Genetic Engineering in Insulin Production as a Potential Cure for Diabetes Type 1 – CRISPR, Stem Cell Programing, and Nanofibrous Devices

Abstract

    Type 1 Diabetes is a serious chronic disease characterized by lack of insulin  production by the beta cells of the pancreas. Despite the great efforts that have been made to find a cure for this disease, one is yet to be proven efficient in order for the affected individuals to give up on insulin-dependent treatments. However, with recent molecular genetic advancements, promising results were pointed to by genetic engineering techniques that might allow for the production of functional beta cells and implicitly the discovery of a permanent cure. Accurate manipulation of stem cells genes using accessible techniques such as CRISPRs allow scientists to give rise to pluripotent cells that will eventually differentiate into insulin producing cells. 

Introduction

    Type 1 Diabetes affects about 8% of Diabetes patients compared to 90% of Type 2 Diabetes. However, DT1 is a more severe disease, with the beta cells within the Islet of Langerhans of the pancreas producing little to no insulin. For this particular reason, patients diagnosed with this disease almost always become insulin-dependent, may it be with injectable insulin or insulin pumps. With no insulin production, individuals suffering from DT1 fail to regulate the high blood sugar and to use glucose for energy purposes. The cause of the disease is still unclear to scientists, however, it is known that cytotoxic T cells attack the pancreatic beta cells (Fig. 1). Research points to causes such as viral infections and predisposition of certain genetic pools possessing haplotypes of human leukocyte antigen (HLA) complexes (Diabetes mellitus type 1).  

                                            (Fig. 1 Cytotoxic T cell attacks a beta cell of the pancreatic islet)

    Ever since the discovery of expression vectors and mass production of insulin, life expectancy and quality of individuals affected by DT1 increased significantly to even normal life expectancy. However, complications were encountered in administering this kind of treatment such as hypoglycemia and immune responses against foreign antigens (Diabetes mellitus type 1). These issues call for further investigation and technique proposals that will finally bring up a cure for the disease. These solutions have recently been identified as genetic engineering of human embryonic stem cells, pluripotent cells that will eventually divide into pancreatic beta cells capable of suitable insulin secretion and self regulation. 

The Use of Induced Pluripotent Stem Cells (iPSC) as a Promising Treatment

    Human embryonic stem cells have been largely engineered to mature into iPSC. A study conducted by D’Amour group developed pancreatic progenitor cells rather than islet cells. These were implanted into diabetic mice (Fig. 2). After about 3 months, the cells matured into islet cells and the mice began to show regulated blood glucose levels (Kroon et al., 2008). Ever since this staggering result, the project showed promising results in human clinical trials. However, just like the case of autoimmune responses that cause the disease, destroying the beta cells, the iPSC implants pose the same issue. Individuals with high risk of developing DT1 were linked to about 18 loci in their genome. These loci contain several genes that have been labeled IDDM1 to IDDM18. The most relevant of these regions is IDDM1 which contains HLA genes accounting for immune response proteins attacking beta cells. There are also non-HLA genes: IDDM2 and another close to the CTLA4 gene that is responsible for immune responses (Dean & McEntyre, 2004). Therefore, we notice how the genetic map of DT1 is tremendously complex, with genes overlapping in regions not necessarily related to insulin, but rather to immune control. The same issue does not apply to stem cell implants, as they pose a different issue: the major histocompatibility complexes (MHC) that will activate immune responses of the host regardless of the nature of the implant. Efforts have been made to knock out genes coding for MHC2 in iPSC, but they proved to be ineffective. As a result, scientists have engaged different delivery methods such as credit card-sized devices filled with cells or micro islets individually coated with shielding polymers (Drew, 2021). The efforts for a better immune tolerance of these foreign bodies are meant to take patients of the life-long immunosuppression treatments.  

