Efficient electroporation of neuronal cells using synthetic oligonucleotides: identifying duplex RNA and antisense oligonucleotide activators of human frataxin expression

Abstract

Oligonucleotide drugs are experiencing greater success in the clinic, encouraging the initiation of new projects. Resources are insufficient to develop every potentially important project, and persuasive experimental data using cell lines close to disease target tissue is needed to prioritize candidates. Friedreich's ataxia (FRDA) is a devastating and currently incurable disease caused by insufficient expression of the enzyme frataxin (FXN). We have previously shown that synthetic nucleic acids can activate FXN expression in human patient-derived fibroblast cells. We chose to further test these compounds in induced pluripotent stem cell-derived neuronal progenitor cells (iPSC-NPCs). Here we describe methods to deliver oligonucleotides and duplex RNAs into iPSC-NPCs using electroporation. Activation of FXN expression is potent, easily reproducible, and potencies parallel those determined using patient-derived fibroblast cells. A duplex RNA and several antisense oligonucleotides (ASOs) with different combinations of 2 ′ ′ ′ ′ ′-methoxyethyl (2 ′ ′ ′ ′ ′-MOE), 2 ′ ′ ′ ′ ′-fluoro (2 ′ ′ ′ ′ ′-F), and constrained ethyl (cEt) were active, providing multiple starting points for further development and highlighting improved potency as an important goal for preclinical development. Our data support the conclusion that ASO-mediated activation of FXN is a feasible approach for treating FRDA and that electroporation is a robust method for introducing ASOs to modulate gene expressions in neuronal cells.

Targeting 3′ and 5′ untranslated regions with antisense oligonucleotides to stabilize frataxin mRNA and increase protein expression

“…FRDA patient induced pluripotent stem cells (iPSCs) were cultured in mTeSR1™ medium (STEMCELL Technologies, Cat# 05850) as described earlier ( 42–44 ). Monolayer NPCs were generated and cultured by using the STEMdiff™ Neural System (STEMCELL Technologies, Cat# 08582, 05833) as described in ( 27 ). Briefly, on day 1, the iPSCs were treated with Accutase ® (STEMCELL Technologies, Cat# 07920) and plated in wells precoated with Corning™ Matrigel™ hESC-qualified matrix (Thermo Fisher, Cat# 08774552) at a density of 2 × 10 5 cells/cm 2 in STEMdiff™ Neural Induction Medium + SMADi + 10 μM Y27632.…”

“…It has been demonstrated that formation of stable R-loops at expanded GAA tracts may be responsible for inhibiting transcription progression ( 29 ). Systematic studies of ONs that target GAA–TTC regions clearly demonstrated that both single-stranded and double-stranded ONs reduce formation of R-loops, reverse chromatin changes and elevate FXN transcription in FRDA patient-derived fibroblasts and neuronal progenitor cells (NPCs) ( 27 , 28 , 39 ). Moreover, gapmers targeting GAA repeats for transcript degradation, likely via targeting FXN pre-mRNA engaged in R-loop formation, also augmented levels of FXN mRNA and protein ( 26 ).…”

A Single-Neuron: Current Trends and Future Prospects

“…Cells 2020, 9, x 26 of 45 frataxin expression [171]. Using fluorescent Ca 2+ indicator-loaded brain slices and in vivo samples, the morphology of the apical dendrites of several pyramidal neurons was found to be normal, indicating that the neurons had recovered from the electroporation procedure [172].…”

“…Electroporation was also tested on an organotypic culture of hippocampal slices to introduce plasmids into single-neurons [170]. The approach has been used to demonstrate synthetic oligonucleotides delivery to identify duplex RNA and antisense oligonucleotide activators of human frataxin expression [171]. Using fluorescent Ca 2+ indicator-loaded brain slices and in vivo samples, the morphology of the apical dendrites of several pyramidal neurons was found to be normal, indicating that the neurons had recovered from the electroporation procedure [172].…”

Molecular Mechanisms and Therapeutics for the GAA·TTC Expansion Disease Friedreich Ataxia

“…ChIP experiments indicate that the activating siRNAs induce small changes in chromatin histone posttranslational modifications on the FXN gene, but have no effect on the recruitment of RNA polymerase II, suggesting that the oligonucleotides relieve a block to transcription elongation through the repeats rather than by increasing pol II occupancy at the promoter [41]. A recent study has also shown efficacy of duplex RNAs and antisense oligonucleotides in neural progenitor cells derived from FRDA induced pluripotent stem cells [86]; however, electroporation of these cells was necessary to see FXN mRNA induction. Future studies will undoubtedly test this approach in animal models for the disease, but a major hurdle will be delivery of nucleic acid therapeutics to the desired target tissues.…”