Striatal readministration of rAAV vectors reveals an immune response against AAV2 capsids that can be circumvented.

Recombinant adeno-associated virus (rAAV) expresses no viral genes after transduction. In addition, because the brain is relatively immunoprivileged, intracranial rAAV transduction may be immunologically benign due to a lack of antigen presentation. However, preexposure to AAV allows neutralizing antibodies (nAbs) to block brain transduction and rAAV readministration in the brain leads to an inflammatory response in the second-injection site. In this study, we replicate our striatal rAAV2/2-GDNF readministration results and extend this effect to a second transgene, green fluorescent protein (GFP). Unlike rAAV2/2-GDNF readministration, striatal rAAV2/2-GFP readministration leads to a loss of transgene in the second site in the absence of detectable circulating nAbs. In order to determine whether the transgene or the AAV2 capsid is the antigenic stimulus in brain for the immune response in the second site, we readministered rAAV2/2-GFP using two different rAAV serotypes (rAAV2/2 followed by rAAV2/5). In this case, there was no striatal inflammation or transgene loss detected in the second-injection site. In addition, striatal readministration of rAAV2/5-GFP also resulted in no detectable immune response. Furthermore, delaying rAAV2/2 striatal readministration to a 11-week interval abrogated the immune response in the second-injection site. Finally, while striatal readministration of rAAV2/2 leads to significant loss of transgene in the second-injection site, this effect is not due to loss of vector genomes as determined by quantitative real-time PCR. We conclude that intracellular processing of AAV capsids after transduction is the immunogenic antigen and capsid serotypes that are processed more quickly than rAAV2/2 are less immunogenic.

Analysis of the PINK1 gene in a large cohort of cases with Parkinson disease.

BACKGROUND: Mutations in the PTEN-induced kinase (PINK1) gene located within the PARK6 locus on chromosome 1p35-p36 have recently been identified in patients with recessive early-onset Parkinson disease. OBJECTIVE: To assess the prevalence of PINK1 mutations within a series of early- and late-onset Parkinson disease patients living in North America. DESIGN: All coding exons of the PINK1 gene were sequenced in a series of 289 Parkinson disease patients and 80 neurologically normal control subjects; the mutation frequencies were evaluated in additional controls (100 white and 50 Filipino subjects). RESULTS: We identified 27 variants, including the first reported compound heterozygous mutation (Glu240Lys and Leu489Pro) and a homozygous Leu347Pro mutation in 2 unrelated young-onset Parkinson disease patients. CONCLUSION: Autosomal recessive mutations in PINK1 are a rare cause of young-onset Parkinson disease.

Time course of transgene expression after intrastriatal pseudotyped rAAV2/1, rAAV2/2, rAAV2/5, and rAAV2/8 transduction in the rat.

In vivo recombinant adeno-associated viral vector (rAAV)-mediated transduction of various tissues including brain has been characterized by slow onset and gradual increase in gene expression before reaching stable long-term protein levels. The early time course of transgene expression has not been quantified using newly available rAAV capsid serotypes. In this experiment, the onset of expression of green fluorescent protein (GFP) after intrastriatal injection of rAAV2-based pseudotyped vectors (rAAV1, rAAV5, and rAAV8 capsids) was quantified. Native GFP fluorescence displayed a delayed onset of expression of at least 7 days for all the pseudotyped rAAV vectors. However, GFP immunohistochemical staining revealed significant transgene expression by 4 days after transduction for all serotypes and stable GFP(+) neuronal populations mediated by all serotypes within 14 days post transduction at the latest. rAAV2/1 and rAAV2/2 displayed no time-dependent increase of GFP(+) striatal neurons; reaching maximal striatal cell GFP(+) counts at 4 days after injection. All serotypes displayed peak transgene expression by 4 weeks post injection where native GFP(+) neurons were equal to immunostained striatal GFP(+) neurons. The inflammatory response to these rAAV vectors was present up to 4 weeks after transduction but was not apparent 9 months post injection. Thus, rAAV-mediated transgene expression begins earlier than previously thought.

Comprehensive screening of a North American Parkinson's disease cohort for LRRK2 mutation.

