1. 1A. Describe the assembly of the transcription machinery on a eukaryotic promoter.
TFIID binds to the TATA box through TBP
TFIIA and TFIIB bind to the promoter
RNA Pol II, TFIIF, TFIIE and TFIIH bind to form the
transcription initiation complex
1B. What are the functions of TFIID and TFIIH?
TFIID contains TBP – the TATA binding protein.
It binds the TATA box and marks the promoter
as an active promoter for transcription
TFIIH contains the protein kinase that phosphorylates the
C-terminus of RNA pol II so that it releases the promoter
to begin elongation.
TFIIH contains a DNA helicase that separates the DNA strands.

2. 1C. Explain why the TG mutation shown in the diagram results in the decreased
production of the β-chain of hemoglobin?
The T to G mutation occurs in the promoter region and appears to destroy the TATA box.
This will inhibit binding of TFIID through the TBP and decrease transcription.

3. 2. List the major classes of genes in a eukaryotic cell and the RNA polymerases
that are transcribe them. What cellular functions are performed by the products
of each gene type?
Ribosomal RNA (rRNA) genes are transcribed by RNA polymerase I.
rRNAs form the basic structure of the ribosome and catalyze protein synthesis.
Transfer RNA (tRNA) genes are transcribed by RNA polymerase III.
tRNAs are central to protein synthesis as adaptors between mRNA and amino acids.
Messenger RNA (mRNA) genes transcribed by RNA polymerase II.
mRNAs encode for proteins.

4. 3. Which of the following is associated with the processing of a pre-mRNA?
A. Addition of a polyA tail to the 5’end of the mRNA
B. Covalent modification of specific bases to form dihydrouridine, pseudouridine and
inosine
Deposition of special “guide” proteins on the pre-mRNA to help define exon-intron
boundaries.
D. Addition of an N7-methylguanine cap to the 3’end of the mRNA
E. Phosphorylation of polyadenylation factors to promote addition of the polyA tail

5. 4. Duchenne Muscular Dystrophy and Eteplirsen
4A. What type of mutation is shown? Insertion; frameshift
4B. Predict the consequence of the mutation.
Frameshift mutation resulting in a truncated protein missing exons

6. 4C. Describe the results. What is the purpose of GAPDH?
The first gel shows a positive control sample. The band indicates dystrophin mRNA can be amplified with the primers and conditions used in the experiment.
The second gel contains samples from DMD cells. No band is detected at day 0 indicating there is no dystrophin mRNA expressed in untreated DMD cells.
A faint band is detected for dystrophin at day 3 that gradually increases in intensity suggesting AO-treatment promotes dystrophin mRNA expression.
The mRNA appears to be slightly smaller than the normal transcript in the first gel.
Amplification of GAPDH confirms the presence of amplifiable cDNA in all samples. This is a positive control.

7. 4D. Describe the results
Immunofluorescent staining for dystrophin (red).
Nuclei are blue (DAPI)
Normal cells show positive staining for dystrophin (red). They have an elongated morphology with dystrophin expression along the length of the cell. Nuclei are present [blue or pink].
The AO-treated DMD cells are also staining red indicating dystrophin is expressed. The cells have an elongated morphology and dystrophin is expressed along the length of the cell. Nuclei are present.
The non-treated DMD cells show a cluster of nuclei with positive staining, but the distribution of the dystrophin is not normal – it is around the nucleus and not along the length of the cell.

8. 4E. Describe the results:
Results of a 6MWT (expressed in meters) over 36 months.
At 12 months, there was no difference in the distance walked between treated patients and historical controls.
At 24 months, patients treated with Eteplirsen appear to have walked further than historical controls, but the results were not statistically different.
At 36 months, the difference between the groups was greater and is statistically significant.

9. 4F. Describe the results:
The bar graph shows the proportion of patients who lost ambulation over a three-year period.
Only 2 of 12 Eteplirsen-treated patients lost ambulation over 36 months compared to 6/13 historical controls

10. 4G. Speculate on how Eteplirsen works
4G. Speculate on how Eteplirsen works? Alters splicing; exon skipping therapy
Pre-mRNA
Pre-mRNA AO 49 50 51 52 53 54 49 50 51 52 53 54
normal splicing
altered splicing
mRNA
mRNA 49 50 51 49 50 53 54
Protein synthesis
Protein synthesis
Truncated, non-functional protein
Truncated protein, partial or fully functional?
DMD
Milder dystrophinopathy phenotype

11. Eteplirsen alters splicing. The insertion of a base in exon 52 causes a frame shift resulting in expression of a truncated dystrophin protein composed of 52 exons instead of 79 exons. This truncation is devastating. The dystrophin produced is non-functional, and the patient is afflicted with DMD, the most severe form of muscular dystrophy.
Eteplirsen binds to the pre-mRNA at the exon-intron junction between intron 51 and exon 52. By preventing utilization of this splice acceptor site, the spliceosome will activate cryptic acceptor splice sites and / or use the next available acceptor site. Figures 2 and 3 suggest that at least of portion of splicing events result in the removal of intron 51, exon 52 and intron 52 in on piece. The protein produced is missing exon 52, but this deletion removes the mutation, prevents the frame shift and eliminates the in-frame stop codon.
In frame mutations tend to lead to milder forms of the disease, thus, we expect Eteplirsen treated patients will synthesize a mixture of non-functional and functional dystrophin. As untreated patients express no functional dystrophin, this treatment may benefit the patient. This is suggested by Figures 4 and 5. .

12. 4H. Post-approval Data. Describe the results
Western blot analysis of dystrophin expression.
Lanes 1-5 are a standard curved on known amounts of dystrophin.
Lane 1 is 4% of normal tissue dystrophin expression levels.
Patient G after 180 weeks of treatment expresses ~1% of normal tissue levels.
BMD patient shows expression in a patient with a milder form of disease.
*The comparison of Patient G with the standard curve reveals the treatment results in
very low levels of dystrophin expression. Yet, the BMD sample indicates that expression
of only ~3% of normal dystrophin levels is sufficient to provide clinical benefit

13. 4I. Describe the results:
Immunostaining demonstrates no dystrophin expression in untreated patients or
patient samples prior to treatment (baseline). Following treatment,
immunoreactive dystrophin is expressed.

14. 4J. Speculate on why control patient DMD1 is not amenable Eteplirsen treatment:
Only DMD patients with mutations in Exon 52 would be amenable to treatment. Thus, Eteplirsen will only benefit a subset of DMD patients.

15. 4K. Do you think Eteplirsen should remain FDA-approved?
4L. What additional information would convince you (or further support your conclusion)?
They have not demonstrated that the level of dystrophin expressed in treated patients is beneficial (improve quality of life or extend life).
They have not demonstrated that the truncated dystrophin produced as a result of exon skipping therapy has functional activity.
There is no evidence that Eteplirsen has any effect on dystrophin in cardiac muscle. DMD patients succumb to respiratory or cardiac failure. As patients can be placed on a ventilator to overcome respiratory issues, preventing cardiac failure is critical to prolonging survival in these patients.