Intracerebroventricular Injection of Adeno-Associated Virus 6 and 9 Vectors for Cell Type–Specific Transgene Expression in the Spinal Cord

Abstract

In the context of motoneuron diseases, gene delivery as an experimental or therapeutic approach is hindered by the challenge to specifically target cell populations that are widely distributed along the spinal cord. Further complicating the task, transgenes often need to be delivered to motoneurons and/or glial cells to address the non-cell-autonomous mechanisms involved in disease pathogenesis. Intracerebroventricular (ICV) injection of recombinant adeno-associated viruses (AAVs) in newborn mice allows distributing viral vectors throughout the central nervous system while limiting undesired transduction of peripheral organs. Here, we show that by combining the appropriate set of AAV serotype and promoter, specific transgene expression can be achieved in either motoneurons or astrocytes along the whole mouse spinal cord. ICV injection of recombinant AAV6 with the cytomegalovirus (cmv) promoter preferentially targets motoneurons, whereas AAV9 particles combined with the astrocyte-specific gfaABC₁D promoter lead to significant transgene expression selectively targeted to astrocytes. Importantly, ICV coinjection of both AAV6-cmv and AAV9-gfaABC₁D results in segregated expression of two different transgenes in motoneurons and astrocytes, respectively. Relevance of viral vector delivery via the cerebrospinal fluid was further investigated in young nonhuman primates. Intracisternal injection of recombinant AAV6-cmv led to robust cervical transduction of motoneurons, highlighting the potential of this approach for gene therapy and modeling of motoneuron diseases.

NH4+ triggers the release of astrocytic lactate via mitochondrial pyruvate shunting

“…The adeno-associated virus (AAV9) expressing Laconic under the control of the short gfaABC1D promoter was generated at the École Polytechnique Fédérale de Lausanne. Design, production, and titration of the AAV9 vector for transgene expression in astrocytes have been described previously (56). Fluo-4 AM, 2,7-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) AM, tetramethyl rhodamine methyl ester (TMRM), and Calcein AM were from Invitrogen.…”

Gene Therapy for the Nervous System: Challenges and New Strategies

“…The CNS cellular tropism of numerous AAV capsids has been examined extensively in the context of intra-parenchymal delivery to different structures, and the universal finding is that most AAVs are exceptionally neurotropic with little evidence for transduction of glia, microglia or oligodendrocytes [16,[99][100][101][106][107][108][109]. These studies have been carried out with AAV vectors carrying the cytomegalovirus (CMV) [16,104,111, CBA [99,106], Rous sarcoma virus (RSV) [109], or human GUSB promoters [101,108] to drive transgene expression.…”

“…This could considerably influence the pre-clinical development process of therapeutic interventions for neurodegenerative diseases, which determines the AAV vectors that will be tested in human clinical trials. As discussed below in "New Strategies", the apparent discrepancy in CNS transduction profiles of AAV vectors with the same capsid but carrying different CNS cell specific promoters or the CBA promoter [102,[110][111][112] suggests that the latter promoter may not mediate detectable transgene expression in all cell types in the brain.…”

“…Apparently, AAV vectors carrying the original 2.2 kb Gfap promoter [150], or a smaller truncated version (gfaABC 1 D) [151], transduce astrocytes in the mouse brain effectively and with a great degree of specificity [102,111]. Similarly, AAV vectors carrying the myelin basic protein (MBP) promoter transduce oligodendrocytes at high efficiency and with excellent cellular specificity [102,110,112].…”

“…These findings with cell-type specific promoters raise the possibility that our current knowledge on the CNS tropism of systemically delivered AAVs is most likely incomplete as the studies carried out thus far have used scAAV vectors encoding GFP under different forms of the CBA promoter [21,36,49,97]. For new AAVs that display strong CNS tropism after systemic delivery using some version of the CAG/CBA promoter, it may be useful to extend studies to include scAAV vectors encoding a marker gene (GFP most likely) under different cell specific promoters for neurons [152], astrocytes [102,110,111], microglia [153], and oligodendrocytes [102,110,112] to develop a more complete understanding of its potential for global gene therapy interventions.…”