains expressed through adolescence in non-affected non-human primate and human brain vasculature. Combined results from IV administration of BMN 250 in Naglu−/−mice and IV and ICV administration in healthy juvenile non-human primates suggest a limitation to therapeutic benefit from IV administration because enzyme distribution is restricted to brain vascular endothelial cells: enzyme does not reach target neuronal cells following IV administration, and pharmacological response following IV administration is likely restricted to clearance of substrate in endothelial cells. In contrast, ICV administration enables central nervous system enzyme replacement with biodistribution to target cells.

Background: Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder caused by mutations in the arylsulfatase A gene (ARSA) and categorized into three subtypes according to age of onset. The functional effect of most ARSAmutants remains unknown; better understanding of the genotype–phenotype relationship is required to support newborn screening (NBS) and guide treatment. Results: We collected a patient data set from the literature that relates disease severity to ARSAgenotype in 489 individuals with MLD. Patient-based data were used to develop a phenotype matrix that predicts MLD phenotype given ARSAalleles in a patient’s genotype with 76% accuracy. We then employed a high-throughput enzyme activity assay using mass spectrometry to explore the function of ARSAvariants from the curated patient data set and the Genome Aggregation Database (gnomAD). We observed evidence that 36% of variants of unknown significance (VUS) in ARSAmay be pathogenic. By classifying functional effects for 251 VUS from gnomAD, we reduced the incidence of genotypes of unknown significance (GUS) by over 98.5% in the overall population. Conclusions: These results provide an additional tool for clinicians to anticipate the disease course in MLD patients, identifying individuals at high risk of severe disease to support treatment access. Our results suggest that more than 1 in 3 VUS in ARSAmay be pathogenic. We show that combining genetic and biochemical information increases diagnostic yield. Our strategy may apply to other recessive diseases, providing a tool to address the challenge of interpreting VUS within genotype–phenotype relationships and NBS.

The Leishmania mexicana PFR2 locus encodes a component of the paraflagellar rod (PFR), a flagellar structure found only in the insect stage of the life cycle. PFR2 mRNA levels are 10-fold lower in the mammalian stage than in the insect stage. Nuclear run-on experiments indicate that the change in PFR2 mRNA abundance is achieved posttranscriptionally. Deletion and block substitution analysis of the entire 1,400-nucleotide 3' untranslated region (UTR) of PFR2C led to the identification of a regulatory element contained within 10 nucleotides of the 3' UTR, termed the PFR regulatory element (PRE), that is necessary for the 10-fold regulation of PFR2 mRNA levels. Comparison of the half-lives of PFR2 transcripts, identical except for the presence or absence of the PRE, revealed that the PRE acts by destabilizing the PFR2 mRNA in amastigotes. The PRE was inserted into a construct which directs the constitutive expression of a chimeric PFR2 transcript. Insertion of the PRE resulted in regulated expression of this transcript, demonstrating that the regulatory element is sufficient for promastigote-specific expression. Since the PRE is present in the 3' UTR of all L. mexicana PFR genes examined so far, we propose that it serves a means of coordinating expression of PFR genes.

ABSTRACTThe Leishmania mexicanaPFR2 locus encodes a component of the paraflagellar rod (PFR), a flagellar structure found only in the insect stage of the life cycle. PFR2mRNA levels are 10-fold lower in the mammalian stage than in the insect stage. Nuclear run-on experiments indicate that the change in PFR2mRNA abundance is achieved posttranscriptionally. Deletion and block substitution analysis of the entire 1,400-nucleotide 3' untranslated region (UTR) of PFR2Cled to the identification of a regulatory element contained within 10 nucleotides of the 3' UTR, termed the PFR regulatory element (PRE), that is necessary for the 10-fold regulation of PFR2mRNA levels. Comparison of the half-lives of PFR2transcripts, identical except for the presence or absence of the PRE, revealed that the PRE acts by destabilizing the PFR2mRNA in amastigotes. The PRE was inserted into a construct which directs the constitutive expression of a chimeric PFR2transcript. Insertion of the PRE resulted in regulated expression of this transcript, demonstrating that the regulatory element is sufficient for promastigote-specific expression. Since the PRE is present in the 3' UTR of all L. mexicanaPFR genes examined so far, we propose that it serves a means of coordinating expression of PFR genes.

The paraflagellar rod (PFR) is a unique network of cytoskeletal filaments that lies alongside the axoneme in the flagella of most trypanosomatids. While little is known about how two major Leishmania mexicana PFR protein components, PFR1 and PFR2, assemble into this complex structure, previous analysis of PFR2 null mutants demonstrated that the PFR is essential for proper cell motility. The structural roles of PFR1 and PFR2 are now examined through comparison of PFR2 null mutants with new PFR1 null mutant and PFR1/PFR2 double null mutant parasites. Both PFR1 and PFR2 were essential for PFR formation and cell motility. When elimination of one PFR gene prevented assembly of a native PFR structure, the other PFR protein accumulated at the distal flagellar tip. Comparison of PFR substructures remaining in each mutant revealed that: (1) fibers that attach the PFR to the axoneme did not contain PFR1 or PFR2, and assemble in the absence of a PFR. (2) PFR1 was synthesized and transported to the flagella in the absence of PFR2, where it formed a stable association with the axoneme attachment fibers. (3) PFR2 was synthesized and transported to the flagella in the absence of PFR1, though it was not found associated with the axoneme attachment fibers. (4) PFR1 and PFR2 were located throughout the subdomains of the PFR. These data suggest that while PFR filaments contain both PFR1 and PFR2, the PFR is attached to the axoneme by interaction of PFR1 with the axoneme attachment fibers.

A soluble enzyme system that specifically initiates λdy plasmid DNA replication at a bacteriophage λ replication origin [Wold et al. (1982) Proc. Natl. Acad. Sci. USA 79, 6176–6180] is also capable of replicating the single–stranded circular chromosomes of phages M13 and øX174 to a duplex form. This chain initiation on single–stranded templates is novel in that it is absolutely dependent on the λ O and P protein chromosomal initiators and on several Escherichia coli proteins that are known to function in the replication of the λ chromosome in vivo, including the host dnaB, dnaG (primase), dnaJ and dnaK replication proteins. Strand initiation occurs at multiple sites following an O and P protein–dependent pre–priming step in which the DNA is converted into an activated nucleoprotein complex containing the bacterial dnaB protein. We propose a scheme for the initiation of DNA synthesis on single–stranded templates in this enzyme system that may be relevant to strand initiation events that occur during replication of phage λ in vivo.