The major advantage of gene gun is precise delivery of DNA doses. Gold, tungsten and silver are used as metal particles and they are typically 1 μm diameter. Gas pressure, particle size, dose frequency are the critical parameters in determining the efficiency of gene transfer. The sufficient speed is achieved by high voltage electronic discharge, spark discharge or helium pressure discharge. The method is based on the principle of delivering DNA coated heavy metal particles by crossing target tissue at a certain speed. This method was first used as gene transfer technique to plants. Ballistic DNA: Particle bombardment, micro projectile gene transfer or gene gun are the other terms used for ballistic DNA. However, the efficiency is low due to rapid degradation by nucleases in serum and cleared by mononuclear phagocyte system. Attractive candidate tissues are muscle, skin, liver, cardiac muscle and solid tumours. Without any carrier it is the simplest and safest method of gene transfer. Needle: The genetic material of interest is administered through a needle carrying syringe into tissue or systemic injection from a vessel. These methods employ physical force to counteract the membrane barrier of the cells thus facilitating intracellular delivery of the genetic material.ġ. Physical Methods: Gene therapy researchers are more attracted towards physical means of transferring gene material as it is simpler.
This has led to research focus on suitable ideal vector delivery system. Unfortunately none of the currently available non-viral vectors fulfills ideal vector properties. Advances in efficiency, specificity, gene expression duration and safety led to an increased number of non-viral vector products entering clinical trials. Use of non-viral vectors in clinical trials increased from 2004 to 2013 while that of viral vector saw significant decrease. Non-viral vectors have drawn significant attention due to its less immunotoxicity. However the application of non-viral gene transfer have been ignored for a long time in past because of their poor efficiency of delivery thereby low transient expression of their transgenes. The major advantage of using non-viral vectors is its bio-safety. Due to its demonstrated reduced pathogenicity, low cost and ease of production, non-viral vectors have important safety advantage over viral approaches. ectopic chromosomal integration of viral DNA disrupts the expression of tumour suppression gene or activates oncogene leading to the malignant transformation of cells. Additional cause of concern over using viral gene transfer vehicle is the phenomenon known as insertional mutagenesis i.e.
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The first related fatality of gene therapy clinical trial was related to the inflammatory reaction to the viral vector (Adenovirus). The main drawbacks of using virus vectors are its immunogenicity and cytotoxicity. The efficiency of transfecting host cells is relatively high with viral vectors compared to non-viral methods. Sequential key steps in gene therapy are shown in.
The most critical step in achieving gene therapy is choosing the vectors.
In the nucleus the genetic material gets integrated into DNA and corrects the defective or mutated gene. After the vector reaches the target cell, it delivers the genetic material to the nucleus. The function of the vector is to deliver the therapeutic gene to the patient target cell. Once the therapeutic gene is produced it is loaded in a vehicle called vector. The therapeutic gene is tailored to the need i.e. This is called therapeutic gene or transgene. The next step is cloning the identical healthy gene. In spite of various methods or types of gene therapy, the therapy starts with the identification of mutant gene which is responsible for the cause of the disease. The disadvantages like in vivo clearance and manufacturing cost faced by the replacement therapy makes gene therapy a potential alternative for various rare genetic disorders. Gene therapy is considered as an alternative for enzyme /protein replacement therapy. It is well documented that almost all human diseases occur due to defect in either a single gene or set of genes due to mutation. It provides an unique approach to treat both inherited and acquired diseases by delivering a therapeutic gene material and its associated regulatory elements into the nucleus in order to correct the loss of function caused by mutation or to express the deficient gene product at physiologic levels. Gene therapy is defined as the procedure used to treat or improve the health condition of the patient by modifying the patient’s cells genetically.
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