Menkes disease is a lethal infantile neurodegenerative disorder of copper metabolism caused by mutations in a P-type ATPase, ATP7A. Currently available treatment (daily subcutaneous copper injections) is not entirely effective in the majority of affected individuals. The mottled-brindled (mo-br) mouse recapitulates the Menkes phenotype, including abnormal copper transport to the brain owing to mutation in the murine homolog, Atp7a, and dies by 14 days of age. We documented that mo-br mice on C57BL/6 background were not rescued by peripheral copper administration, and used this model to evaluate brain-directed therapies. Neonatal mo-br mice received lateral ventricle injections of either adeno-associated virus serotype 5 (AAV5) harboring a reduced-size human ATP7A (rsATP7A) complementary DNA (cDNA), copper chloride, or both. AAV5-rsATP7A showed selective transduction of choroid plexus epithelia and AAV5-rsATP7A plus copper combination treatment rescued mo-br mice; 86% survived to weaning (21 days), median survival increased to 43 days, 37% lived beyond 100 days, and 22% survived to the study end point (300 days). This synergistic treatment effect correlated with increased brain copper levels, enhanced activity of dopamine-β-hydroxylase, a copper-dependent enzyme, and correction of brain pathology. Our findings provide the first definitive evidence that gene therapy may have clinical utility in the treatment of Menkes disease.

Background: Members of the Burkholderia cepacia complex (Bcc) cause significant morbidity and mortality in patients with chronic granulomatous disease (CGD) and cystic fibrosis (CF). Many Bcc strains are antibiotic resistant requiring the exploration of novel antimicrobial approaches including antisense technologies, such as phosphorodiamidate morpholino oligomers (PMOs). Methods: Peptide-conjugated PMOs (PPMOs) were developed to target the acpP gene, encoding an acyl carrier protein thought to be essential for growth. Their antimicrobial activities were tested against different strains of Bcc in vitro and in infection models. Results: PPMOs targeting acpP were bactericidal against clinical isolates of Bcc (> 4 log reduction), whereas a PPMO with a scrambled base sequence (Scr) had no effect on growth. Human neutrophils (PMN) were infected with B. multivorans, and treated with AcpP PPMO. AcpP PPMO augmented killing compared to PMN alone ± Scr PPMO. CGD mice infected with B. multivorans were treated with AcpP PPMO, Scr PPMO or water at 0, 3 and 6 hours post-infection. Compared to water treated controls, the AcpP PPMO treated mice showed a ~80% reduction in the risk of dying by day 30 and relatively little pathology. Conclusions: AcpP PPMO is active against Bcc infections in vitro and in vivo.

The alb1 (pksP) gene has been reported as a virulence factor controlling the pigmentation and morphology of conidia in Aspergillus fumigatus. A recent report suggested that laeA regulates alb1 expression and conidial morphology but not pigmentation in the A. fumigatus strain AF293. laeA has also been reported to regulate the synthesis of secondary metabolites, such as gliotoxin. We compared the role of laeA in the regulation of conidial morphology and the expression of alb1 and gliP in strains B-5233 and AF293, which differ in colony morphology and nutritional requirements. Deletion of laeA did not affect conidial morphology or pigmentation in these strains, suggesting that laeA is not involved in alb1 regulation during conidial morphogenesis. Deletion of laeA, however, caused down-regulation of alb1 during mycelial growth in a liquid medium. Transcription of gliP, involved in the synthesis of gliotoxin, was drastically reduced in B-5233laeA⌬, and the gliotoxin level found in the culture filtrates was 20% of wild-type concentrations. While up-regulation of gliP in AF293 was comparable to that in B-5233, the relative mRNA level in AF293laeA⌬ was about fourfold lower than that in B-5233laeA⌬. Strain B-5233laeA⌬ caused slower onset of fatal infection in mice relative to that with B-5233. Histopathology of sections from lungs of infected mice corroborated the survival data. Culture filtrates from B-5233laeA⌬ caused reduced death in thymoma cells and were less inhibitory to a respiratory burst of neutrophils than culture filtrates from B-5233. Our results suggest that while laeA is not involved in the regulation of alb1 function in conidial morphology, it regulates the synthesis of gliotoxin and the virulence of A. fumigatus.Aspergillus fumigatus, a ubiquitous saprophyte, is the predominant cause of aspergillosis throughout the world. This species most often causes life-threatening invasive aspergillosis (IA) in individuals with dysfunctional phagocytes or with prolonged neutropenia resulting from immunosuppressive drug therapy (9,19). In previous studies, we have dissected the molecular basis of conidial pigment formation and identified a cluster of six genes involved in the biosynthesis of dihydroxynaphthalene (DHN)-like melanin in A. fumigatus strain B-5233 (34-37). The alb1 (pksP) gene in this cluster encodes a polyketide synthase that catalyzes the first step of DHN-like melanin synthesis during conidial formation. Conidia of A. fumigatus are bluish green and coarsely echinulated due to the protrusions on their surfaces as revealed by scanning electron microscopy (34). Deletion of the alb1 gene from strain B-5233 resulted in two simultaneous changes in conidial morphology: albino instead of bluish green color and a smooth rather than echinulated surface. Complementation of the alb1⌬ strain with the wild-type alb1 gene restored the bluish green pigment as well as the echinulation on the surface (17, 34), indicating that the alb1 gene is involved in conidial morphology as well as in conidial pigment synthesis. Fur...