Gene Therapy


  1. The Basics
  2. Genetic Disorders
  3. Applications of Gene Therapy
  4. Applications of Tissue Engineering in Gene Therapy
  5. Plasmid Banks and other repositories
  6. Research Methods used in Gene Therapy
  7. Methods to Analyze RNA
  8. Methods to Analyze Proteins

1.The Basics

1.1. Genes

A human cell (except for Red Blood Cells) has 23 pairs of chromosomes. Each chromosome houses numerous genes. Genes are basically the sequences of DNA within the chromosomes. Genome is an organism’s complete set of DNA.

Nucleus chromosome to DNA

Each DNA is made up of sequences of nucleotides which are in turn made from nucleoside bases or nucleobases. There are 4 primary nucleobases in DNA and several other secondary bases. These primary bases form the genetic code. Hence, genetic codes usually consisist of Adenosine (A), Guanine (G), Cytosine (C), and T (Thymine). A stretch of the FKRP gene looks like this:

…C T G G T C C T C T T C T A T G T C T C G T G G C T G…

1.2. Proteins

Each gene consists of a set of DNA sequences that direct the synthesis of a protein. Proteins are biomolecules which are composed of amino acid chains.

Gene to protein formation using transcription and translation.

In the transcription process, the DNA sequences are copied to mRNA. mRNA is then decoded by ribosomes to produce specific chains of amino acids which then form polypeptide chains and undergo complex folding processes to form proteins.

Proteins have many different forms and functions. Based on the functions, some common types of proteins are shown in the image below.

Various types of proteins. Structural proteins collagen and elastin are missing in the figure.

Ferritin is responsible for the storage of iron in the spleen. Structural proteins include glycoproteins, collagen, and elastin. Collagen and elastin are found in the ligaments, articular cartilage, and tendons. Hormones like insulin are responsible for carbohydrate metabolism while enzymes like amylase take part in digestion. RuBisCO is an enzyme that takes part in carbon dioxide fixation in plants (not much relevant here).

2. Genetic Diseases

3. Gene Therapy

In gene therapy, the cells are modified in a variety of ways to correct the gene. Take an example of the use of gene therapy in hepatocellular carcinoma. Hepatocarcinoma or liver cancer is the second leading cause of cancer-related death worldwide. The typical methods used in gene delivery are illustrated below.

Gene therapy in hepatocarcinoma (Source: Reghupaty and Sarkar et al., 2019)

There are two types of gene delivery systems, in vivo gene therapy and ex vivo therapy. In vivo gene therapy involves the direct injection of the therapeutic transgene via a carrier virus into the donor site, the liver in this case. In the ex vivo gene therapy, autologous cells like hMSCs are isolated from the patient, the therapeutic transgene is introduced into the hMSCs and the modified cells are then injected back into the patient.

The ex vivo therapy has high specificity and high transfection efficiency. However, it is more time consuming, involves invasive procedures to derive cells and expensive. Moreover, the ex vivo therapeutic gene delivery is not much suitable for organs like brain and lungs.

4. Applications of Gene Therapy in Tissue Engineering

Gene delivery from the scaffold represents a versatile approach to manipulating the local environment for directing cell function. 

5. Plasmid Repository

Addgene is a repository of 85,000+ plasmids which one use for gene therapy applications.

HIF-Plasmid from Addgene for Gene Therapy applications

6. GFP: Research Methods used in Gene Therapy

When reporter genes are taken by cells, they express fluorescent proteins that are easy to observe or quantify. Green Fluorescent Proteins (GFPs) are one such protein that can be easily measured or quantified. GFPs can be used to tag proteins that allow us to study various changes in the cell.

7. Methods to Analyze RNA

Polymerase Chain Reaction (PCR) and microarrays are used to analyze RNA. For PCR, RNA is isolated from the cells. Heat is applied to separate the DNA strands. On each of these two strands, primers (complimentary short DNA strands) bind to the DNA sequences. The primer is usually chosen as the gene of interest.

8. Methods to Analyze Proteins


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