The technological advancements of today have led to leaps and bounds in the improvement in human health and ways in which restoration of health can be achieved. Successful gene mapping has led to the identification of the various genetic causes and predisposition of diseases. Scientific knowledge was never this vast before. It can be called a dream come true for physicians and health professionals around the world, who can now inject the right sequences and codons into the genetic system of the host to generate a healthier gene, thereby remedying the disease as well as conferring the patient a lifetime of immunity to that condition.
Background and significance
The role of genetics in the causation and predisposition towards certain diseases has been well known for many years. Yet improper and incomplete knowledge led to gaps in understanding and utilizing this knowledge in practical means. With the mapping of the genes of the human genome, scientists now had a complete map with which to work on. This knowledge was perhaps the beginning of the true gene therapy procedures and the advent of this field. Now gene therapy procedures have created many possibilities. The most common is the placement of a normal functional gene to replace a nonfunctional gene. Other combinations include the swapping of an abnormal gene with a normal one, repairing of the abnormal gene, and the regulation of gene/s.
There are essentially two methods of gene delivery in use. These are categorized as the physical methods and the chemical method, respectively. The chemical methods employ various chemicals for the introduction of DNA into the body in order to transfer substances from one place to another. These include the DEAE Dextran, which is a polycationic derivative of dextran, calcium phosphate, cationic lipids or lipofection, polymers, dendrimers, and targeting proteins and peptides.
The initial method of gene delivery that is still widely used today is the viral method. Viruses are used as vectors due to their inherent properties to combine and reconfigure the human genome. Instead of infecting, these viruses are prepared and inserted to carry out a particular task, as mentioned above.
The researchers are, however, still looking for novel ways with which to introduce these genes into the body due to the health and safety concerns as well as the biological and ethical issues involved. The use of polymers, peptides, etc., are all in line with the use of nonvirus vectors in the delivery of these genes.
For a successful gene vehicle, it must possess four qualities. It should have the ability to tightly compact and protect the DNA injected, the vector should be recognized by the target-specific cell-surface receptors, and it should be able to disrupt the endosomal membrane, and finally, it should be able to deliver the DNA unharmed to the nucleus. Any substance that can accomplish these four goals is an ideal substance for gene delivery.
Of the viral technique, the four viruses currently in use for the gene therapy include the retroviruses, the adenoviruses, the adeno-associated viruses, and the Herpes Simplex viruses. Some other viruses now being used also include Lentiviruses, adeno associated viruses, and vaccinia viruses.
The ultrasound method of gene delivery is another method with which transfers have been successfully achieved. This method is one of the successful physical methods of gene delivery, which have shown good results and a 10 to 20 fold increase in the permeation of the genetic material. Although it is dependant on certain factors such as frequency and the strength of the ultrasound and the amount of plasmid used with the DNA, the results are almost always beneficial. It is a good noninvasive method used in the transfer of genetic material and therefore is an area of immense interest.
Ultrasound is an example of the physical methods of introduction of genetic material into the body. Many other physical, genetic methods exist, which include microinjection, electroporation, gene gun, and naked DNA.
Current state-of-the-Art
The gene delivery method is primarily chosen on many factors regarding the location and the site of DNA injection. The first and foremost concern is the type of cell environment where these DNA molecules will be injected into. In this regard, the main concern lies in the selective uptake of the molecules and not letting absorption take place in other cells. This is an area that has limited the full potential use of gene therapy. In this situation, some of the more popular methods are the use of peptides, viral vectors, retroviruses, and cationic lipids.
The second problem is the correct identification of the DNA uptake capability of the tissues with which one is working. This is because some tissues take up the DNA with more difficulty than others. And therefore, prior knowledge about the tissue nature should be known before any therapy is carried out. Delivery efficiency must be calculated, which is usually the best with the viral vectors used for DNA transfer.
Next, the type of gene delivery is important, whether it is stable or transient, and what kind of nucleic acid is to be introduced into the body. The selection of the right method can help prevent the death of many cells during the procedure; for example, the electrophoresis and the gene gun delivery systems are extremely harmful to the cells and cause cell death in a majority of these. The suspension cells are more difficult to gene transfer than the adherent cells, and other considerations such as expertise, time, and cost of the procedure also govern the outcomes of the gene therapy success.
