Assignment: The Hutchinson Gilford Progeria Syndrome a Disease Question

Assignment: The Hutchinson Gilford Progeria Syndrome a Disease Question

Brief Communication https://doi.org/10.1038/s41591-018-0338-6 Development of a CRISPR/Cas9-based therapy for Hutchinson–Gilford progeria syndrome Olaya Santiago-Fernández1, Fernando G. Osorio1, Víctor Quesada 1,2, Francisco Rodríguez1, Sammy Basso1, Daniel Maeso1, Loïc Rolas3, Anna Barkaway3, Sussan Nourshargh3, Alicia R. Folgueras1, José M. P. Freije 1,2* and Carlos López-Otín 1,2* CRISPR/Cas9-based therapies hold considerable promise for the treatment of genetic diseases. Among these, Hutchinson– Gilford progeria syndrome, caused by a point mutation in the LMNA gene, stands out as a potential candidate. Here, we explore the efficacy of a CRISPR/Cas9-based approach that reverts several alterations in Hutchinson–Gilford progeria syndrome cells and mice by introducing frameshift mutations in the LMNA gene. Hutchinson–Gilford progeria syndrome (HGPS) is a rare disease characterized by aging-like manifestations emerging in childhood1. Most cases (80–90%) result from a de novo point mutation in the LMNA gene—encoding the nuclear lamins A and C—which activates a cryptic splice site in exon 11 (c.1824C >​  T; p.Gly608Gly)2,3. This event leads to the expression of progerin, a truncated lamin A variant with an internal deletion of 50 amino acids, which remains farnesylated, inducing morphological and functional alterations of the nuclear envelope4. A mouse model—LmnaG609G/G609G—recapitulating the mutation and many of the clinical features of these children5, confirmed that HGPS is caused by progerin accumulation and not by the loss of normal lamin A5,6.

Assignment: The Hutchinson Gilford Progeria Syndrome a Disease Question

Several approaches against this syndrome were tested in preclinical models7, including farnesyltransferase inhibitors, which provided clinical benefits to HGPS patients8,9. CRISPR/Cas9 gene-editing tools constitute promising alternatives for diseases such as Duchenne muscular dystrophy10, metabolopathies11 and deafness12. This system involves a Cas9 endonuclease directed by a single-guide RNA (sgRNA) that recognizes its target region, plus a protospacer-adjacent motif (PAM). The nuclease generates double-strand breaks in the DNA, repair of which through non-homologous end-joining produces insertions and deletions (indels)13. The finding that mosaic mice with both normal and prelamin A-producing progeroid cells have a completely normal phenotype14 indicates that a partial reduction in the accumulation of farnesylated lamin A products could be sufficient for an important phenotype relief. On this basis, we developed a CRISPR/Cas9-based strategy against HGPS aimed at blocking the accumulation of lamin A and progerin. The LMNA gene encodes lamin C (exons 1–10) and lamin A (exons 1–12) through alternative splicing and polyadenylation. Since lamin A appears to be dispensable5,6, our strategy would disrupt the last part of the LMNA gene, impeding lamin A/progerin production without affecting lamin C. We first designed an sgRNA (sgRNA-LCS1) with the 5′​-NGG PAM sequence of Streptococcus pyogenes Cas9 to target LMNA exon 11 upstream of the HGPS mutation, in a region conserved across both humans and mice (Fig. 1a). To test the efficacy of this approach, we cloned sgRNA-LCS1 or sgRNA-control in a lentiviral vector containing S. pyogenes Cas9 (lentiCRISPRv2) and used these to transduce Lmna+/+ and LmnaG609G/G609G murine fibroblasts. As a result, indels of variable length were produced in sgRNA-LCS1-transduced cells, as assessed by capillary electrophoresis-based fragment analysis (Extended Data Fig. 1). Immunoblot analysis showed a significant decrease in the accumulation of progerin and lamin A, while lamin C levels were not affected (Fig. 1b).

