Transduction Profiles of Recombinant Adeno-Associated Virus Vectors Derived from Serotypes 2 and 5 in the Nigrostriatal System of Rats

Abstract

We compared the transduction efficiencies and tropisms of titer-matched recombinant adeno-associated viruses (rAAV) derived from serotypes 2 and 5 (rAAV-2 and rAAV-5, respectively) within the rat nigrostriatal system. The two serotypes (expressing enhanced green fluorescent protein [EGFP]) were delivered by stereotaxic surgery into the same animals but different hemispheres of the striatum (STR), the substantia nigra (SN), or the medial forebrain bundle (MFB). While both serotypes transduced neurons effectively within the STR, rAAV-5 resulted in a much larger EGFP-expressing area than did rAAV-2. However, neurons transduced with rAAV-2 vectors expressed higher levels of EGFP. Consistent with this result, EGFP-positive projections emanating from transduced striatal neurons covered a larger area of the SN pars reticulata (SNr) after striatal delivery of rAAV-5, but EGFP levels in fibers of the SNr were higher after striatal injection of rAAV-2. We also compared the potentials of the two vectors for retrograde transduction and found that striatal delivery of rAAV-5 resulted in significantly more transduced dopaminergic cell bodies within the SN pars compacta and ventral tegmental area. Similarly, EGFP-transduced striatal neurons were detected only after nigral delivery of rAAV-5. Furthermore, we demonstrate that after striatal AAV-5 vector delivery, the transduction profiles were stable for as long as 9 months. Finally, although we did not target the hippocampus directly, efficient and widespread transduction of hippocampal neurons was observed after delivery of rAAV-5, but not rAAV-2, into the MFB.With the exception of rare inherited mutations in specific genes that segregate with familial forms of Parkinson's disease (PD) (24,29,30,42), the molecular causes of PD remain unknown. Pathologically, PD is caused by the progressive degeneration of the dopaminergic neurons in the substantia nigra pars compacta (SNc), which is accompanied by a corresponding decline of the neurotransmitter dopamine in the striatum (STR). As a consequence, PD patients present with resting tremor, bradykinesia, rigidity, and postural instability. Dopamine replacement therapy using L-dopa is the most frequent treatment for PD, but its beneficial effects wear off over time and severe side effects often occur. Surgical treatments such as pallidotomy, thalamotomy, or deep brain stimulation as well as cell transplantation therapies ameliorate some of the symptoms but do not inhibit ongoing neurodegeneration. With increasing knowledge about molecular mechanisms of neuron death (19), gene therapy may offer the potential to interfere with the pathophysiological mechanisms causing cell death in PD. Previous gene therapy studies in rodent and nonhuman primate models of PD assessed the protective potential of neurotrophic, antiapoptotic, antioxidative, and dopamine-restorative genes delivered by recombinant adenovirus (rAV) (8), lentivirus (28), herpes simplex virus (13), adeno-associated virus (rAAV) (22), or hybrid viral (9) vectors. Besides the cho...

AAV2-mediated gene transfer of GDNF to the striatum of MPTP monkeys enhances the survival and outgrowth of co-implanted fetal dopamine neurons

“…These results showing sustained overexpression of GDNF in normal monkeys striatum justified an investigation of the potential of AAV2-GDNF to enhance the survival and outgrowth of grafted fetal DA neurons in MPTP-treated monkeys in a relatively long-term study. In fact, the volume of striatum occupied by transduced cells in the monkey was larger than predicted by rodent studies, in which rAAV serotype 2 vectors have delivered genes only to a restricted region surrounding the injection needle (Paterna et al, 2004;Taymans et al, 2007). While this apparent difference in distribution of GDNF may be due to species or methodological differences, the observation of substantial spread of GDNF promised adequate exposure of the graft to GDNF.…”

Administration-route and gender-independent long-term therapeutic correction of phenylketonuria (PKU) in a mouse model by recombinant adeno-associated virus 8 pseudotyped vector-mediated gene transfer

“…A rAAV vector was constructed by inserting the murine Pah-cDNA into an AAV2 vector plasmid (provided by H Bü eler 17 ). This vector, pAAV2-PKU-5, contained the Pah-cDNA under the control of a modified CMV enhancer chicken b-actin (CBA) promoter, the woodchuck post-transcriptional regulatory element (WPRE) sequence to enhance gene expression, and an SV40 polyadenylation signal sequence flanked by inverted terminal repeats from AAV2 ( Figure 1).…”

“…The Pah-cDNA fragment was then excised by BamHI and EcoRI, filled-in by Klenow polymerase, and cloned into the PmlI site of the AAV2 vector plasmid (provided by H Bü eler 17 ) to generate plasmid pAAV2-PKU-5. Orientation of the Pah-cDNA and in-frame cloning were confirmed by DNA sequencing.…”

“…10 Preclinical experiments with AAV in animal models for different diseases have proven the long-term stable transgene expression in liver, muscle, and central nervous system. [11][12][13][14][15][16][17] Several early-phase clinical trials with AAV vectors have been initiated and shown to be safe. [18][19][20][21] Among the at least eight different AAV natural serotypes discovered so far, AAV vector derived from serotype 2 (AAV2) has been the most extensive studied and widely used AAV vector in the past 20 years.…”

AAV9-mediated erythropoietin gene delivery into the brain protects nigral dopaminergic neurons in a rat model of Parkinson's disease

“…Transduction profiles of the AAV9 vectors after striatal injections were similar to previous findings using AAV2 or AAV5 vectors. 32 It has been suggested that EPO exerts its neuroprotective effects on CNS neurons through multiple mechanisms, including anti-apoptosis, 5,33 anti-inflammation, 34 inhibition of glutamate release, inhibition of reactive oxygen species formation, 35 activation of Akt/ protein kinase B through the phosphoinositide 3-kinase pathway, 5 and activation of Janus kinase-2 and nuclear factor-kB signaling pathways. 36 We have recently showed that intrastriatal administration of EPO protects nigral DA neurons from cell death induced by 6-OHDA in part through an anti-inflammatory mechanism.…”