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    Introduction To BioTech Powerpoint

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    Tee Bee
    This document summarizes a study that tested delivering genome editing agents via lipid nanoparticles to treat autosomal dominant hearing loss in mice. The study found that injecting Cas9-guide RNA complexes targeting a mutant allele into the cochlea of neonatal mice with that mutation significantly reduced their progressive hearing loss. The treatment enhanced hair cell survival and improved neural responses compared to untreated mice. There were indels found at the target locus, showing the treatment disrupted the mutant allele specifically. However, more testing would be needed before applying this approach to humans.

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    Introduction To BioTech Powerpoint

    Uploaded by

    Tee Bee
    56 views10 pages
    This document summarizes a study that tested delivering genome editing agents via lipid nanoparticles to treat autosomal dominant hearing loss in mice. The study found that injecting Cas9-guide RNA complexes targeting a mutant allele into the cochlea of neonatal mice with that mutation significantly reduced their progressive hearing loss. The treatment enhanced hair cell survival and improved neural responses compared to untreated mice. There were indels found at the target locus, showing the treatment disrupted the mutant allele specifically. However, more testing would be needed before applying this approach to humans.

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    Introduction to BioTech Powerpoint

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    This document summarizes a study that tested delivering genome editing agents via lipid nanoparticles to treat autosomal dominant hearing loss in mice. The study found that injecting Cas9-guide RNA complexes targeting a mutant allele into the cochlea of neonatal mice with that mutation significantly reduced their progressive hearing loss. The treatment enhanced hair cell survival and improved neural responses compared to untreated mice. There were indels found at the target locus, showing the treatment disrupted the mutant allele specifically. However, more testing would be needed before applying this approach to humans.

    Copyright:

    © All Rights Reserved
    Download as PPTX, PDF, TXT or read online from Scribd
    Download as pptx, pdf, or txt
    56 views10 pages

    Introduction To BioTech Powerpoint

    Uploaded by

    Tee Bee
    This document summarizes a study that tested delivering genome editing agents via lipid nanoparticles to treat autosomal dominant hearing loss in mice. The study found that injecting Cas9-guide RNA complexes targeting a mutant allele into the cochlea of neonatal mice with that mutation significantly reduced their progressive hearing loss. The treatment enhanced hair cell survival and improved neural responses compared to untreated mice. There were indels found at the target locus, showing the treatment disrupted the mutant allele specifically. However, more testing would be needed before applying this approach to humans.

    Copyright:

    © All Rights Reserved
    Download as PPTX, PDF, TXT or read online from Scribd
    Download as pptx, pdf, or txt
    of 10

    TREATMENT OF AUTOSOMAL DOMINANT

    HEARING LOSS BY IN VIVO DELIVERY OF


    GENOME EDITING AGENTS.
    Twanie Kifani Fergus
    105025611
    ABSTRACT

    ➤ A genome-editing approach
    targets a dominantly inherited
    form of deafness.
    ➤ Cationic lipid-mediated in-vivo
    delivery of Cas9-guide RNA
    complexes can improve hearing
    loss in a mouse model of
    human genetic deafness.
    ➤ Injection of Cas9-guide RNA-
    lipid complexes targeting the
    mutant TmcBth allele into the
    cochlea of neonatal TmcBth/+
    mice substantially reduce
    progressive hear loss.
    Method
    1. Primary cell culture.
    2. Delivery of proteins complexed with cationic lipids into the mouse fibroblasts.
    3. GUIDE-seq and data analysis.
    4. High-thoughput DNA sequencing of genomic DNA samples.
    5. Microinjection into the inner ear of neonatal mice.
    6. Microinjection into adult inner ear by canalstomy.
    7. Acoustic testing.
    8. Acoustic startle reflex.
    9. Immunohistochemistry and histology.
    10. Haircell transduction current recording.
    11. Inner ear tissue dissection for HTS.
    12. Hair cells solution for HTS.
    13. Statistical analysis.
    14. Code availability.
    15. Data availability.
    RESULTS
    RESULTS CONTINUED
    RESULTS CONTINUED
    DISCUSSION
    ➤ Cas-9 based genome editing agents can mediate targeted gene disruption.
    ➤ Mutations in TMC1 have been linked to recessive and dominant genetic deafness in
    humans.
    ➤ Three candidates sgRNAs that place the Bth mutation at position 11,12 and 15 of
    the spacer were identified, this allowed the PAM to be counted as position 21-23.
    ➤ Lipid-mediated delivery of Cas-sgRNA RNP complexes into cultured primary
    fibroblasts derived from wild type Tmc1Bth/Bth mice was used to evaluate the allele
    specificity of genomic DNA modification in mouse cells.
    ➤ A significant decline in transduction current amplitudes in Tmc1Bth/ΔTmc2Δ/Δ mice was
    observed following injection with Cas9-Tmc-mut3-lipid complexes, consistent with
    the disruption of Tmc1Bth allele in sensory hair cells in vivo, but not with Cas-GFP
    sgRNA-lipid complexes.
    DISCUSSION CONTINUED
    ➤ Un-injected ears exhibited substantial loss of IHCs and partial degeneration of
    OHCs compared with wild type ears. On the other hand in injected ears, survival
    of IHCs and OHCs was enhanced significantly.
    ➤ In uninjected ears of the TmcBth/+ mice, profound attenuation of cochlear neural
    responses, with ABR thresholds ranging from 70-90 dB at four weeks of age
    compared with 30-50dB for wild-type C3H mice.
    ➤ No significant startle responses was detected following stimulation at 120dB in
    the un-injected mice while in the injected mice a significant startle response was
    detected.
    ➤ There were unambiguous indels at the Tmc1Bth locus in cochlear tissue from
    treated mice.
    LIMITATIONS

    ➤ Not every mice will react the


    same to the genome editing.
    ➤ This experiment was done on
    rodents, this means that it
    would not necessarily work for
    humans.
    CONCLUSION
    It was presented in this research that cationic lipid mediated
    Cas9-sgRNA complex delivery in vivo can attain in allele-specific
    gene disruption in a mouse model of a human genetic disease
    (deafness) resulting in amelioration of the disease phenotype.

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