Targeting microglia with lentivirus and AAV: Recent advances and remaining challenges

Abstract

Microglia have emerged as a critical component of neurodegenerative diseases. Genetic manipulation of microglia can elucidate their functional impact in disease. In neuroscience, recombinant viruses such as lentiviruses and adeno-associated viruses (AAVs) have been successfully used to target various cell types in the brain, although effective transduction of microglia is rare. In this review, we provide a short background of lentiviruses and AAVs, and strategies for designing recombinant viral vectors. Then, we will summarize recent literature on successful microglial transductions in vitro and in vivo, and discuss the current challenges. Finally, we provide guidelines for reporting the efficiency and specificity of viral targeting in microglia, which will enable the microglial research community to assess and improve methodologies for future studies.

Keywords: Adeno-associated virus (AAV); Brain; In-vivo transduction; Lentivirus; Microglia.

Fig. 1

Lentiviral production (Step I) and transduction (Step II). Virus packaging cell line is transfected with envelope, transfer, and packaging vectors (1). (2) The transcribed mRNAs for the envelope, transfer, and packaging vectors are translated into: (2a) viral envelope proteins that are sorted to the cell membrane via the endoplasmic reticulum; (2b) single-stranded RNA viral genome; (2c) viral structural proteins and enzymes, respectively. All three components are assembled into viral particles (3a), which bud from the host cell membrane (3b). (4) Viral particles attach to the host cell surface receptors using the envelope protein. (5) Fusion of viral with host plasma membrane releases structural, enzymatic proteins, and viral core. (6) Viral RNA is reverse transcribed to double-stranded DNA that then forms a pre-integration complex with the integrase (7), which passes the nuclear pore complex (8), and catalyzes viral DNA integration into the host genome (9). (10) The transfer vector promoter drives transgene expression.

Fig. 2

Schematic of HIV genome in (a) proviral form, and lentiviral packaging system for the 2^nd (b) and 3^rdgeneration (c), which consists of transfer, packaging, and envelope vector. CMV: cytomegalovirus. cPPT: central polypurine tract, which initiates second DNA strand synthesis. Env: envelope protein. Gag: capsid components. HIV: human immunodeficiency virus. LTR: long terminal repeat. PBS: primer binding site for host cell tRNAs to start reverse transcription. Pol: reverse transcriptase and integrase. ψ: packaging signal for nucleocapsid assembling. R: tat-binding region. Rev: facilitates nuclear RNA genome export. RRE: rev responsive element, which serves as binding site for the viral rev protein. RSV: tat-independent transcription of viral genome. Tat: initiates transcription of the viral genome. U3: RNA polymerase II promoter for transcription of the viral genome during replication. U3^Δ: mutated U3. Vif, Vpr, Vpu, Nef: virulence factors. Double arrows indicate splicing events.

Fig. 3

Adeno-associated virus (AAV) production (Step I) and transduction (Step II). (1) Virus packaging cell line expressing the adenovirus E1+ is transfected with packaging, transfer, and helper vectors. (2) Transcribed mRNA from packaging, transfer, and helper vectors are translated into (2a) viral capsid (VP1, 2, 3) and Rep, which replicates the viral genome from the ITR (2b). (2c) The helper proteins support final virus assembling. (3) Viral particles are released into the supernatant. (4) Viral particles attach through interaction of the capsid with host glycan moieties, which triggers endocytosis of the particle (5). AAVs escape to the endosome by an unknown mechanism (6), and enter the nucleus via the nuclear core complex. (7) The viral genome is released from the capsid, the double-stranded genome synthesized (8), and an episomal circular DNA is formed (9). (10) The promoter of the transfer vector drives the expression of the transgene. ITR: inverted terminal repeats.

Fig. 4

Schematic of (a) wildtype AAV genome and (b) AAV viral vectors system consisting of transfer, packaging, and helper vectors. AAV: adeno-associated virus. Cap: open reading frames for proteins (VP1, VP2, VP3) which assemble into a capsid protein shell. E4, E2A, and VA: adenoviral genes necessary for AAV lifecycle. ITR: inverted terminal repeats, which form hairpin structures. ITR^Δtrs: on self-complementary genome, prevents rep-mediated nicking resulting in double-stranded viral genome. p5, p10, p40: promoter sequences to initiate transcription. Rep: open reading frames for proteins involved in genome replication (rep78, rep68) and packing into viral particles (rep52, rep40). trs: terminal resolution site. Double arrows indicate splicing events.