AAV8, 9, Rh10, Rh43 Vector Gene Transfer in the Rat Brain: Effects of Serotype, Promoter and Purification Method

Abstract

We compared adeno-associated virus (AAV) serotypes for expression levels of green fluorescent protein (GFP) in the adult rat hippocampus by biophotonic imaging. Preparations of AAV serotypes 8, 9, Rh10, and Rh43 incorporating cytomegalovirus (CMV) promoter-driven GFP were purified by a CsCl method. Neither AAV Rh10 nor AAV Rh43 produced greater levels of GFP than AAV8, which was used as a reference. For AAV9, there was an increase relative to AAV8. The CsCl-purified AAV8 displayed an astroglial transduction pattern in contrast to the expected neuronal expression of other AAVs. After preparing the same CMV-GFP plasmid in AAV8 with an iodixanol purification method, the expected neuronal pattern resulted. The astroglial expression with the CsCl AAV8 was probably due to relatively high levels of protein impurities. We compared the CMV promoter with the CMV/chicken beta-actin (CBA) promoter in the context of AAV8, both prepared by iodixanol, and found the CBA promoter to produce stronger GFP expression. At two doses of vectors optimized for serotype, promoter and purification, we did not observe serotype differences among AAV8, AAV9, or AAV Rh10. The purification method can therefore impact the transduction pattern as well as the results when comparing serotype strengths.

Schwann cell targeting via intrasciatic injection of AAV8 as gene therapy strategy for peripheral nerve regeneration

“…Although AAV8 vectors purified by iodixanol gradient infected exclusively neurons, CsCl purification changed AAV8 tropism to glial cells. 15 We performed intrasciatic injections using 8Â10 6 green fluorescent units of CsCl-or iodixanol-purified AAV8-GFP vectors to compare their tropism in the PNS. Three weeks later, GFP mRNA was mostly detected in the sciatic nerve, where nuclei of Schwann cells but not neurons are located, regardless of the purification method employed.…”

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

“…The AAV9 vector produced widespread neuronal expression of either EPO or GFP transgenes in the striatum, which recapitulated the high-efficiency neuronal transduction observed previously with this vector in the rat hippocampus or SN. 19,20 The expression seemed to be stable between 3 and 10 weeks after gene transfer. In addition to the widespread striatal transduction, either the expressed EPO or GFP was detected in the SNpr, an anterograde terminal area of striatal output neurons, and for EPO, expression was observed in DA neuronal perikarya in the SNpc.…”

“…14 Numerous studies have shown that recombinant AAV with newer serotypes have improved gene transfer into the brain. [17][18][19][20] AAV9 vectors are of particular interest because of their high gene transfer efficiency in the brain. [18][19][20] Adeno-associated virus-mediated EPO gene delivery into skeletal muscle has been previously reported to result in sustained EPO gene expression and systemic delivery of therapeutic protein in mice [21][22][23][24] and nonhuman primates.…”

“…[17][18][19][20] AAV9 vectors are of particular interest because of their high gene transfer efficiency in the brain. [18][19][20] Adeno-associated virus-mediated EPO gene delivery into skeletal muscle has been previously reported to result in sustained EPO gene expression and systemic delivery of therapeutic protein in mice [21][22][23][24] and nonhuman primates. [25][26][27][28] However, it is not clear whether AAV-mediated EPO gene delivery into the brain can produce sustained EPO gene expression and elicit functional benefits in an animal model of PD.…”

Efficient transduction of non-human primate motor neurons after intramuscular delivery of recombinant AAV serotype 6

“…Indeed, it was demonstrated that the purification method and presence of contaminating proteins in vector preparations alter the tropism or rAAV2/8 for glial cells and neurons after injection into the rat brain. 13 We have examined the two vector preparations used for this study by protein gel electrophoresis and observed low levels of non-viral protein contaminants that may have accounted for the enhanced uptake from nerve terminals (Supplementary Figure 1). Future work will be required to examine these issues more directly and determine the respective contributions of serotypes, production and purification methods in the capacity of rAAV vectors to be retrogradely transported in neurons.…”