Likewise, immunofluorescence analysis demonstrated that numbers of progerin-positive nuclei were reduced by 74% in sgRNA-LCS1-transduced cells compared to sgRNA-control-transduced cells (Fig. 1c). Accordingly, we found a 65% decrease in the number of nuclear alterations in LmnaG609G/G609G cells transduced with sgRNA-LCS1compared to sgRNA-controltransduced cells (Fig. 1c). To test this system in human cells, we infected LMNAG608G/+ fibroblasts from HGPS patients and LMNA+/+ fibroblasts with these lentiviral vectors. Similar to mouse fibroblasts, we observed different indels in the DNA (Extended Data Fig. 2), a decrease in progerin and lamin A by immunoblot (Fig. 1d), an 83% decrease in progerin-positive nuclei and a 39% reduction in the number of aberrant nuclei in sgRNA-LCS1- versus sgRNA-control-transduced HGPS cells (Fig. 1e). We next tested, in vivo, this editing approach using LmnaG609G/G609G mice as an HGPS animal model. We chose an adeno-associated virus 9 (AAV9) delivery vector due to its safety and broad tissue tropism. Given the packaging limit of these viruses (approximately 5 kb), we used Staphylococcus aureus Cas9 nuclease15 and designed a new sgRNA against the same region in exon 11 with the 5′​-NNGRRT PAM sequence (sgRNA-LCS2). After packaging the vectors, with either sgRNA-LCS2 or the sgRNA-control, we injected intraperitoneally 2 ×​  1011 AAV9 genome copies in P3 LmnaG609G/G609G mice (Fig. 2a). To assess editing efficiency, we performed Illumina sequencing of the target region in DNA from AAV9 target organs— liver, heart, muscle and lung—of injected mice. Notably, Lmna contained indels in 13.6 ±​ 2.6% of the genome copies in liver, 5.3 ±​  1.0% in heart, 4.1 ±​ 0.6% in muscle and 1.1 ±​ 0.2% in lung (Fig. 2b,c; Extended Data Fig. 3; Supplementary Tables 1–4). Given the modest fraction of cells edited in vivo, the global decrease in progerin messenger (RNA) was too low for reliable detection by quantitative reverse transcription polymerase chain reaction (RT–qPCR) (Extended Data Fig. 4). However, immunohistochemical analysis revealed a significant reduction in progerin-positive nuclei in liver, heart and skeletal muscle from sgRNA-LCS2-transduced mice 1 Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Instituto Universitario de Oncología del Principado de Asturias, Universidad de Oviedo, Oviedo, Spain. 2

Assignment: The Hutchinson Gilford Progeria Syndrome a Disease Question

Centro de Investigación Biomédica en Red de Cáncer, Madrid, Spain. 3William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, UK. *e-mail: jmpf@uniovi.es; clo@uniovi.es Nature Medicine | www.nature.com/naturemedicine Brief Communication NAtuRE MEDICInE a SD PolyA Lamin C CTR LCS1 Exon 11 C >T (G608G) LCS1 Lamin A Progerin Lamin C β-Actin 4 4 3 2 1 0 37 P = 0.0084 5 P = 0.7002 P = 0.9539 3 2 1 CTR LCS1 CTR LCS1 +/+ G609G/G609G Progerin-positive nuclei (%) sgRNA-LCS1 80 60 40 20 P = 0.0018 40 30 20 10 LCS1 CTR β-Actin 37 sgRNA-CTR Progerin sgRNA-LCS1 LMNAG608G/+ e 4 3 2 1 0 P = 0.0014 8 4 Lamin C/β-actin kD 75 3 2 1 0 P = 0.9216 P = 0.3023 6 4 2 0 CTR LCS1 CTR LCS1 +/+ G608G/+ DAPI LCS1 G609G/G609G CTR LCS1 CTR LCS1 +/+ P = 0.0006 G608G/+ P = 0.0070 40 100 Nuclear defects (%) Lamin A Progerin Lamin C LCS1 5 5 Progerin/β-actin CTR P = 0.0370 Progerin-positive nuclei (%) LCS1 Lamin A/β-actin CTR G609G/G609G 0 CTR 6 1 50 0 LMNAG608G/+ 2 +/+ P = 0.0008 100 LMNA+/+ 3 CTR LCS1 CTR LCS1 G609G/G609G d 4 0 0 DAPI sgRNA-CTR Progerin LmnaG609G/G609G kD 75 Exon 12 P = 0.0015 5 PolyA Lamin A Nuclear defects (%) c 6 LmnaG609G/G609G CTR SA Lamin C/β-actin Lmna+/+ Exon 10 SD Progerin/β-actin b Exon 9 Lamin C Lamin A Progerin Lamin A/β-actin Exon 8 SD SA 80 60 40 20 0 CTR LCS1 G608G/+ 30 20 10 0 CTR LCS1 G608G/+ Fig. 1 | CRISPR/Cas9 testing in HGPS cellular models. a, sgRNA-LCS1 directs Cas9 nuclease against exon 11 of LMNA gene upstream of the HGPS mutation, disrupting lamin A and progerin without altering lamin C. b, Cropped immunoblot of lamin A, progerin and lamin C from WT and LmnaG609G/G609G mouse embryonic fibroblasts (MEFs) transduced with sgRNA-control or sgRNA-LCS1 (n =​3 independent infections and MEF lines; two-tailed Student’s t-test). c, Immunofluorescence analysis of progerin-positive nuclei and quantification of nuclear alterations by 4′​,6-diamidino-2-phenylindole (DAPI) staining (n =​3 independent infections and MEF lines; two-tailed Student’s t-test). Arrowheads indicate nuclear aberrations. d, Cropped immunoblot of lamin A, progerin and lamin C from WT and LMNAG608G/+ human fibroblasts transduced with sgRNA-control or sgRNA-LCS1 (n =​3 independent infections; two-tailed Student’s t-test). e, Progerin immunofluorescence and analysis of nuclear aberrations by DAPI staining (n =​3 independent infections; two-tailed Student’s t-test). Arrowheads indicate blebbings and invaginations.