                                                (Fig. 2 Development of cell lines in vitro and implantation in vivo)

Clustered Regularly Interspaced Palindromic Repeats (CRISPR) Used in Gene Therapy for Type 1 Diabetes

    Research conducted at University of Alicante, Spain pointed to the discovery of what later was established by Jennifer Doudna and Emmanuelle Charpentier as the CRISPR Cas9 complex (Fig. 3), a molecular tool used by bacteria to gain adaptive immunity against viral infections. When the viral RNA or DNA is present in the cytosol of a bacteria, the latter uses this complex to digest the foreign genetic material. The Cas (CRISPR associated protein) has a DNA binding domain and a nuclease domain that digests the DNA. In addition, a complex of two RNAs aids in the recognition of sites that need to be digested. This site is usually 20 base-pairs long, giving the mechanism high specificity. Immediately downstream of the recognition site, called a protospacer, bound by the CRISPR RNA (crRNA) is a three base-pair long protospacer adjacent motif (PAM) that stabilizes the binding of the crRNA to the protospacer. The other RNA found in the complex is a trans-activating RNA (tracrRNA)  that also plays a role in guiding the Cas9 nuclease to the specific site. The complex has been thus engineered to consist of a single-guide RNA (sgRNA), containing both the crRNA and tracrRNA. When activated, the complex recognizes the protospacer and produces double-stranded breaks in the DNA that can be repaired via homology direct repair (HDR) or non-homologous end joining (NHEJ) (Fig. 2). This characteristic allows for exact knock-in or knock-out using a template that will insert or delete desired sequences or even single base-pairs. Previously encountered sequences of viral genetic material are integrated in the bacterial genome and are highly methylated so digestion is prevented. 

    (Fig. 3 structure of the CRISPR Cas9 complex, Homology Direct Repair and Non-Homologous End Joining mechanisms)

    This entire mechanism can be molecularly engineered to modify the nuclease domain of the Cas9 protein so mutations can be corrected in diseases such as cancer, sickle cell disease, diabetes, and many more. Considering these aspects, we are going to explore the nature of the DT1 genetics and the transcription factors involved in it that are often mutated, leading to abnormal protein expression and islet differentiation. As mentioned in the previous section, cellular reprogramming can be a crucial tool in regaining the endocrine function of the islets and this can be done using the powerful tool that CRISPR offers. 

    There are several genes that code for transcription factors required for cell differentiation of the pancreatic islets. However, the most extensively studied genes include those coding for early pancreatic progenitor formation: pancreas/duodenum homeobox protein 1 (PDX1), forkhead box A2 (FOXA2), and sex determining region Y-box 17 (SOX17); and those coding for lineage specification and differentiation, such as Neurogenin 3 (NEUROG3) and neurogenic differentiation 1 (NEUROD1). Moreover, maturation of beta cells is realised through factors such as V-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MAFA), V-maf musculoaponeurotic fibrosarcoma oncogene homolog B (MAFB), paired box gene 6 (PAX6), and estrogen-related receptor gamma (Zhu et al., 2017). PDX1 in particular is required for early embryonic pancreatic development, as a study shows in a 5 year old female diagnosed with pancreatic agenesis due to a point mutation deletion in the PDX1 gene (Stoffers et al.). In addition to that, Bevacqua et al. conducted thorough research demonstrating the applicability of CRISPR systems in treatment of DT1. The scientists targeted a point mutation in the exon 1of the PDX1 gene via lentiviral transduction in mice. Fluorescent cells were marked as controls and results showed that insertion-deletion mutations were observed in as high as 66% of sequences by TIDE PCR and in 48% of sequences with ddPCR. Moreover, 72% reduction of PDX1+, Green Fluorescent Protein+ cells was noted by immunostaining. Another exciting result within this research showed that indels were absent in seven out of seven expected off-targets, reassuring that CRISPR is a highly effective and precise tool that has potential to cure the disease and reprogram stem cells (Bevacqua et al., 2021). 

    The next step that takes us further in the journey to finding a safe and effective cure for DT1 turns out to be implant nanodevices that contain stem cells genetically engineered using CRISPR that will differentiate and fully act as pancreatic islets and maintain normoglycemia in an organism. Efforts have been made in this sense in two comprehensive studies by McCloskey (McCloskey et al., 2020) and Wang (Wang et al., 2021) that show success in safe delivery of insulin by nanofibrous encapsulation devices in mice. However, this method is still in its infancy and is yet to be designed to be tried in human organisms. 

