Efficient gene delivery and selective transduction of astrocytes in the mammalian brain using viral vectors

Abstract

Astrocytes are now considered as key players in brain information processing because of their newly discovered roles in synapse formation and plasticity, energy metabolism and blood flow regulation. However, our understanding of astrocyte function is still fragmented compared to other brain cell types. A better appreciation of the biology of astrocytes requires the development of tools to generate animal models in which astrocyte-specific proteins and pathways can be manipulated. In addition, it is becoming increasingly evident that astrocytes are also important players in many neurological disorders. Targeted modulation of protein expression in astrocytes would be critical for the development of new therapeutic strategies. Gene transfer is valuable to target a subpopulation of cells and explore their function in experimental models. In particular, viral-mediated gene transfer provides a rapid, highly flexible and cost-effective, in vivo paradigm to study the impact of genes of interest during central nervous system development or in adult animals. We will review the different strategies that led to the recent development of efficient viral vectors that can be successfully used to selectively transduce astrocytes in the mammalian brain.

Keywords: CNS; astrocytes; gene therapy; tropism; viral vectors.

FIGURE 1

Strategies to target astrocytes. Three steps of viral cycle are used to modify the tropism of viral vectors: (1) the entry, (2) the transcriptional and (3) post-transcriptional regulations. After binding to their respective receptors, LV, AAV, and Ad enter into host cells via receptor-mediated endocytosis. Viral DNA (AAV and Ad) or RNA (LV) are uncoated in the cytoplasm. The viral DNA remains as extrachromosomal episomes in the nucleus while viral RNA is integrated into the host genome after reverse transcription. For non-replicative vectors, in most cases only the transgene is expressed. In the case of oncolytic viruses, viral genes encoding structural proteins are necessary for the encapsidation and production of replicative particles. PIC, pre-integration complex.

FIGURE 2

Mechanisms used to restrain the transgene expression of AAV and LV in astrocytes. (1) To modify the entry, various AAV serotypes or LV pseudotyping with heterologous VSV-G (green) and MOK-G (blue) envelopes were used. The tropism of LV is mainly neuronal (green cells) with the VSV-G envelope and a partial shift toward astrocytes (blue cells) is observed with the MOK-G envelope. AAV1, 2, 5, 7, and 8 mainly transduce neurons (green) while AAV4, 9, rh43 display a partial astrocytic tropism. (2) To restrict transgene expression, astrocytic promoters were investigated (cells in the upper part). Transgene expression under the control of a PGK promoter (pPGK, green mRNA) leads to a preferential expression in neurons, whereas a gfa2 promoter (pgfa2, blue mRNA) results in an astrocytic expression. (3) To block the transgene expression in unwanted cells (lower part), miRNA target (miRT) sequences are integrated in the 3′-UTR of the vector (red signal on the green mRNA). The miR124 is exclusively expressed in neurons. As a consequence the miR124T is only recognized in neurons and the transgene expression is blocked (mRNA degraded). miR124, microRNA 124; miR124T, miR-124 target sequence; Tg. transgene.

FIGURE 3

Effects of the envelope/serotype, promoter, and miRT detargeting on the cellular tropism of LV, AAV and Ad. Overview depicting the tropisms of viral vectors in the CNS. References used for this figure are detailed and cited in the text.