TREATMENT OF AUTOSOMAL DOMINANT HEARING LOSS BY IN

VIVO DELIVERY OF GENOME EDITING AGENTS.

Why this topic? - I choose this topic merely because I had an Aunty who specialized in teaching children
with special needs such as children who are slow or deaf and dumb. I got attached to these as she used to
take me to work with her and I used to try and interact with these children by using the few sign language
that my aunty taught me. Knowing that this method that I am about to discuss shows promise to help
people at risk for hearing loss makes me gravitate to share it with you.

In this experiment they used rodents to help us to arrive at our promising conclusion. Somme of us may
wonder why rats? The answer to this question is that the rodent’s cochlea (organ of inner ear which sends
sounds to the brain) is similar to that of humans.

Read first sentence

What is dominant inheritance?- So if trait is dominant, only one allele is required for the trait to be
observed. The denotations for dominant alleles are AA or Aa.

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What is Cas9-guide RNA?- This RNA is developed for functional knockout and not just cutting. This
method was done when they prepared both in vitro and in primary fibroblast, genome editing agents that
preferentially disrupt the dominant deafness associated allele in tmc1.

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This causes high hair cell survival rates and lower auditory brainstem response thresholds in injected ears
when compared to the uninjected ear ( the control). You will see in this presentation that the genetic
mutations affect the sensory hair cells that amplify acoustic vibrations into electric nerve signals. Without
these hair cells we wont be able to hear properly and progressively leads to deafness. When the mutation
damages these hairs they do not generate spontaneously ( they will be damaged forever).

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1. This step is where the fibroblasts were cultured for about 2-3 days to reach ~90% confluence. This
was done by removing the fibroblasts from P5 pups and digested with 0.5mg/ml Liberase DL.
2. Delivery of Cas9-sgRNA was performed by combining 100nM RNP complex with 3microliter
cationic lipid in 50microliter OPTIMEM medium.
3. For GUIDE-seq off-target DNA cleavage analysis, pCas9, pTmc1-mut3 sgRNA, pmaxGFP, and
dsODN were nucleofected into Tmc bth/+ heterozygous mouse fibroblasts. The retrieved sequences
were aligned to the Cas9 target sequence using a Smith Waterman local alignment algorithm.
4. Treated cells were collected after four days and genomic DNA was isolated. In this step indels might
occur, if the index window was one or more bases longer or shorter than the reference sequence, this
suggested that there was a deletion or insertion.
5. This is pretty straight forward. It suggests that Cas9-sgRNA lipid complexes were injected into the
mice.
6. This step was done on three week old mice ( adult)
7. Acoustic testing merely means the testing for sounds and this is measured in kHz
8. This is the measurement of how the mice reacted to the sound.
9. Immunohistochemistry and histology.- viewing under microscope
10. Hair cell transduction current recording
11. HTS- high throughput screening
12. HTS- high throughput screening
13. Statistical analysis
14. Code availability

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Third point- mis matches between the sgRNA and Genomic DNA that are close to the PAM are poorly tolerated by Cas910, this
increases the chances that the bth mutant allele will be selectively edited.

Fourth point- the highest rate of targeted insertions and deletions (indels) in mutant fibroblasts was observed in Cas9-Tmc1-
mut3RNPs, while lower indel frequencies were observed using the other sgRNAs.

Omit fifth point on slide

In vivo taking place in a living organism.
IHC- immunohistochemistry
ABR- auditory brain stem responses

The analysis of intel containing Tmc1 sequencing reads from treated mice allowed direct assessment of the allele specifity of
Cas9-Tmc1-mut3 in vivo.

Delivering the RNP complex instead of DNA into the cochleae showed
significantly fewer off-target effects.
The treatment also helped preserve the injected animals' acoustic behavioral
reflexes.

Conclusion
This genome editing strategy developed may inform the future development of DNA-free, virus-free, one-time treatment for
certain genetic hearing loss disorders.