The new possibility of the introduction of polymers in the gene delivery methods is under investigation. This is a deviation from the road in which only live vectors or viruses have been considered to be probable vectors into the body. The polymers are the first of the kind to be introduced into the body, which is synthetic and nonliving. This is specifically being done so to reduce concerns about injecting viruses into the body. For this purpose, not every polymer can be used, and those that could be must be further modified to enhance the properties and outcomes. The current blue-eyed polymers for the investigators include the beta-amino esters, which carry the potential of becoming gene carriers. This is because they behave in a somewhat similar manner as do viruses when they enter the bloodstream. Again, these researchers believe that by changing the morphology and sequence arrangement of the molecules, they can improve the outcomes of the performance levels. In vivo studies in mice have been successful so far, and therefore, they hold promise in their use in human models.
This is because these polymers are able to retain themselves with the DNA, enter the cells successfully, and instead of being broken down by any enzymes, enter the nucleus and exert their genetic effects and results.
Some of the other methods of gene delivery currently in use include the direct injection of the DNA into the target cells and thereby achieving effects. Others can include the use of liposomes within aqueous mediums, which carry the therapeutic DNA, and binding the DNA molecule to specific molecules so as to attach itself through specific target receptors.
Another possibility of using nonviral vectors is the use of peptide agents in the delivery of the gene sequences. This area of research again was introduced to gain independence from the viral methods of gene delivery and has already shown promise in this regard. Peptides have been able to show almost the same qualities of gene deliverance as in the cases of the virus, without the need to use the virus, making it an active area of research. Lysine and arginine-based peptides are able to make themselves more compact, which can be stable for a long time within the serum. Similarly, targeting and attachments can be carried out with precision, which was not possible with many of the other new agents developed. The reduced cytotoxicity and immunogenicity and increased biodegradability make these substances ideal for gene therapy procedures.
With the advent of gene therapy, many new procedures have been introduced. Each one has shown positive contribution and results. For example, Pathway IV gene delivery mechanism is a very good system introduced for patients who have any muscular problems such as muscular dystrophy. It has become a popular method due to its ease of administration and the long effect on the single dose. Intravenous plasmid DNA is injected into the venous system into the area of muscle to be treated. This plasmid gains residence in the targeted tissues, whereby it causes changes in the muscle by producing proteins locally or systemically to cause repair. The complexity may vary to the level of including antigenic and immune responses. While this is a good method of delivery, the only problem is the somewhat inability to specifically target the diseased tissue only.
A somewhat recent method of overcoming this problem was introduced to increase the vascular pressure so as to aid the delivery of the substance into the specific tissue. This has two benefits. Firstly the nucleic acids, which in normal circumstances may not be absorbed by the cells, are done so quickly, and secondly, to the target tissue. This can be easily done with the help of a tourniquet and diseases such as muscular dystrophy, anemia, cancer, etc. The applications are variable, and either local or systemic, and the outcomes are positive.
This method was an example of gene transfer by needle injection of the naked DNA(5). This technique is primarily carried out in tissues such as the lungs, liver, muscle, and skin. However, they show low gene expression. Again the issue here is the nonspecific mechanism of binding to the tissues and target cells. While this therapy is very useful in some tissues, it is not feasible due to the hydrophilic nature of the DNA, which prevents its entry into the cells. A technique to increase this permeability is the introduction of various other substances that enhance the DNA’s permeability. These include substances like transferrin, water-immiscible solvents, non-ionic polymers, surfactants, hypotonic solutions, and nuclease inhibitors.
There are many challenges that are causing problems in the progression of gene therapy at the moment. All of these are concerned with the complexity of the human body and the mechanism and the way with which it responds to external stimuli. The first challenge that scientists are facing is the short-term results that are achieved in the gene therapy introduction. The genetic results anticipated are hard to achieve in the dynamic environments of the dividing cells. Therefore, the results are achievable for a limited time frame only. In order to truly succeed in gene therapy, it is important that the genes introduced become fully integrated into the cell’s genome and act as if it was the body’s own.
The second issue is the immune response the body may elicit towards the foreign material. Third, the viral vectors have the potential to achieve their potential for causing disease within the patient and may cause other body defense systems to get activated. Lastly, while single gene dependant diseases have shown promise in the treatment and cure of the condition, not the same could be said for those conditions that are dependant on the multiple genetic variations or discrepancies. This complicates the treating of such diseases considerably and is an area of intense research for scientists.
