Shikimoyl Functionalised Block Polypeptides for Targeted CRISPR/Cas9 Delivery

Panda, Sidharth (2021) Shikimoyl Functionalised Block Polypeptides for Targeted CRISPR/Cas9 Delivery. Masters thesis, Indian Institute of Science Education and Research Kolkata.

Text (MS Dissertation of Sidharth Panda (16MS024)) 16MS024_Thesis_file.pdf - Submitted Version Restricted to Repository staff only Download (2MB)

Abstract

Genetic disorders have plagued the humankind since time immemorial. While some of these disorders manifest as innocuous phenotypic alterations, others can be pernicious and cause life-threatening damage to the organism. Genetic disorders related to the blood are the most common type and include Sickle cell disease and thalassemia as the most prevalent ones. Though several medications that can ameliorate the detrimental effect of the disorders are available, almost all of them are transient solutions and provide no permanent cure. Bone marrow transplants can successfully negate the malevolency of the diseases. However, the transplants themselves are torturous, and the process compromises the immunity of the patient. Recently genome editing tools have emerged as propitious therapeutic methods for ensuing a permanent amelioration of these diseases. With the help of these tools, the faulty genomic sequence can be rectified permanently, thus alleviating the translation of the harmful phenotypes. CRISPR/Cas9 systems are one such category of gene editing tools that provide for a versatile hand at amending erroneous genomic sequences. These systems consist of a guide RNA, which directs an endonuclease to the defective sequence on the genome, where it can make the necessary corrections. These systems have been widely used for treating genetic disorders through ex vivo approaches. However, this approach requires for isolation of stem cells from the patient, which is then later corrected with the help of CRISPR/cas9 and then transplanted autologously into the patient. This circuitous method is quite expensive and has higher rates of failure. In vivo approach involves the direct administration of the requisite therapeutic cargo into the patient with the help of a vector and addresses the drawbacks posed by ex vivo approaches. However, a significant bottleneck herein, is to design a suitable vector capable of shuttling both the Cas9 endonuclease and the sgRNA together to the target site. Moreover, the vector should also be able to deliver the Cas9/sgRNA complex to the nucleus, which is its primary site of action. Our group has recently demarcated the synthesis of a novel shikimoyl functionalised polypeptide capable of internalising into the nucleus in a wide variety of cell lines. Taking this work forward, we envision the design and synthesis of a novel shikimoyl functionalised block polypeptide, capable of encapsulating Cas9/sgRNA complex and delivering the same specifically to the nucleus in vivo.

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