BACKGROUND: Recently, mutations in LRRK2 encoding the protein dardarin have been linked to an autosomal dominant form of parkinsonism. OBJECTIVE: To identify mutations causing Parkinson's disease (PD) in a cohort of North Americans with familial PD. METHODS: We sequenced exons 1-51 of LRRK2 in 79 unrelated North American PD patients reporting a family history of the disease. RESULTS: One patient had a missense mutation (Thr2356Ile) while two others had the common Gly2019Ser mutation. In addition, 1 patient had a 4-bp deletion in close proximity to the exon 19 splice donor (IVS20+4delGTAA) that in vitro abrogates normal splicing. CONCLUSIONS: Our observations in the 79 North American patients indicate that mutations in LRRK2 are associated with approximately 5% of PD cases with a positive family history. The results also show that G2019S represents approximately half of the LRRK2 mutations in United States PD cases with a family history of the disease. We have identified two novel mutations in LRRK2.

Differential modulation of ovine fetal ACTH secretion by PGHS-1 and PGHS-2.

BACKGROUND/AIMS: We have previously demonstrated that prostaglandin generation within the fetal brain augments or partially mediates fetal reflex responsiveness to hypotension. The present study was performed to test the relative roles of prostaglandin endoperoxide synthases-1 and -2 (PGHS-1 and 2, or COX-1 and 2) as potential mediators of this interaction. METHODS: Chronically catheterized and instrumented fetal sheep were subjected to transient brachiocephalic occlusion (BCO) after intracerebroventricular injection of resveratrol (PGHS-1 or COX-1 inhibitor), nimesulide (PGHS-2 or COX-2 inhibitor), or vehicle. RESULTS: BCO decreased arterial pressure perfusing the fetal brain and stimulated increases in systemic blood pressure and heart rate as well as in circulating concentrations of ACTH. Inhibition of PGHS-1 and PGHS-2 had differential effects on fetal ACTH secretion. Pre-BCO concentrations of plasma ACTH increased in response to nimesulide, while the fetal ACTH response to BCO was delayed by resveratrol. Prior to the BCO, nimesulide also increased fetal blood pressure and decreased fetal heart rate. The injections of resveratrol and nimesulide did not alter placental biosynthesis of prostaglandins and therefore acted within the fetal brain. CONCLUSION: We conclude that prostaglandin generated in the fetal brain by the action of PGHS-1 augments fetal ACTH reflex responses to BCO but that, in contrast, the action of PGHS-2 is inhibitory to ACTH secretion.

Recombinant adeno-associated viral vectors as therapeutic agents to treat neurological disorders.

Recombinant adeno-associated virus (rAAV) is derived from a small human parvovirus with an excellent safety profile. In addition, this viral vector efficiently transduces and supports long-term transgene expression in the nervous system. These properties make rAAV a reasonable candidate vector for treating neurological disorders. Indeed, rAAV is currently being used in five early stage clinical trials for various neurodegenerative disorders. Therefore, we will review the currently available preclinical data using rAAV in animal models of central nervous system (CNS) disorders. Moreover, potential caveats for rAAV-based gene therapy in the CNS are also presented.

Does reduction of circulating prostaglandin E2 reduce fetal hypothalamic-pituitary-adrenal axis activity?

OBJECTIVE: Placental production of prostaglandin E2 (PGE2) increases in fetal sheep as term approaches. It has been suggested that placental PGE2 may act as a hormone to activate the fetal hypothalamic-pituitary-adrenal (HPA) axis. Alternatively, we have proposed that local generation of prostaglandins in the fetal brain and/or pituitary might play a more prominent role in the stimulation of fetal adrenocorticotropin (ACTH) secretion. We performed the present experiments to test the hypothesis that the elevated concentrations of PGE2 in fetal plasma do not tonically stimulate fetal ACTH secretion. METHODS: We studied chronically catheterized late-gestation fetal sheep. Selective inhibitors of prostaglandin synthase-1 and -2 (PGHS-1 and PGHS-2) were injected intravenously. Fetal blood pressure and heart rate were monitored continuously, and circulating concentrations of PGE2, ACTH, and cortisol were measured by specific immunoassay. RESULTS: Injection of vehicle did not have an effect on circulating levels of PGE2 or ACTH, but it did have a mild stimulatory effect on cortisol. The selective PGHS-2 inhibitor, Nimesulide (Cayman Chemical, Ann Arbor, MI), significantly decreased plasma PGE2 concentrations. The selective PGHS-1 inhibitor, Resveratrol (Cayman Chemical), produced smaller decreases in plasma PGE2 concentrations and significantly increased mean arterial blood pressure. Neither inhibitor significantly altered plasma ACTH or cortisol concentrations. CONCLUSION: These results demonstrate that reduction of circulating PGE2 concentrations in response to intravenous injection of PGHS-1 and PGHS-2 inhibitors does not reduce fetal HPA axis activity. We conclude that PGE2 in late-gestation ovine fetal plasma does not tonically stimulate fetal ACTH secretion.