Improved Lentiviral Transduction of ALS Motoneurons <i>in Vivo</i> via Dual Targeting
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Abstract
Treatment
of amyotrophic lateral sclerosis (ALS), a progressive
neurodegenerative disease, is hampered by its complex etiology and
lack of efficient means for targeted transfer of therapeutics into
motoneurons. The objective of this research was engineering of a versatile
motoneuron targeting adaptera full-length atoxic tetanus toxin
fused to core-streptavidin (CS-TeTIM)for retro-axonal transduction
of viral vectors; validation of the targeting efficiency of CS-TeTIM <i>in vivo</i>, by expression of green fluorescence protein (GFP)
reporter in motoneurons of presymptomatic and symptomatic ALS-like
SOD1<sup>G93A</sup> mice, and comparison with age-matched controls;
and appraisal of lentiviral transduction with CS-TeTIM relative to
(1) a H<sub>C</sub> binding fragment of tetanus toxin CS-TeTx(H<sub>C</sub>), (2) rabies glycoprotein (RG), and (3) a CS-TeTIM-RG dual
targeting approach. CS-TeTIM and CS-TeTx(H<sub>C</sub>) were engineered
using recombinant technology and site-directed mutagenesis. Biotinylated
vectors, pseudotyped with vesicular stomatitis virus glycoprotein
(VSV-G) or RG, were linked to these adaptors and injected intraperitoneally
(ip) into presymptomatic (12 weeks old), symptomatic SOD1<sup>G93A</sup> (22 weeks old) or wild type control mice, followed by monitoring
of GFP expression in the spinal cord and supraspinal motor structures
with quantitative PCR and immuno-histochemistry. Transcripts were
detected in the spinal cord and supraspinal motor structures of all
mice 2 weeks after receiving a single ip injection, although in symptomatic
SOD1<sup>G93A</sup> animals reporter RNA levels were lower compared
to presymptomatic and wild-type controls irrespective of the targeting
approach. GFP transduction with CS-TeTIM proved more efficient than
CS-TeTx(H<sub>C</sub>) across all groups while CS-TeTIM-RG dual-targeted
vectors yielded the highest transcript numbers. Importantly, in both
wild-type and presymptomatic SOD1<sup>G93A</sup> mice strong colabeling
of choline-acetyltransferase (ChAT) and GFP was visualized in neurons
of the brain stem and spinal cord. CS-TeTIM, a versatile adaptor protein
for targeted lentiviral transduction of motoneurons, has been engineered
and its competence assessed relative to CS-TeTx(H<sub>C</sub>) and
RG. Evidence has been provided that highlights the potential usefulness
of this novel recombinant tool for basic research with implications
for improved transfer of therapeutic candidates into motoneurons for
the amelioration of ALS and related diseases
