Premium
Gene delivery to mid‐cervical dorsal root ganglia using AAV9
Author(s) -
Benevides Ethan S.,
Sunshine Michael D.,
Fuller David D.
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.06542
Subject(s) - titer , green fluorescent protein , transgene , paraformaldehyde , virus , medicine , andrology , pathology , microbiology and biotechnology , virology , anatomy , biology , gene , biochemistry
Delivery of viruses (e.g., adeno‐associated virus, AAV) or small molecule tracers (e.g., cholera toxin) to the pleural space between the lung and chest wall (i.e., “intrapleural delivery”) can effectively target phrenic motor neurons (see Mantilla et al., 2009, Journal of Neuroscience Methods). In ongoing experiments we are evaluating the effectiveness of intrapleural delivery of AAV serotype 9 (AAV9) for driving transgene expression in diaphragm sensory neurons in the cervical dorsal root ganglia (DRG) in C57/bl6 mice. Initial experiments used AAV9‐CBA‐GFP (titer: 1.42–8.11×10 12 vg/ml) with relatively low total volume (6 μl of virus diluted in 6 μl of 1x PBS) for bilateral intrapleural injection; this approach failed to produce noticeable DRG transgene expression. We next tried larger unilateral doses (33 μl virus) diluted into 1x PBS to make a larger total volume (400 μl); this approach produced cervical DRG GFP expression, but positive DRG somata were sparse. Ultimately we utilized a higher titer AAV9‐CBA‐GFP (1.48×10 13 ) at several doses: 33 μl, 66 μl, 100 μl, or 200 μl (n=2–3 per group). All doses were diluted up to 400 μl total volume in 1x PBS. After 6–8 weeks mice were perfused with 4% paraformaldehyde (PFA) and the central nervous system was harvested, post‐fixed in 4% PFA, and cryo‐protected in 30% sucrose. Brains and spinal cords were cryo‐sectioned (40 μm) and imaged using a fluorescence microscope for GFP expression. All the doses showed unequivocally positive GFP expression in afferent fibers in the mid‐cervical dorsal roots as well as DRG somata. Initial qualitative evaluation indicates a dose‐dependent increase in total cervical DRG labeling progressing from 33–100 μl, with no additional labeling at 200 μl. Interestingly, GFP expression was detected in very few phrenic motoneurons, even at the high doses. Our preliminary conclusion is that intrapleural injection of relatively large volumes of high titer AAV9 can drive transgene expression in mid‐cervical DRG neurons. Support or Funding Information Support: SPARC OT2 OD023854, F31 HL145831‐01 (MDS)