Intrathecal administration of AAV/GALC vectors in 10-11-day-old twitcher mice improves survival and is enhanced by bone marrow transplant.

Globoid cell leukodystrophy (GLD), or Krabbe disease, is an autosomal recessive neurodegenerative disease caused by the deficiency of the lysosomal enzyme galactocerebrosidase (GALC). Hematopoietic stem cell transplantation (HSCT) provides modest benefit in presymptomatic patients but is well short of a cure. Gene transfer experiments using viral vectors have shown some success in extending the survival in the mouse model of GLD, twitcher mice. The present study compares three single-stranded (ss) AAV serotypes, two natural and one engineered (with oligodendrocyte tropism), and a self-complementary (sc) AAV vector, all packaged with a codon-optimized murine GALC gene. The vectors were delivered via a lumbar intrathecal route for global CNS distribution on PND10-11 at a dose of 2 × 10(11) vector genomes (vg) per mouse. The results showed a similar significant extension of life span of the twitcher mice for all three serotypes (AAV9, AAVrh10, and AAV-Olig001) as well as the scAAV9 vector, compared to control cohorts. The rAAV gene transfer facilitated GALC biodistribution and detectable enzymatic activity throughout the CNS as well as in sciatic nerve and liver. When combined with BMT from syngeneic wild-type mice, there was significant improvement in survival for ssAAV9. Histopathological analysis of brain, spinal cord, and sciatic nerve showed significant improvement in preservation of myelin, with ssAAV9 providing the greatest benefit. In summary, we demonstrate that lumbar intrathecal delivery of rAAV/mGALCopt can significantly enhance the life span of twitcher mice treated at PND10-11 and that BMT synergizes with this treatment to improve the survival further. © 2016 Wiley Periodicals, Inc.

Disruption of wave-associated Rac GTPase-activating protein (Wrp) leads to abnormal adult neural progenitor migration associated with hydrocephalus.

Hydrocephalus is the most common developmental disability and leading cause of brain surgery for children. Current treatments are limited to surgical intervention, as the factors that contribute to the initiation of hydrocephalus are poorly understood. Here, we describe the development of obstructive hydrocephalus in mice that are null for Wrp (Srgap3). Wrp is highly expressed in the ventricular stem cell niche, and it is a gene required for cytoskeletal organization and is associated with syndromic and psychiatric disorders in humans. During the postnatal period of progenitor cell expansion and ventricular wall remodeling, loss of Wrp results in the abnormal migration of lineage-tagged cells from the ventricular region into the corpus callosum. Within this region, mutant progenitors appear to give rise to abnormal astroglial cells and induce periventricular lesions and hemorrhage that leads to cerebral aqueductal occlusion. These results indicate that periventricular abnormalities arising from abnormal migration from the ventricular niche can be an initiating cause of noncommunicating hydrocephalus.

WRP/srGAP3 facilitates the initiation of spine development by an inverse F-BAR domain, and its loss impairs long-term memory.

The WAVE-associated Rac GAP, WRP, is thought to regulate key aspects of synapse development and function and may be linked to mental retardation in humans. WRP contains a newly described inverse F-BAR (IF-BAR) domain of unknown function. Our studies show that this domain senses/facilitates outward protrusions analogous to filopodia and that the molecular basis for this is likely explained by a convex lipid-binding surface on the WRP IF-BAR domain. In dendrites the IF-BAR domain of WRP forms a bud on the shaft from which precursors to spines emerge. Loss of WRP in vivo and in vitro results in reduced density of spines. In vivo this is primarily a loss of mushroom-shaped spines. Developmentally, WRP function is critical at the onset of spinogenesis, when dendritic filopodia are prevalent. Finally, because WRP is implicated in mental retardation, behaviors of WRP heterozygous and null mice have been evaluated. Results from these studies confirm that loss of WRP is linked to impaired learning and memory.

Novel oligodendroglial alpha synuclein viral vector models of multiple system atrophy: studies in rodents and nonhuman primates.

