6 Resist - Notes, video links, questions on HIV and gene editing with Dr Linda Martin-Morris PDF

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Notes, video links, questions on HIV and gene editing with Dr Linda Martin-Morris and Dr Clemens Cabernard...

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Biology 355

Project 6 – RESIST! Autumn 2017

Learning objectives of this project: - Relate GPCR and chemokine signaling from project 3 with HIV infection. - Apply information about the pathway of infection to predict potential therapeutic opportunities. - Understand how cell surface molecules can be used for identification, functionality, and be co-opted by pathogens. - Understand genome editing techniques – and how DNA recognition, DNA digestion, and innate DNA repair are being turned into therapeutic potential. - Tackle consideration of ethical nuance in genome editing.

Part 1 – Genome Editing Therapy A) - PREPARATION (in advance) 1 – Start with your textbook. Topic Immunology – T cells Virology – HIV DNA repair

Alberts 6th edition Figure 24-11, pages 1326-1332 Pages 1279-1280 273-276

Karp 7th edition Figure 17.2, pages 707-710 Pages 23-26 Pages 564-568

2 – Examine this model figure (source: PMID 20609987). We are looking at a model figure that describes the HIV retroviral “life cycle”. Within the boxes are several antiretroviral medications used by HIV infected individuals. The --| symbol indicates that these drugs are blocking the specified aspect of viral function. Guiding questions for figure:

  

  infection?

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What is a retrovirus and how does their genome get integrated into the host cell’s genome? Which antiretroviral drug blocks this (previous question) step? Which antiretroviral drug is in the category of “protease inhibitors” and what viral process is not happening when they are used? What type of a molecule is CCR5 or CXCR4 and what is its role in HIV infection? What would distinguish between a treatment that reduces viral impact vs. a cure that would eliminate

Biology 355

Project 6 – RESIST! Autumn 2017

3 – Watch to the “Method of the Year” video (2011) on Genome Editing. https://youtu.be/zDkUFzZoQAs Guiding questions: What is a gene editing nuclease? What can one do to blood cells to help them “not let the [HIV] virus in”? What are the two parts to a gene editing nuclease? What are TALENs? How precise are the gene edits accomplished by engineered gene editing nucleases? 4 – Watch the video at https://youtu.be/2pp17E4E-O8 on CRISPR-Cas9. Guiding questions:  Why do bacteria have Cas9?  Gene editing with CRISPR-Cas9 substitutes the “guide RNA” with an engineered RNA for what?  CRISPR-Cas9 is good at making mutations when the double strand break is repaired. But what is needed in order to FIX mutations?  What cell types can be edited with CRISPR-Cas9 system? 5 – Obtain PMID 27741222 and read the abstract. Guiding questions:  What must a T cell have on its surface to be infected by HIV?  Gene disruption was tested in cell lines and primary cells. What are these and what did the disruption accomplish?  What is engraftment and why do we care? B) LECTURE (55 minutes). We’ll have a pre-project lecture that introduces you to the immune system and T-cells specifically, virology and HIV specifically, and genome editing. C) DISCUSSION (15 minutes) Meet with your group and work out your understanding of the model figure, guiding questions, and the abstract D) SUMMARIZE (5 minutes) Part 2 – How CCR5 Became VERY Interesting to HIV Investigators! A) PREPARATION (In advance) 1 – Read the abstracts for the following articles: PMID 10383387, 19213682, 21994649, and 24927590. From the abstracts, build a timeline of what we have learned about the role of CCR5 and HIV infection. Write ONE take-home lesson for each abstract.

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Biology 355

Project 6 – RESIST! Autumn 2017

2 – Read up a bit on FACS: Video https://www.youtube.com/watch?v=TDRhCWaYRsg Overview http://www.antibodies-online.com/resources/17/1247/what-is-flow-cytometry-facs-analysis/ FACS data http://www.bioinformin.net/cytometry/flow_plots.php Super-“simple” summary https://www.linkedin.com/pulse/flow-cytometry-facs-simple-method-guidekarthik-raman-phd B) INTRODUCE (15 minutes) – Timeline of research toward potential HIV cure. C) DISCUSS (25 minutes) – Methods – Flow Cytometry Worksheet D) SUMMARIZE (20 minutes) – Flow summary and how to look at gels for editing confirmation E) WRAP UP (10 minutes)

Part 3 – A Recent, and Local Paper – Edit CCR5 – RESIST HIV! A) PREPARATION (In advance) 1 – Read PMID 27741222 (you previously read the abstract). Focus on figures 2a, 2b&c, 4c-f, and – 5. Your TA will notify you which figure you are responsible to present to the section. Unlike previous presentations, this time, tell the BIG PICTURE story and use the data to support it (rather than explain the data to build to the big picture).

