Handout 8-Pre-mRNA/DNA Gene Therapy for Tazswana 5 Exon 23mm 5 5\' Exon3 (dotted
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Handout 8-Pre-mRNA/DNA Gene Therapy for Tazswana 5 Exon 23mm 5 5' Exon3 (dotted rectangles) Exon 2 Exon 3 Normal mRNA Exon Abnormal mRNA Exon 3 ) Figure 8-I. Gene Therapy for Tazwana. Alternative splice patterns are shown. arrow) results in exon 2 juxtaposed to exon 3. The alternative splice pattern (solid lines and arrow of a portion of the intron between exons 2 and 3. Oligonudleotides (xo nt long) an affect -globin pre mRNA. The -globin pre-mRNA (top offigure) that causes Tmwanas Adalasemia is depicted. The mutation causing the aberrant s' splicing sequence is represented by a checkered-bor. The crypdc s splicing sequence Wild-type splicing (dashed line and Figure modified from "Therapeutic potential of antisense oligonucleotides as modulators of akternative splicing" Sazani, P and R. Kole. (2003) The Journal of Clinical Investigation 112(4)481-486 Questions I. What is "alternative splicing" and how does it apply to Tazswana's case 2. Design Tazswana's gene therapy to force the splicesome into the "normal" alternative splice pattern. a. Propose a complementary DNA sequence to interact (hybridize) with Tazswana's pre-mRNA. Draw the 1o nt DNA oligonucleotide that will complementary base-pair to the pre-mRNA to specifically "mask the extra s' splice site (underlined). Don't mask the normal splice sites! Assume her intron sequence for the pre mRNA is as shown here 5'GUAAGUCUAAUGUCCCCUCAGAAGAAGGUCACUCUAACGACUCUUCAG3' b. Discuss the type of cell the oligonucleotides need to be delivered to in order to affect alternative splicing of the -globin pre-mRNA. . Recall that splicesomes are composed of snRNA and protein. Explain how hybridization (complementary base-pairing) of the DNA oligonucleotides to the cryptic and aberrant pre-mRNA sequences would correct the splicing defect 4. If Taswana's s' splice site at the correct location (exonz/intronz boundary) had a point mutation from G to U so that the original 5' splice site read "UU" instead of "GU," would the gene therapy correct her B-thalassemia? Explain your answer. s. (Optional) Since the oligonucleotides target splice site consensus sequences that are common to all introns, wouldn't they potentially cause side-effects in many cell types? Discuss within your group and come up wich a. Potential side-effects b. Plan to limit side-effects. 6. If you were in Tazswanas family, would you want Tazswana to undergo gene therapy treatmente Explain why or why not.Explanation / Answer
1. Alternative splicing is the mechanism of RNA splicing variation in which the pre-mRNA's exons are detached and rejoined so as to generate arrangement of alternative ribonucleotide. These linear combinations then bear the translation process where amino acid's particular and unique sequences are stated, causing in isoform proteins. Alternative splicing promote the production of a large variety of proteins. In Tazswana’s case, introns part that was known to be spliced out was maintained, finaly designing a new exon, and a new sequence of mRNA.
2.
a. An oligonucleotide GTCTTCTTCC that could complementary base pair to the sequence of mRNA sequence so that the aberrant splice site would be masked. This would permit the spliceosome to splice out the original intron.
b.The oligonucleotides have to be brought to the cells that produce the red blood cells. These hematopoetic progenitor cells are situated in the red bone marrow. These cells must be aimed because they, dissimilar red blood cells, have nuclei where processing of pre-mRNA is occurring.
3.. The DNA oligonucleotides hybridization would rectify the splicing defect so the join both exons once again, As if the process never occured. When these oligonucleotides do this, they revise the nuclear information therefore, the gene splicing would be carried out correctly in the accurate spots, particularly the 5’ splice site.
4. The gene therapy would rectify beta thalassemia if the 5’ splice site is spliced in the accurate, having a point mutation from G to U so that it can read “UU” instead of “GU”. The gene therapy would essentially revise the sequence, operating the splicing correctly and removing the gene defect of beta thalassemia from her genetic code.
5.
a. The engineered oligonucleotide could finish influencing body's another cells obstructing with splicing processes. This could generate potentially many defective proteins and many influence the cell functioning.
b.To limit side effects, we might take aTazswana’s bone marrow sample and exposed the sample to the treatment of oligonucleotide. These treated hematopoetic cells could then be reinjected into her bone to give healthy blood cells.
6. If we were inTazswana's family, we would confidently want her to undergo the gene therapy treatment. Since it would recover a disease that results in many side effects as well as a poor quality of life. The gene therapy would recover her from the genetic defect, therefore helping her in a normal life.
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