NAME_____________________________________________________________TA__________________ SEC____7.012 Problem Set 7 FRIDAY December 3, 2004Not due unless you have had a prior agreement with Claudette Gardel Solutions will be posted on the web.Question 1Leukemia is type of cancer characterized by the uncontrolled proliferation of white bloodcells (leukocytes). Chronic Myelogenous Leukemia (CML) is a type of leukemia primarilycaused by a translocation that relocates an oncogene, called abl, from the long arm ofchromosome 9 to the long arm of chromosome 22 in the bcr region (breakpoint clusterregion). The resulting bcr-abl fusion protein encodes a constitutively active tyrosinekinase, which when expressed, leads to the CML phenotype.a) Gleevec is an effective drug treatment of CML. What design principles were applied tothe development of Gleevec? How is this approach different from that used to developconventional cancer therapies?Rational drug design was applied to the development of Gleevec. The idea is to design a drug thatspecifically targets the underlying molecular defect, rather than nonspecifically target alldividing cells, as do many conventional cancer therapiesb) How does Gleevec work at the molecular level?Gleevec works by binding to the ATP binding site of bcr-abl thus inhibiting its tyrosine kinaseactivity.c) How many protein targets does Gleevec have? Name each target.Gleevec does bind to two other tyrosine kinases: kit and the PDGF receptor.d) Besides target specificity, what other characteristics of a drug should you considerwhen designing a therapy?Toxicity, absorption efficiency, is it metabolized, possible reactivity of metabolites, etc.In contrast to drug treatments, gene therapy attempts to correct the defect byintroducing a functional copy of the malfunctioning gene that is responsible for thedisease phenotype. The functional gene copy can be introduced directly into the diseasedorgan or can be used to genetically modify isolated tissue that is later re-introduced intothe patient.e) What do you need to know about the target disease in order to apply gene therapy?You need to know the gene involved in the disease. In particular, the disease should be caused bya defect in a single gene.MIT Biology Department7.012: Introductory Biology - Fall 2004Instructors: Professor Eric Lander, Professor Robert A. Weinberg, Dr. Claudette Gardel2f) How could you deliver the functional gene into the diseased cells?A viral vector could be used (example: adenovirus).g) What do you think are some of the challenges facing gene therapy?Challenges include efficient uptake of the functional gene, stability of the gene,long term expression, side effects of viral vector.h) Based on your understanding of oncogenes, why would bcr-abl be a particularlychallenging target for gene therapy?You would have to replace the fused bcr-abl gene with a functional copy of theabl tyrosine kinase, not simply introduce the functional copy into the cell.3Question 2Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder caused by mutations in thegene encoding dystrophin, a protein involved in maintaining membrane integrity in muscle cells.The dystrophin gene spans roughly 2.5 Mb and is spliced to form a 14 kb mRNA transcriptconsisting of 79 exons.a) Is dystrophin is a typical human gene in terms of its size and exon count?Dystrophin is encoded by one of the largest human genes. Although there is great variation bothin gene size and exon count in the human genome, the average human gene length is roughly10-15 kb and average exon count is approximately 4-8 exons per gene.b) Why is DMD much more common in boys than in girls?The gene is on the X chromosome and confers a recessive phenotype. Females have 2 X chromosomesc) The DMD phenotype results from mutations that disrupt the reading frame of the dystrophinmRNA. What is the impact of such mutations on the dystrophin protein?A frameshift mutation in the gene would result in a truncated protein productd) Based on what you know of dystrophin’s normal function described above, what do you think arecharacteristics of the DMD disease phenotype?Muscle cells would be severely damaged in response to the normal pressures exerted on them.Symptoms of DMD include severe muscle damage and muscle weakness.The dystrophin protein contains several functionally distinct regions (domains). The N-terminal domaininteracts with proteins inside the muscle cell, while the C-terminal domain enables dystrophin to bind tomembrane-associated proteins. The long rod-like central part of the protein is composed of 24 identicalrod domains (internal repeats). As a consequence of the modular structure of dystrophin, proteins missingsome of the internal repeats can be fully functional or at least partly active.One therapeutic strategy involves excluding, or skipping, an internal exon during splicing. Inparticular, skipping the exon that contains the frame-shift mutation in the DMD individual canresult in a shorter than wild type but functional dystrophin protein. This exon-skipping therapyinvolves introducing a small RNA molecule that is complementary to the exon to be skipped.Binding of the RNA to the pre-mRNA prevents proper recognition of the exon by the splicingmachinery and results in specific exon skipping.e) In order for this therapy to work, what properties must this small RNA have? (Think aboutcellular localization.)The therapeutic RNA would need to localize to the site of splicing to cause efficient exon skipping.Not only does the RNA need to enter the nucleus, but it must bind to its target exon on the pre-mRNA. In fact, the small RNA can be engineered to bind to a specific protein that localizes to thespliceosome! It is also important the RNA be stable and resistant to rapid degradation in the cell.f) How would you deliver the RNA molecule into the target cell?You could use a viral vector to introduce a gene encoding the therapeutic RNA into the cell(allows for efficient uptake and expression).N-terminaldomainC-terminaldomain4g) How is this therapeutic approach different from gene therapy?This approach is different from gene therapy in that it modifies the spliced mRNA rather than justcounteracting the genomic defect by introducing a functional copy of the whole gene.h) What challenges do gene therapy and exon-skipping therapy have in common?Efficient uptake, stability and expression of the foreign molecule, side effects due to viral vector,etc.Question 3a) What
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