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Covers the use of biotechnology in diagnosing diseases, producing pharmaceuticals, and treating genetic disorders.
What if we could treat a disease not by swallowing a pill, but by rewriting the very code that caused it? Imagine turning your own cells into high-tech factories that produce the medicine you need to survive.
Before gene therapy, patients with diabetes relied on insulin extracted from cows or pigs, which often caused allergic reactions. Today, we use recombinant DNA technology to turn bacteria into insulin factories. Scientists isolate the human insulin gene and insert it into a bacterial plasmid (a circular loop of DNA) using restriction enzymes to 'cut' and DNA ligase to 'paste'. This transgenic bacterium, usually E. coli, then divides rapidly, expressing the human gene to produce identical insulin proteins. Similarly, monoclonal antibodies (mAbs) are produced by fusing a specific immune cell with a cancer cell to create a 'hybridoma' that churns out identical antibodies to target specific diseases like cancer or autoimmune disorders.
1. Isolation: The human insulin gene is identified on Chromosome 11. 2. Vector Preparation: A plasmid is removed from E. coli and cut with the same restriction enzyme used on the human DNA to create 'sticky ends'. 3. Ligation: The human gene and plasmid are mixed; they bind together to form recombinant DNA. 4. Transformation: The recombinant plasmid is inserted back into the bacteria. 5. Fermentation: Bacteria are grown in large vats, and the insulin they produce is harvested and purified.
Quick Check
Why is it necessary to use the same restriction enzyme on both the human gene and the bacterial plasmid?
Answer
Using the same enzyme ensures that the 'sticky ends' (overhanging DNA sequences) are complementary, allowing them to base-pair and join correctly.
To fix a genetic disorder, we must deliver a functional gene into a patient's cells. Viral vectors (like Adenoviruses or Lentiviruses) are the most common 'delivery trucks'. They are highly efficient because viruses have evolved for millions of years to inject DNA into host cells. However, they can trigger dangerous immune responses. Non-viral systems, such as liposomes (fatty bubbles) or nanoparticles, are safer and less likely to be attacked by the immune system, but they are significantly less efficient at entering the cell nucleus. Choosing a vector is a balance between the 'payload' size, the target cell type, and the risk of an inflammatory reaction.
Imagine you are treating a patient with Cystic Fibrosis. 1. Option A (Viral): Use an Adeno-associated virus (AAV). Pros: High infection rate of lung cells. Cons: Potential for the body to develop antibodies against the virus, making future doses ineffective. 2. Option B (Non-Viral): Use Lipid Nanoparticles (LNPs). Pros: No viral proteins, so no immune rejection. Cons: Many particles are destroyed by lysosomes before they reach the nucleus, requiring a much higher dose.
Quick Check
Which delivery system would you choose if you were worried about a patient having a severe allergic reaction to the treatment?
Answer
Non-viral delivery systems (like liposomes or nanoparticles) are generally safer regarding immune reactions.
Delivering genes in vivo (inside the living body) is like trying to deliver a package to a specific apartment in a giant city without a map. The first hurdle is the immune system, which may destroy the vector before it reaches the target. The second is tissue specificity: how do we ensure a gene for liver function doesn't end up in the brain? Physical barriers like the Blood-Brain Barrier (BBB) prevent most vectors from entering the central nervous system. Furthermore, if a gene integrates into the wrong spot in the genome (insertional mutagenesis), it could accidentally activate an oncogene, leading to cancer. Scientists are now engineering 'tropism'—modifying the surface proteins of vectors to act like keys that only fit specific cellular locks.
Consider a gene therapy for a muscular disorder. If the efficiency of your vector is (only 5% of cells are successfully transduced) and the muscle mass requires functional cells to restore movement, calculate the total number of vectors () needed if each vector targets one cell.
1. Use the formula: 2. 3. vectors.
Challenge: If the liver sequesters 90% of all injected vectors, you must multiply your dose by 10, increasing the risk of liver toxicity. This illustrates why 'off-target' effects are the biggest hurdle in gene therapy.
What is the primary role of DNA ligase in producing recombinant insulin?
Which of the following is a major disadvantage of using viral vectors for gene therapy?
In vivo gene therapy involves removing cells from the patient, modifying them in a lab, and then returning them to the patient.
Review Tomorrow
In 24 hours, try to sketch the 5 steps of recombinant insulin production and list one pro and one con for viral vectors.
Practice Activity
Research the 'Blood-Brain Barrier' and write a short paragraph on why it makes treating Alzheimer's disease with gene therapy particularly difficult.