Molecular Biotechnology: Principles and Applications - Glick B., Pasternak J. 2002

Molecular Biotechnology of Microbiological Systems
Gene Therapy
Conclusion

For many Hereditary diseases, no sufficiently effective treatments exist, which is largely due to the difficulties in obtaining and targeted delivery of the corresponding Gene product. Following The Development of Methods for identifying and cloning normal variants of defective genes (often as cDNA), attempts were made to use them to correct genetic defects. Two main approaches are used to treat diseases at THE MOLECULAR LEVEL: Ex Vivo Gene therapy and In Vivo Gene therapy.

In ex vivo gene therapy, the "therapeutic" gene is transferred into isolated patient Cells using retroviral vectors or other delivery systems; the transduced cells are then cultured and administered to the patient. While this approach does not trigger an unwanted Immune Response in the recipient, the Procedure itself is quite expensive and labor-intensive. An alternative method of ex vivo gene therapy employs Introduction/32.html">Genetic Engineering modification of non-autologous cells encapsulated within a membrane, which prevents an adverse immune response without hindering the release of the "therapeutic" gene product.

In in vivo gene therapy, the "therapeutic" gene is introduced directly into the target tissue Cells of the patient. Various delivery systems have been developed for this purpose: viral systems (retroviral, adenoviral, adeno-associated vectors, and Herpes simplex virus-based vectors) and non-viral systems (injection of naked DNA, bombardment of target tissue with DNA-conjugated particles, and cellular uptake of lipid-encapsulated DNA). In addition, Cell-specific viral and non-Viral Gene Delivery systems have been engineered.

A highly promising approach for destroying rapidly dividing Cancer cells is gene-directed enzyme prodrug therapy. The most widely used approach involves the sequential introduction of the herpes simplex virus thymidine kinase gene (HSVtk) and ganciclovir into tumor cells. This results in The formation of ganciclovir triphosphate, which is toxic to rapidly dividing cells. Non-transformed cells in contact with the HSVtk-modified cells are also destroyed (the "bystander effect").

Not only gene products, but also oligonucleotides can be used as therapeutic agents. So-called "antisense" oligonucleotides can be used to completely or partially suppress the expression of a gene responsible for a particular hereditary disease. In one variation of this therapy, the cloned gene is inserted into an expression vector in the reverse orientation, generating a DNA transcript complementary to the normal mRNA; this transcript pairs with the mRNA and inhibits Translation. In a more common approach, the "antisense" oligonucleotide introduced into the target cell hybridizes with a specific mRNA and blocks its translation. The efficacy and half-life of "antisense" oligonucleotides are enhanced by modifications involving the phosphodiester bond, Pyrimidines, and sugar residues. Currently, researchers are investigating the therapeutic potential of various oligonucleotides: genetically engineered ribozymes that cleave specific mRNAs; aptamers that bind to specific Proteins and block their Functions; and oligonucleotides capable of correcting single nucleotide pair substitutions and Mutations leading to aberrant splicing.

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Review Questions

1. What is ex vivo gene therapy?

2. Describe The Use of genetically engineered non-autologous cells for ex vivo gene therapy. Why does this approach appear to be highly promising?

3. What is in vivo gene therapy?

4. Describe in detail at least two viral systems for targeted gene delivery.

5. Describe in detail at least three non-viral systems for gene delivery.

6. What is antisense oligonucleotide therapy?

7. What modifications can be used to increase the half-life and enhance the efficacy of antisense oligonucleotides as therapeutic agents?

8. Describe a method for treating malignant neoplasms using the HSVtk gene.

9. How should a ribozyme be modified so that it can be used as a therapeutic agent?

10. How is a "therapeutic" aptamer isolated? What are the advantages of aptamers over Other types of drugs?