Improvements
A novel possibility of creating a 47 chromosome cell with an extra chromosome to already present 46 chromosomes is not a very new idea and has been in processing for some time now. Scientists claim that it will take an autonomous chromosome not able to elicit anybody’s response and will contain the genetic information as a vector. There is, however, much research going on in this area.
Of the current technologies, the use of transposons is becoming the next method of delivery of genetic material. Also nicknamed the jumping genes, the transposons are superior to either viruses or a plasmid, which are currently underuse. These have the advantage of not being infectious as in the case of viruses and getting to the target tissue, which is not as efficient in the case of plasmids. These transposons can be used to carry one desired DNA sequence to another one and thereby enzymatically activate or deactivate it. A somewhat newer area of research, it has to undergo the same scrutiny procedures to make it safe to use in man successfully.
The current advancement in genetic transfer therapy is being investigated by the Innovation Corporation. The use of genetic vaccines is in studies and researches. Although not exactly a gene therapy, this method is essentially a protein recombination technique. This method does not use gene transfer and therefore prevents the risk of getting any mutations or reactions from the therapy. This therapy has shown a longer duration of action when compared to standard gene therapy procedures and can be effective for as long as six months. These vaccines have the advantage of using the genetic component of the disease only, which causes the production of proteins. These proteins then cause the activation of the body’s immune response that acts to kill the diseased or cancerous cells.
Gene delivery methods have been of immense interest in the fields of cancer chemotherapy. Here poses a great challenge for the researchers; for a while, they know which genes need to be altered to cause the death of the cancer cells, the methods of delivering these DNA sequences to the cells remain a feat to be achieved. In solid tumors, this becomes an exceptionally difficult issue. The extracellular barriers include the nature of the solid tumor, wherein diffusion becomes dependant on the properties and nature of the molecules, the kind of virus used for the transfer, and the composition and structure of the tumor. The higher than normal intracellular pressure of the cells of the tumor mass increases the difficulty of penetration into the tissues. In summary, these barriers are the interstitial penetration and transportation, and cellular targeting.
In this situation where the intracellular pressure is high, the use of intratumoral injection has been considered a good alternative. Injection directly into the tumor site with high pressure aids and increases the chances of the delivery of the molecules into the cells and thereby achieving effect. The only problem in this method is the leaking of the viruses injected into the systemic circulation, which can give rise to systemic symptoms. For this purpose, attempts are being made to inject the viruses in a substance that is highly viscous, so as not to allow the diffusion of the viruses into the circulation. This has been successfully achieved through the alginate solutions, which have an extremely high viscosity, and decreased rates of diffusion. Th se findings have given scientists much to hope for, in an attempt to treat cancer patients with permanent outcomes. However, the technical limitations are still holding outcomes that are desired. (10)
Conclusions
Gene therapy holds much promise for the future treatment of many conditions that are genetic in nature, as well as cancer patients. The present research has been able to identify the technical difficulties that are associated with this method. However, given the growing understanding of the process, it is now ascertained that the future will be an era for the genetic therapies, and cures.
Works cited
- Gene Therapy, 2007. Human Genome Project Information. Web.
- Genlantis 2007. How to Choose The Optimal Gene Delivery Method.
- Massacheussetes Institute of Technology. Polymers Hold Promise for Safer Gene Delivery. Web.
- Molly E. Martin and Kevin G. Rice, 2006. Peptide Guided Gene Delivery. AAPS Journal, 2007; 9(1): E18-E29
- Xiang Gao, Keun Sik-Kim, and Dexi Liu, 2007. Nonviral Gene Delivery: What We Know and What is Next. AAPS Journal, 2007 9(1): E92-E 104
- Science Daily, 1999. Researchers Track Path of Safer Gene Delivery Method. Web.
- Pathway IV Gene Delivery. Web.
- Science Daily 2007. “Jumping Genes” Could Make for Safer Gene Delivery System. Web.
- Innovio Biomedical Corporation. Immunotherapy DNA Vaccine/Gene Delivery. Web.
- Dan Luo, 2004 A New Solution for Improving Gene Delivery. Trends in Biotechnology Vol 22, No. 3.