Citation
- Authors: Marcovich I. et al.
- Year: 2022
- Journal: Biomolecules 12 914
- Applications: in vitro / DNA / PEIpro
- Cell type: HEK-293
Description: Human embryonic kidney Fibroblast
Known as: HEK293, 293
Method
Human TMC1 codon-optimized sequence (hTMC1) was cloned into AAV2 plasmids downstream of a cytomegalovirus or chicken beta-actin-derived (CB6) promoter and followed by a woodchuck hepatitis post-transcriptional regulatory element (WPRE), unless otherwise indicated.
Vector was produced by triple transfection of suspension HEK293 cells grown in Expi293 media.
Briefly, cells at ~2 × 106 cells/mL were transfected with pALDX-80 helper plasmid, AAV2/AAV9PHP.B rep/cap plasmid, and hTMC1 plasmid using PEIpro (Polyplus, Illkirch-Graffenstaden, France) formulated in OptiPRO SFM.
Seventy-two hours after transfection, cells were harvested by addition of Benzonase and 10× lysis buffer (500 mM Tris, 20 mM Magnesium Chloride, 10% Polysorbate 20, pH 8.0) followed by addition of 5 M Sodium Chloride to a final concentration of 500 mM. Lysates were clarified by centrifugation and stored at −80 °C until purification.
Immediately prior to chromatography, lysates were filtered across a 0.45/0.22 µm polish filter and then passed over POROS CaptureSelect AAV9 affinity resin using an AKTA Pure instrument. Elution fractions with A280 values > 100 mAU were pooled and immediately neutralized with 10% volume of neutralization buffer (500 mM Bis-Tris Propane, 200 mM NaCl, 1% w/v Sucrose, 0.001% Poloxamer 188, pH 9.0). Following purification, the pooled affinity neutralized eluate was buffer-exchanged into final formulation buffer (dPBS, 5% w/v Sorbitol, 0.001% w/v Poloxamer 188, pH 7.0).
Titer was determined using ddPCR as previously described [18].
The following titers of genome-containing particles were used:
AAV2/9-PHP.B-CMV-hTMC1-WPRE: 3.26 × 1013 gc/mL or 6.3 × 1013 gc/mL;
AAV2/9-PHP.B-CB6-hTMC1 No WPRE: 3.14 × 1012 gc/mL;
AAV2/9-PHP.B-CB6-hTMC1-WPRE: 3.14 × 1012 gc/mL, 1.57 × 1013 gc/mL, 3.14–4.30 1013 gc/mL or 7.16 1013 gc/mL.
Virus aliquots were stored at −80 °C.
Abstract
Gene therapy for genetic hearing loss is an emerging therapeutic modality for hearing restoration. However, the approach has not yet been translated into clinical application. To further develop inner-ear gene therapy, we engineered a novel mouse model bearing a human mutation in the transmembrane channel-1 gene (Tmc1) and characterized the auditory phenotype of the mice. TMC1 forms the mechanosensory transduction channel in mice and humans and is necessary for auditory function. We found that mice harboring the equivalent of the human p.N199I mutation (p.N193I) had profound congenital hearing loss due to loss of hair cell sensory transduction. Next, we optimized and screened viral payloads packaged into AAV9-PHP.B capsids. The vectors were injected into the inner ears of Tmc1Δ/Δ mice and the new humanized Tmc1-p.N193I mouse model. Auditory brainstem responses (ABRs), distortion product otoacoustic emissions (DPOAEs), cell survival, and biodistribution were evaluated in the injected mice. We found broad-spectrum, durable recovery of auditory function in Tmc1-p.N193I mice injected with AAV9-PHP.B-CB6-hTMC1-WPRE. ABR and DPOAE thresholds were equivalent to those of wild-type mice across the entire frequency range. Biodistribution analysis revealed viral DNA/RNA in the contralateral ear, brain, and liver but no overt toxicity. We conclude that the AAV9-PHP.B-CB6-hTMC1-WPRE construct may be suitable for further development as a gene therapy reagent for treatment of humans with genetic hearing loss due to recessive TMC1 mutations.