Comprehensive evaluation of human-derived anti-poly-GA antibodies in cellular and animal models of C9orf72 disease.

Hexanucleotide G4C2 repeat expansions in the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Dipeptide repeat proteins (DPRs) generated by translation of repeat-containing RNAs show toxic effects in vivo as well as in vitro and are key targets for therapeutic intervention. We generated human antibodies that bind DPRs with high affinity and specificity. Anti-GA antibodies engaged extra- and intra-cellular poly-GA and reduced aggregate formation in a poly-GA overexpressing human cell line. However, antibody treatment in human neuronal cultures synthesizing exogenous poly-GA resulted in the formation of large extracellular immune complexes and did not affect accumulation of intracellular poly-GA aggregates. Treatment with antibodies was also shown to directly alter the morphological and biochemical properties of poly-GA and to shift poly-GA/antibody complexes to more rapidly sedimenting ones. These alterations were not observed with poly-GP and have important implications for accurate measurement of poly-GA levels including the need to evaluate all centrifugation fractions and disrupt the interaction between treatment antibodies and poly-GA by denaturation. Targeting poly-GA and poly-GP in two mouse models expressing G4C2 repeats by systemic antibody delivery for up to 16 mo was well-tolerated and led to measurable brain penetration of antibodies. Long-term treatment with anti-GA antibodies produced improvement in an open-field movement test in aged C9orf72450 mice. However, chronic administration of anti-GA antibodies in AAV-(G4C2)149 mice was associated with increased levels of poly-GA detected by immunoassay and did not significantly reduce poly-GA aggregates or alleviate disease progression in this model.

CUG initiation and frameshifting enable production of dipeptide repeat proteins from ALS/FTD C9ORF72 transcripts.

Expansion of G4C2 repeats in the C9ORF72 gene is the most prevalent inherited form of amyotrophic lateral sclerosis and frontotemporal dementia. Expanded transcripts undergo repeat-associated non-AUG (RAN) translation producing dipeptide repeat proteins from all reading frames. We determined cis-factors and trans-factors influencing translation of the human C9ORF72 transcripts. G4C2 translation operates through a 5'-3' cap-dependent scanning mechanism, requiring a CUG codon located upstream of the repeats and an initiator Met-tRNAMeti. Production of poly-GA, poly-GP, and poly-GR proteins from the three frames is influenced by mutation of the same CUG start codon supporting a frameshifting mechanism. RAN translation is also regulated by an upstream open reading frame (uORF) present in mis-spliced C9ORF72 transcripts. Inhibitors of the pre-initiation ribosomal complex and RNA antisense oligonucleotides selectively targeting the 5'-flanking G4C2 sequence block ribosomal scanning and prevent translation. Finally, we identified an unexpected affinity of expanded transcripts for the ribosomal subunits independently from translation.