Multiple system atrophy (MSA) is a horrible and unrelenting neurodegenerative disorder with an uncertain etiology and pathophysiology. MSA is a unique proteinopathy in which alpha-synuclein (α-syn) accumulates preferentially in oligodendroglia rather than neurons. Glial cytoplasmic inclusions (GCIs) of α-syn are thought to elicit changes in oligodendrocyte function, such as reduced neurotrophic support and demyelination, leading to neurodegeneration. To date, only a murine model using one of three promoters exist to study this disease. We sought to develop novel rat and nonhuman primate (NHP) models of MSA by overexpressing α-syn in oligodendroglia using a novel oligotrophic adeno-associated virus (AAV) vector, Olig001. To establish tropism, rats received intrastriatal injections of Olig001 expressing GFP. Histological analysis showed widespread expression of GFP throughout the striatum and corpus callosum with >95% of GFP+ cells co-localizing with oligodendroglia and little to no expression in neurons or astrocytes. We next tested the efficacy of this vector in rhesus macaques with intrastriatal injections of Olig001 expressing GFP. As in rats, we observed a large number of GFP+ cells in gray matter and white matter tracts of the striatum and the corpus callosum, with 90-94% of GFP+ cells co-localizing with an oligodendroglial marker. To evaluate the potential of our vector to elicit MSA-like pathology in NHPs, we injected rhesus macaques intrastriatally with Olig001 expressing the α-syn transgene. Histological analysis 3-months after injection demonstrated widespread α-syn expression throughout the striatum as determined by LB509 and phosphorylated serine-129 α-syn immunoreactivity, all of which displayed as tropism similar to that seen with GFP. As in MSA, Olig001-α-syn GCIs in our model were resistant to proteinase K digestion and caused microglial activation. Critically, demyelination was observed in the white matter tracts of the corpus callosum and striatum of Olig001-α-syn but not Olig001-GFP injected animals, similar to the human disease. These data support the concept that this vector can provide novel rodent and nonhuman primate models of MSA.

Improved MECP2 Gene Therapy Extends the Survival of MeCP2-Null Mice without Apparent Toxicity after Intracisternal Delivery.

Intravenous administration of adeno-associated virus serotype 9 (AAV9)/hMECP2 has been shown to extend the lifespan of Mecp2-/y mice, but this delivery route induces liver toxicity in wild-type (WT) mice. To reduce peripheral transgene expression, we explored the safety and efficacy of AAV9/hMECP2 injected into the cisterna magna (ICM). AAV9/hMECP2 (1 × 1012 viral genomes [vg]; ICM) extended Mecp2-/y survival but aggravated hindlimb clasping and abnormal gait phenotypes. In WT mice, 1 × 1012 vg of AAV9/hMECP2 induced clasping and abnormal gait. A lower dose mitigated these adverse phenotypes but failed to extend survival of Mecp2-/y mice. Thus, ICM delivery of this vector is impractical as a treatment for Rett syndrome (RTT). To improve the safety of MeCP2 gene therapy, the gene expression cassette was modified to include more endogenous regulatory elements believed to modulate MeCP2 expression in vivo. In Mecp2-/y mice, ICM injection of the modified vector extended lifespan and was well tolerated by the liver but did not rescue RTT behavioral phenotypes. In WT mice, these same doses of the modified vector had no adverse effects on survival or neurological phenotypes. In summary, we identified limitations of the original vector and demonstrated that an improved vector design extends Mecp2-/y survival, without apparent toxicity.

No evidence for parental imprinting of mouse 22q11 gene orthologs.

Non-Mendelian factors may influence central nervous system (CNS) phenotypes in patients with 22q11 Deletion Syndrome (22q11DS, also known as DiGeorge or Velocardiofacial Syndrome), and similar mechanisms may operate in mice carrying a deletion of one or more 22q11 gene orthologs. Accordingly, we examined the influence of parent of origin on expression of 25 murine 22q11 orthologs in the developing and mature CNS using single nucleotide polymorphism (SNP)-based analysis in interspecific crosses and quantification of mRNA in a murine model of 22q11DS. We found no evidence for absolute genomic imprinting or silencing. All 25 genes are biallelically expressed in the developing and adult brains. Furthermore, if more subtle forms of allelic biasing are present, they are very small in magnitude and most likely beyond the resolution of currently available quantitative approaches. Given the high degree of similarity of human 22q11 and the orthologous region of mmChr16, genomic imprinting most likely cannot explain apparent parent-of-origin effects in 22q11DS.