Outcomes of progranulin gene therapy in the retina are dependent on time and route of delivery.

Neuronal ceroid lipofuscinosis (NCL) is a family of neurodegenerative diseases caused by mutations to genes related to lysosomal function. One variant, CNL11, is caused by mutations to the gene encoding the protein progranulin, which regulates neuronal lysosomal function. Absence of progranulin causes cerebellar atrophy, seizures, dementia, and vision loss. As progranulin gene therapies targeting the brain are developed, it is advantageous to focus on the retina, as its characteristics are beneficial for gene therapy development: the retina is easily visible through direct imaging, can be assessed through quantitative methods in vivo, and requires smaller amounts of adeno-associated virus (AAV). In this study we characterize the retinal degeneration in a progranulin knockout mouse model of CLN11 and study the effects of gene replacement at different time points. Mice heterologously expressing progranulin showed a reduction in lipofuscin deposits and microglia infiltration. While mice that receive systemic AAV92YF-scCAG-PGRN at post-natal day 3 or 4 show a reduction in retina thinning, mice injected intravitreally at months 1 and 6 with AAV2.7m8-scCAG-PGRN exhibit no improvement, and mice injected at 12 months of age have thinner retinas than do their controls. Thus, delivery of progranulin proves to be time sensitive and dependent on route of administration, requiring early delivery for optimal therapeutic benefit.

Cystatin B: mutation detection, alternative splicing and expression in progressive myclonus epilepsy of Unverricht-Lundborg type (EPM1) patients.

Progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1) is an autosomal recessive neurodegenerative disorder caused by mutations in the cystatin B gene (CSTB) that encodes an inhibitor of several lysosomal cathepsins. An unstable expansion of a dodecamer repeat in the CSTB promoter accounts for the majority of EPM1 disease alleles worldwide. We here describe a novel PCR protocol for detection of the dodecamer repeat expansion. We describe two novel EPM1-associated mutations, c.149G > A leading to the p.G50E missense change and an intronic 18-bp deletion (c.168+1_18del), which affects splicing of CSTB. The p.G50E mutation that affects the conserved QVVAG amino acid sequence critical for cathepsin binding fails to associate with lysosomes. This further supports the previously implicated physiological importance of the CSTB-lysosome association. Expression of CSTB mRNA and protein was markedly reduced in lymphoblastoid cells of the patients irrespective of the mutation type. Patients homozygous for the dodecamer expansion mutation showed 5-10% expression compared to controls. By combining database searches with RT-PCR we identified several alternatively spliced CSTB isoforms. One of these, CSTB2, was also present in mouse and was analyzed in more detail. In real-time PCR quantification, CSTB2 expression was less than 5% of total CSTB expression in all human adult and fetal tissues analyzed. In patients homozygous for the minisatellite mutation, the level of CSTB2 was reduced similarly to that of CSTB implicating regulation from the same promoter. The physiological significance of CSTB2 remains to be determined.

Amnionless, essential for mouse gastrulation, is mutated in recessive hereditary megaloblastic anemia.

The amnionless gene, Amn, on mouse chromosome 12 encodes a type I transmembrane protein that is expressed in the extraembryonic visceral layer during gastrulation. Mice homozygous with respect to the amn mutation generated by a transgene insertion have no amnion. The embryos are severely compromised, surviving to the tenth day of gestation but seem to lack the mesodermal layers that normally produce the trunk. The Amn protein has one transmembrane domain separating a larger, N-terminal extracellular region and a smaller, C-terminal cytoplasmic region. The extracellular region harbors a cysteine-rich domain resembling those occurring in Chordin, found in Xenopus laevis embryos, and Sog, found in Drosophila melanogaster. As these cysteine-rich domains bind bone morphogenetic proteins (Bmps), it has been speculated that the cysteine-rich domain in Amn also binds Bmps. We show that homozygous mutations affecting exons 1-4 of human AMN lead to selective malabsorption of vitamin B12 (a phenotype associated with megaloblastic anemia 1, MGA1; OMIM 261100; refs. 5,6) in otherwise normal individuals, suggesting that the 5' end of AMN is dispensable for embryonic development but necessary for absorption of vitamin B12. When the 5' end of AMN is truncated by mutations, translation is initiated from alternative downstream start codons.