Important information for writing discussion questions and participation

Welcome to class

Hello class and welcome to the class and I will be your instructor for this course. This is a -week course and requires a lot of time commitment, organization, and a high level of dedication. Please use the class syllabus to guide you through all the assignments required for the course. I have also attached the classroom policies to this announcement to know your expectations for this course. Please review this document carefully and ask me any questions if you do. You could email me at any time or send me a message via the “message” icon in halo if you need to contact me. I check my email regularly, so you should get a response within 24 hours. If you have not heard from me within 24 hours and need to contact me urgently, please send a follow up text to

I strongly encourage that you do not wait until the very last minute to complete your assignments. Your assignments in weeks 4 and 5 require early planning as you would need to present a teaching plan and interview a community health provider. I advise you look at the requirements for these assignments at the beginning of the course and plan accordingly. I have posted the YouTube link that explains all the class assignments in detail. It is required that you watch this 32-minute video as the assignments from week 3 through 5 require that you follow the instructions to the letter to succeed. Failure to complete these assignments according to instructions might lead to a zero. After watching the video, please schedule a one-on-one with me to discuss your topic for your project by the second week of class. Use this link to schedule a 15-minute session. Please, call me at the time of your appointment on my number. Please note that I will NOT call you.

Please, be advised I do NOT accept any assignments by email. If you are having technical issues with uploading an assignment, contact the technical department and inform me of the issue. If you have any issues that would prevent you from getting your assignments to me by the deadline, please inform me to request a possible extension. Note that working fulltime or overtime is no excuse for late assignments. There is a 5%-point deduction for every day your assignment is late. This only applies to approved extensions. Late assignments will not be accepted.

If you think you would be needing accommodations due to any reasons, please contact the appropriate department to request accommodations.

Plagiarism is highly prohibited. Please ensure you are citing your sources correctly using APA 7th edition. All assignments including discussion posts should be formatted in APA with the appropriate spacing, font, margin, and indents. Any papers not well formatted would be returned back to you, hence, I advise you review APA formatting style. I have attached a sample paper in APA format and will also post sample discussion responses in subsequent announcements.

Your initial discussion post should be a minimum of 200 words and response posts should be a minimum of 150 words. Be advised that I grade based on quality and not necessarily the number of words you post. A minimum of TWO references should be used for your initial post. For your response post, you do not need references as personal experiences would count as response posts. If you however cite anything from the literature for your response post, it is required that you cite your reference. You should include a minimum of THREE references for papers in this course. Please note that references should be no more than 5 years old except recommended as a resource for the class. Furthermore, for each discussion board question, you need ONE initial substantive response and TWO substantive responses to either your classmates or your instructor for a total of THREE responses. There are TWO discussion questions each week, hence, you need a total minimum of SIX discussion posts for each week. I usually post a discussion question each week. You could also respond to these as it would count towards your required SIX discussion posts for the week.

I understand this is a lot of information to cover in 5 weeks, however, the Bible says in Philippians 4:13 that we can do all things through Christ that strengthens us. Even in times like this, we are encouraged by God’s word that we have that ability in us to succeed with His strength. I pray that each and every one of you receives strength for this course and life generally as we navigate through this pandemic that is shaking our world today. Relax and enjoy the course!

Hi Class,

Please read through the following information on writing a Discussion question response and participation posts.

Contact me if you have any questions.

Important information on Writing a Discussion Question

• Your response needs to be a minimum of 150 words (not including your list of references)
• There needs to be at least TWO references with ONE being a peer reviewed professional journal article.
• Include in-text citations in your response
• Do not include quotes—instead summarize and paraphrase the information
• Follow APA-7th edition
• Points will be deducted if the above is not followed

Participation –replies to your classmates or instructor

• A minimum of 6 responses per week, on at least 3 days of the week.
• Each response needs at least ONE reference with citations—best if it is a peer reviewed journal article
• Each response needs to be at least 75 words in length (does not include your list of references)
• Responses need to be substantive by bringing information to the discussion or further enhance the discussion. Responses of “I agree” or “great post” does not count for the word count.
• Follow APA 7th edition
• Points will be deducted if the above is not followed

• Remember to use and follow APA-7th edition for all weekly assignments, discussion questions, and participation points.
• Here are some helpful links
• The is a great resource