Conclusion

    Great efforts have been made to find a safe and efficient production and delivery method of insulin. As we noticed in the previous points, there is a great implication of genetic aspects that affect the complexity of Type 1 Diabetes. Scientists have been able to find solutions for the treatment of diabetes, but too few measures were taken towards finding a cure. However, with the new era of genetic engineering we notice that cures for a great range of diseases are on the horizon. CRISPR is a powerful, simple, and accessible tool that might play a pivotal role in reprograming cells, tissues, and ultimately organs to perform their proper function and cure disease. We have also noted how CRISPR in combination with stem cells is currently pointing to finer molecular adjustments that will have a larger and more effective applicability to organisms than TALENs and ZFNs that involve significantly higher cytotoxicity and less specificity. 

    Corroborating these arguments, we are still left with experimental gaps in CRISPR applications that will, however, be covered by future research accompanied by our current promising knowledge in this domain. 

WORKS CITED

Bevacqua, R. J., Dai, X., Lam, J. Y., Gu, X., Friedlander, M. S. H., Tellez, K., Miguel-Escalada, I., Bonàs-Guarch, S., Atla, G., Zhao, W., Kim, S. H., Dominguez, A. A., Qi, L. S., Ferrer, J., MacDonald, P. E., & Kim, S. K. (2021, April 23). CRISPR-based genome editing in primary human pancreatic islet cells. Nature News. Retrieved May 3, 2022, from https://www.nature.com/articles/s41467-021-22651-w 

Dean, L., & McEntyre, J. R. (2004). The genetic landscape of diabetes. NCBI, NIDDK. 

Drew, L. (2021, July 14). How stem cells could fix type 1 diabetes. Nature News. Retrieved April 30, 2022, from https://www.nature.com/articles/d41586-021-01842-x#ref-CR7 

Kroon, E., Martinson, L. A., Kadoya, K., Bang, A. G., Kelly, O. G., Eliazer, S., Young, H., Richardson, M., Smart, N. G., Cunningham, J., Agulnick, A. D., D’Amour, K. A., Carpenter, M. K., & Baetge, E. E. (2008). Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin-secreting cells in vivo. Nature Biotechnology, 26(4), 443–452. https://doi.org/10.1038/nbt1393 

McCloskey, A. G., Miskelly, M. G., Moore, C. B. T., Nesbit, M. A., Christie, K. A., Owolabi, A. I., Flatt, P. R., & McKillop, A. M. (2020). CRISPR/Cas9 gene editing demonstrates metabolic importance of GPR55 in the modulation of GIP release and pancreatic beta cell function. Peptides, 125, 170251. https://doi.org/10.1016/j.peptides.2019.170251 

Stoffers, D. A., Zinkin, N. T., Stanojevic, V., Clarke, W. L., & Habener, J. F. (n.d.). Pancreatic agenesis attributable to a single nucleotide deletion in the human IPF1 gene coding sequence. Nature News. Retrieved May 3, 2022, from https://www.nature.com/articles/ng0197-106 

U.S. Department of Health and Human Services. (n.d.). Diabetes mellitus type 1. Genetic and Rare Diseases Information Center. Retrieved April 30, 2022, from https://rarediseases.info.nih.gov/diseases/10268/diabetes-mellitus-type-1 

Wang, X., Maxwell, K. G., Wang, K., Bowers, D. T., Flanders, J. A., Liu, W., Wang, L.-H., Liu, Q., Liu, C., Naji, A., Wang, Y., Wang, B., Chen, J., Ernst, A. U., Melero-Martin, J. M., Millman, J. R., & Ma, M. (2021). A nanofibrous encapsulation device for safe delivery of insulin-producing cells to treat type 1 diabetes. Science Translational Medicine, 13(596). https://doi.org/10.1126/scitranslmed.abb4601 

Zhu, Y., Liu, Q., Zhou, Z., & Ikeda, Y. (2017, November 2). PDX1, neurogenin-3, and MAFA: Critical transcription regulators for beta cell development and regeneration – stem cell research & therapy. BioMed Central. Retrieved May 3, 2022, from https://stemcellres.biomedcentral.com/articles/10.1186/s13287-017-0694-z

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