A patient with type 1 diabetes has visited you and you had a

A patient with type 1 diabetes has visited you and you had all the technology that you need to conduct an autologous cell based therapy. Genetic analysis of this patient showed that there is defective splicing of the insulin mRNA and you have the abililty to correct this defect by knocking in a cDNA that can encode a fucntional insulin protein.

a) How would you generate the cells needed for this genetic correction in the patient?

b) After you produced the corrected cells how would you test in vitro whether these cells are capable of producing insulin?

c) Suppose you could inject these cells intravenously and that the cells can home into the pancreas. How do you propose to trace these engineered cells in pancreas so that they can be distinguised from the native defection Beta cells?

Solution

A)
The defective cells from the patient can be cultured In Vitro and used for cloning in the functional Insulin gene. Using the patient\'s own beta cells would ensure that the probability of transplant rejection would be at a minimum. There are several methods by which a gene can be introduced in a cell. As of 2017, the most recent and efficient method is called the CRISPR-Cas9 method.

B)
The cells need to be cultured In Vitro for atleast 2-3 generations. This will ensure that the cells get time to adjust to the new environment. Since insulin is produced in response to glucose, one can simply introduce glucose into the culture medium. The culture medium can be tested for the presence of insulin at regular intervals. Since insulin is a protein, one can simply perform a western blot to quantify the amount of insulin. The reduction in glucose levels would be indication of the efficacy of the cloned insulin gene.

C)
Cells can be labelled with fluorophores, nanoparticles or radionuclides for direct tracking. Newer methods of indirect tracking by Bioluminiscence based reporter genes are being studied in smaller animals.