Icon Legend

This session is not in your schedule.

This session is in your schedule. Click again to remove it.

Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session D

Poster I9 - RNA-guided Transcriptional Upregulation of Neuromuscular Disease Modifiers Using CRISPR-DREAM

Friday, October 25, 2024

3:30 PM – 4:30 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Ian Popp (he/him/his)

Undergraduate Student
University of Arkansas
Fayetteville, Arkansas, United States

Primary Investigator(s)

CN

Christopher Nelson, PhD

Assistant Professor
University of Arkansas
Fayetteville, Arkansas, United States

Co-Author(s)

EA

Eliana Adamos

Undergraduate Researcher
University of Arkansas Fayetteville, United States

Introduction: Duchenne Muscular Dystrophy (DMD) is an X chromosome related neuromuscular genetic disease that affects 1 per 5,000 male births. Duchenne Muscular Dystrophy results in progressive wasting of muscle leading to mortality. DMD is caused by a mutation that affects the production of the dystrophin protein [1, 2]. Utrophin (a paralog of dystrophin) can alleviate the impact of dystrophin protein deficiency. Gene therapy techniques have been used to increase the expression of the compensatory protein utrophin (gene name UTRN). When UTRN RNA expression is increased, more utrophin proteins will be produced by the cell, alleviating the negative effects from the dysfunctional dystrophin proteins [3]. In addition, there are a number of other targets that might modify disease progression including myostatin, myostatin receptor, IL-10, and Klotho [4, 5, 6, 7]. These target sequences were transformed into CRISPR-DREAM plasmids for transcriptional regulation. CRISPR-DREAM is a promising version of CRISPR-dCas9 with transcriptional activation domains (TADs) derived from humans [8]. This research investigates the transcriptional efficacy of the regulation of protospacer sequences individually and in combination with each other in the mouse skeletal muscle myoblast C2C12 cell line. Gene targets are combined to harness the multiplex and orthogonal capacity of CRISPR transcriptional regulation. In this study, we will investigate synergistic or competing effects to investigate a more holistic approach to gene therapies in neuromuscular disorders.

Materials and

Methods: Sanger sequencing was used to confirm the identities of the plasmids. Oxford nanopore sequencing was used for full-plasmid sequencing to confirm identity of all cloned plasmids. pDNA midipreps were used to purify the plasmids following sequence verification. Mouse myoblast cells (C2C12) were transfected with Lipofectamine 3000 using the purified plasmid according to manufacturer protocol. The cell media was collected and replaced for all samples every three days and media was stored in -80C for downstream analysis. The cell media was analyzed using enzyme-linked immunoassay (ELISA). Western blots were used to measure the quantity of produced proteins and immunostaining to confirm protein identity. The cells were harvested for RNA and qRT-PCR was used to measure expression generated for each protospacer sequence or combinations of protospacers.

Results, Conclusions, and Discussions: Sanger sequencing confirmed the presence of the protospacer sequences in the respective CRISPR-DREAM plasmids. Whole plasmid nanopore sequencing confirmed the identity of all cloned plasmids. We performed several experiments in C2C12 myoblasts. GFP fluorescence confirmed transfection of DREAM plasmids after 24h. Initial qRT-PCR testing of IL-10 yielded no significant upregulation. Future experiments will screen additional protospacers and synergy between multiple protospacers. Transfection of Utrn targeted protospacers showed high levels of GFP expression and qRT-PCR results will reveal activity of designed protospacers. Future work will continue to investigate gene expression modulation by individual targets and synergy between multiple targets. Synthesis of recombinant AAV encoding each protospacer will be used in mdx mice to investigate improvement in muscle phenotype in vivo.

Acknowledgements (Optional): We would like to acknowledge Isaac Hilton and Barun Mahata for providing plasmids and helpful discussion. This work was supported by the 21st Century Chair in the Department of Biomedical Engineering. Ian Popp was supported by a University of Arkansas Honors College Research Grant.

1. Elangkovan, Nertiyan, and George Dickson. “Gene Therapy for Duchenne Muscular
Dystrophy.” Journal of Neuromuscular Diseases, vol. 8, no. Suppl 2, pp. S303–16. PubMed Central, https://doi.org/10.3233/JND-210678.
2. Venugopal, Vijay, and Steven Pavlakis. “Duchenne Muscular Dystrophy.” StatPearls,
StatPearls Publishing, 2024. PubMed, http://www.ncbi.nlm.nih.gov/books/NBK482346/.
3. Wu, Ruo, et al. “Promising Therapeutic Approaches of Utrophin Replacing Dystrophin in
the Treatment of Duchenne Muscular Dystrophy.” Fundamental Research, vol. 2, no. 6, Nov. 2022, pp. 885–93. ScienceDirect, https://doi.org/10.1016/j.fmre.2022.07.004.
4. Carnac, Gilles, et al. “Myostatin in the Pathophysiology of Skeletal Muscle.”
Current Genomics, vol. 8, no. 7, Nov. 2007, pp. 415–22. PubMed Central, https://doi.org/10.2174/138920207783591672.
5. Rodgers, Buel D., and Christopher W. Ward. “Myostatin/Activin Receptor Ligands in
Muscle and the Development Status of Attenuating Drugs.” Endocrine Reviews, vol. 43, no. 2, Sept. 2021, pp. 329–65. PubMed Central, https://doi.org/10.1210/endrev/bnab030.
6. Iyer, Shankar Subramanian, and Genhong Cheng. “Role of Interleukin 10 Transcriptional
Regulation in Inflammation and Autoimmune Disease.” Critical Reviews in Immunology, vol. 32, no. 1, 2012, p. 23. www.ncbi.nlm.nih.gov, https://doi.org/10.1615/critrevimmunol.v32.i1.30.
7. Castner, Stacy A., et al. “Longevity Factor Klotho Enhances Cognition in Aged
Nonhuman Primates.” Nature Aging, vol. 3, no. 8, Aug. 2023, pp. 931–37. www.nature.com, https://doi.org/10.1038/s43587-023-00441-x.
8. Mahata, Barun, et al. “Compact Engineered Human Mechanosensitive Transactivation
Modules Enable Potent and Versatile Synthetic Transcriptional Control.” Nature Methods, Oct. 2023, pp. 1–13. www.nature.com, https://doi.org/10.1038/s41592-023-02036-1.