Recombinant adeno‐associated viral (rAAV) vectors mediate efficient gene transduction in cultured neonatal and adult microglia

Abstract

Microglia are a specialized population of myeloid cells that mediate CNS innate immune responses. Efforts to identify the cellular and molecular mechanisms that regulate microglia behaviors have been hampered by the lack of effective tools for manipulating gene expression. Cultured microglia are refractory to most chemical and electrical transfection methods, yielding little or no gene delivery and causing toxicity and/or inflammatory activation. Recombinant adeno-associated viral (rAAVs) vectors are non-enveloped, single-stranded DNA vectors commonly used to transduce many primary cell types and tissues. In this study, we evaluated the feasibility and efficiency of utilizing rAAV serotype 2 (rAAV2) to modulate gene expression in cultured microglia. rAAV2 yields high transduction and causes minimal toxicity or inflammatory response in both neonatal and adult microglia. To demonstrate that rAAV transduction can induce functional protein expression, we used rAAV2 expressing Cre-recombinase to successfully excise a LoxP-flanked miR155 gene in cultured microglia. We further evaluated rAAV serotypes 5, 6, 8, and 9, and observed that all efficiently transduced cultured microglia to varying degrees of success and caused little or no alteration in inflammatory gene expression. These results provide strong encouragement for the application of rAAV-mediated gene expression in microglia for mechanistic and therapeutic purposes.

Targeted Gene Editing of Glia Maturation Factor in Microglia: a Novel Alzheimer’s Disease Therapeutic Target

“…One possible explanation could be that our AAV-SaCas9-GMF-sgRNA bears a modified capsid with a higher affinity for the selective transduction of neuronal cells. Previous studies have shown that amongst various AAV serotypes 2, 5, 6, 8 and 9 examined, AAV6 and AAV8 have significantly higher transduction efficiency in microglial cells [56]. Further microglial specificity can be achieved using either novel tropism modified and engineered AAV capsids, robust microglia specific promoter such as TMEM119 [57] and/or by employing neuronal detargeting by incorporating mir-124 target sequences in the AAV 3’UTR [58,59].…”

Pharmacological Tools to Activate Microglia and their Possible use to Study Neural Network Patho-physiology

“…An alternative strategy to modify microglia function is to modulate its phenotype by transgene expression in genetically modified animals (for a recent review see [ 317 ]). Alternatively, microglia can be modified by transfecting them with genes using various techniques [ 318 - 320 ], including viral infection [ 321 ]. It is important to consider that microglia are refractory to most chemical and electrical transfection methods, yielding little or no gene delivery and causing toxicity and/or inflammatory activation [ 320 , 322 ].…”

“…Alternatively, microglia can be modified by transfecting them with genes using various techniques [ 318 - 320 ], including viral infection [ 321 ]. It is important to consider that microglia are refractory to most chemical and electrical transfection methods, yielding little or no gene delivery and causing toxicity and/or inflammatory activation [ 320 , 322 ]. Previous studies with lentiviral vectors have shown effective gene transfer into microglia; however, lentiviral infection was associated with modest toxicity and mild inflammatory activation [ 320 ].…”

“…It is important to consider that microglia are refractory to most chemical and electrical transfection methods, yielding little or no gene delivery and causing toxicity and/or inflammatory activation [ 320 , 322 ]. Previous studies with lentiviral vectors have shown effective gene transfer into microglia; however, lentiviral infection was associated with modest toxicity and mild inflammatory activation [ 320 ]. So far, it has been shown that microglia transfection with recombinant adeno-associated virus (rAAV), serotype 2 (rAAV2, [ 320 ]), yields high transduction and causes minimal toxicity or inflammatory response [ 320 ].…”

“…Previous studies with lentiviral vectors have shown effective gene transfer into microglia; however, lentiviral infection was associated with modest toxicity and mild inflammatory activation [ 320 ]. So far, it has been shown that microglia transfection with recombinant adeno-associated virus (rAAV), serotype 2 (rAAV2, [ 320 ]), yields high transduction and causes minimal toxicity or inflammatory response [ 320 ]. In contrast, rAAV of serotypes 5, 6, 8 and 9 can transduce microglia but might induce an undesired inflammatory reaction [ 320 ].…”