University:
College
Course:
College Rehabilitation Medicine
Academic year:

2023
Views:

201

Pages:

1
Author:

Carlo M.

MOLECULAR BIOLOGY AND DIAGNOSTICS LABORATORY • GENE THERAPY AND GENE SILENCING OUTLINE • • Gene Therapy Gene Silencing • GENE THERAPY & GENE SILENCING GENE THERAPY • • Gene Therapy is the insertion of genes into an individual’s cells to treat hereditary diseases by replacing defective alleles. It differs from standard transgenic practices in that it is intended to transform existing organisms rather than create new ones. Viral vectors are typically used, due to their ability to integrate DNA into the host’s genome. The process involves removing cells from a patient and using a viral vector to introduce a functional copy of the defective gene. When the cells are transplanted back into the patient, they should begin expressing the missing protein to restore normal health. An example of the successful use of gene therapy in humans is in the treatment of adenosine deaminase (ADA) deficiency: o ADA deficiency is an autosomal recessive disease that causes severe combined immunodeficiency (SCID) in sufferers o The disorder results from an inability to synthesise the enzyme ADA, which is needed for the safe breakdown of a toxic chemical o Individuals who have undergone gene therapy have shown a steady increase in the levels of ADA in their cells. • GENE SILENCING • Gene silencing refers to the ability of a cell to prevent the expression of a particular gene, while this occurs naturally, it can also be induced by scientists to study gene interactions and potentially treat inherited diseases. The Cre protein is a site-specific DNA recombinase that can catalyse the recombination of DNA between specific sites in a DNA molecule. These sites, known as loxP sequences, contain specific binding sites for Cre that surround a directional core sequence where recombination can occur. Conditional Knockouts: o A gene knockout is a genetic technique whereby a specific gene is rendered inoperative within an organism. Using the Cre-LoxP system, this gene knockout can be targeted to specific tissues within the organism (conditional knockout). The Cre-LoxP system uses the enzyme Cre recombinase to remove genetic sequences located between two Lox sites: o Using recombinant DNA technology, LoxP sequences are inserted on either side of a gene of interest in a test animal o The Cre gene is inserted next to a tissue-specific promoter in another test animal o When the two animals are bred, the resulting offspring will possess LoxP sequences and a tissue-specific Cre recombinase o The Cre recombinase will remove the gene located between the Lox sites to create a conditional knockout for further study. RNA Interference (RNAi) o Short interfering RNA (siRNA) is a double-stranded RNA molecule that is roughly 20 – 25 base pairs in length, (siRNA) interferes with the expression of genes by causing the mRNA transcripts to be broken prior to translation. o When the siRNA is unwound, one strand (the passenger strand) is degraded and one strand (the guide strand) remains o The guide strand will hybridise to its complementary mRNA sequence and recruit a protein complex called RISC. o RISC (RNA induced silencing complex) will then destroy the targeted mRNA, preventing translation of the associated gene.