B) INTRODUCE (5 minutes) TA will set the stage for this paper. C) DISCUSS (10 minutes) Groups have a little time to solidify their understanding of the figures. D) PRESENT (25 minutes) Explain your solution to the data given to a classmate. E) SUMMARIZE (10 minutes) TA will wrap as time allows.

Part 4 – Host-Pathogen Arms Race A) PREPARE (In advance) 1 – Finish reading the assigned paper (if you have not). 2 – Read abstract to PMID 25939314. What did this paper show about CCR5? B) INTRODUCE (20 minutes) Figure 5 and conclusion. C) BRAINSTORM (15 minutes) What could be the consequences of introducing edited DNA into individuals? What are the ethical concerns and why might this “meddling” fail? 3

Biology 355

Project 6 – RESIST! Autumn 2017

D) WRITING LAY LITERATURE (10 minutes) We will lay out the plan for considering how and why one writes for non-scientists. Your writing teams will be comprised of 2-4 students, from any section and from any group.

Part 5 – Why Should Non-Science People Care? A) PREPARE (In advance) 1 – Read The Economist’s article on CrispR and ethics. https://www.economist.com/news/leaders/21661651-new-technique-manipulating-genes-holdsgreat-promisebut-rules-are-needed-govern-its B) INTRODUCE (10 minutes) C) WRITE (Remaining time) Here are your article’s criteria: 1) Must be accessible. Does it pass the “grandparent test” (could your non-scientist grandparent understand all the terms you use and what the main message is?)? 2) Must be attention-grabbing. Does article (title and beginning) make a reader want to know more? Does article help one feel personally connected to the story (local science, impactful to many who have loved ones with HIV)? 3) Must be descriptive of research on CCR5 and HIV. You should relate to the paper read without getting technical about how they came to their conclusions. You should relate to the abstracts read to build context and history. 4) Any science digest (from ones found in The Economist, to those found in Science News and Science Daily) must be brief enough to keep a reader’s attention. You will write as if you are a contributor to UW’s DAILY newspaper. Your article will therefore need to be brief enough to fit in such a publication. Your word count, including title, will be limited to 600 words. 5) This article will be a do-ahead portion of your second midterm exam. It will be worth 14 points.

AFTERMATH 1 – Critical Thinking Questions a. Why are the authors following only CD4+ cells? b. What different question underlies studying both blood and spleen in this study? c. We saw examples of experimental data and graphs quantifying those outcomes. The graphs had p-values. From an experimental perspective, what was required in order to obtain these p-values (not what statistical tool, but what experimental tool)? d. Antiretrovial therapy (ART) is working but is criticized. e. BFP (blue fluorescent protein) and GFP are both used in this project. What is there GENERAL role and their specific roles? f. These experiments look at cells in culture (primary cell lines) and in vivo (in mice). Why?

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Biology 355

Project 6 – RESIST! Autumn 2017

2 – Do Post-Project practice quizzes. 3 – Write your abstract of the four data figures in this project. Abstracts will be graded based on clarity, correctness, inclusion of BQMOC elements, completeness, and brevity. Do not exceed 275 words. 4 – Study guide: We expect you to know about/how to: Relate GPCR and chemokine signaling from project 2 with HIV infection. Apply information about the pathway of infection to predict potential therapeutic opportunities. Understand how cell surface molecules can both receive signals and mediate cell-cell interactions. Understand genome editing techniques – and how DNA recognition, DNA digestion, and innate DNA repair are being turned into therapeutic potential. Become an expert in the scientific abstract (reading and writing).

Test yourself:  Name the molecules in project 2 and 3 that served analogous roles as well as naming that role (or category of molecule)  Based on the experiments performed in project 2, what would you expect CCR5 does in addition to binding to HIV?  What is ART in the context of HIV?  Name one TARGET of ART in the context of HIV. Name one alternate approach to affecting HIV aside from the ART and CCR5 based approaches.  Describe the cellular localization of GPCRs (be thorough!)  Name TWO different ways one can inhibit a GPCR.  What do CRISPR-Cas9 and megaTAL have in common?  PCR discovery was based on understanding the basic principles of DNA polymerase and replication. What basic principles of cell biology were the inspiration of genome editing research?  Read the abstracts associated with these PMIDs. About ONE of them, we will ask you to identify the QUESTION and explain a new vocabulary term. PMID 26674113 and 25840792  WRITE an article for non-scientists that describes the work in the paper you read and also considers ethical ramifications.

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