What is Gene Therapy?
Gene Therapy simply means artificially changing the genetic information in a cell to alter it’s functioning. The cell may be changed temporarily or permanently. The technology relies upon“genetic engineering” ; that is being able to tinker with the genome. A main problem was how to get foreign DNA into a cell, since they are protected from such large molecules getting in by their cell membranes. In the 1950′s it was found that genetic information could sometimes be transfered from one cell to another by soaking them in a solution of calcium chloride, which opened up “pores” within the cell membrane. Another method was “electroporation”, meaning giving a small electrical shock to the receiving cell to increase it’s membrane permeability. The problem with these techniques was the difficulty in transfering large pieces of DNA, and how to get the new DNA into every cell. Other challenges including getting the DNA specifically into the cells we wanted to treat, getting the new gene(s) into the right place in the “transduced” cell, and getting the new gene(s) to turn on (and stay turned on!). Sometimes the unhealthy human cells were removed from the patient and the DNA transfer was done in the laboratory, a technique called “in vitro” (in glass). The cells that has successfully taken up the new DNA could be isolated and then re-injected back into the patient. The other option was to do the gene transfer within the patient themselves, by directly injecting the with the new DNA carried by a “vector” . The most effective vectors were found to be stripped-out viruses, since these naturally bound to human cells and injected their foreign DNA into them. Placing the new genes directly into the patient is called“in vivo” (in life) gene therapy.
What is Gene Therapy Cont.
It was found that viruses were constructed out of a protein coat surrounding a small amount of DNA. The virus had an actual injection system whereby it could land on a host cell (it has little “legs” making it look like a lunar lander) poking a hole with a hollow tube through the cell’s membrane. Once “docked” onto the host cell, the viral DNA is injected into it. The DNA may just float about in the cell fluid (“cytoplasm”) or may insert itself into the host DNA. Empty viral protein “capsules” could be stuffed with custom DNA and let loose to transduce this DNA into the target cell. Furthermore, certain types of viruses had a predilection to latch on to particular body cells. For example, adenovirus tends to go the lung while hepatitis virus attatches to liver cells. Thus, we could get at least some “specificity” to correctly target the modified viral DNA to the diseased cells.
A further way of getting new gene information into cells using viruses is by linking the information to a class of viruses called “retroviruses”, which include the deadly HIV (AIDS causing) virus as well as less harmful mouse (“murine”) viruses which can be used for therapy. Transfering genetic material this way is called “In Vivo Gene Therapy”, meaning instilling the gene via a “living” entity directly into the Human body. The gene we want to transfer can be “spliced” into the retrovirus, which has the ability to enter a Human cell and integrate directly into the Human genome! This exact capability which makes retroviruses so dangerous also makes them extremely useful tools for gene therapy, as we will see later with specific examples.
On recent idea of a way to get a new gene into a cell is by encapsulating them in a “liposomes”, which are basically storage sacs which float about in the cell cytoplasm to store fats, enzymes, proteins, drugs, or whatever. They appear under the light microscope as little “bubbles” within the cell fluid, and have the ability (sometimes) to pass through the normally poorly permeable cell membranes. Cell membranes have to be very selective in what they let through, otherwise they would serve no purpose in protecting the cell from the hostile outside world. Apparently, things which ordinarily would be too large to pass through cell membranes can be packaged in real or artifical liposomes, and then induced to enter cells. Once inside, the liposome coating may be broken down by intracellular enzymes, and the contents (such as a new gene) released. It is possible (but far from certain) that the new gene will successfully integrate into the “host” DNA, and start functioning! The chances for this happening are greater if many “copies” of the gene are introduced into the host cell, instead of just one or a few . Much research is going on using liposomes as the transporter.
An important development was the ability to isolate the particular genes we wanted
from a genome which might contain 3000 genes! It was easier to get a small bit of foreign genetic material to penetrate through a receiving cells membrane using calcium chloride or electroporation, than to try to get a large chromosome through. Also, it was much easier to pack a small piece of genetic material into an empty virus shell than to incorporate a large piece. Furthermore, we didn’t want to transfer unwanted genes into our “host” but only ones coding for the specific attribute of interest. The ability to “map” genes to particular chromosomes, and then to actual physical areas on the chromosomes, was crucial to isolating, cutting out and transfering genes. The actual method of localizing genes was called“hybridization”, whereby we would work backward from the product that the gene made, looking in the cell for the“messenger RNA” coding sequence that was coming out of the nucleus to attatch to the cell’s ribosomes (in the cytoplasm). These ribosomes represented the actual “machinery” which translated the genetic message into protein and enyzmes which in turn build a living organism. By examining the RNA, we could elucidate the corresponding DNA sequence that producing it. By synthesizing a radioactively labelled “complimentary” DNA sequence and putting it into the cell, we could see where it migrated and attatched to within the nucleus, and viola, know where the gene of interest was located.
Once the gene of interest was localized, we had to know how to “cut it out” (excise it) so as to isolate it. Fortunately, we discovered that the cell itself produced special enzymes to slice and dice up DNA, and that we could use these enzymes to cut up the DNA for our own purposes. These particular enzymes were called “Restriction Endonucleases”, and more are still being discovered today. They acted as “scissors” for cutting the DNA only in a specific area. Now the DNA is composed of four “bases”, which are Adenine, Guanine, Cytosine and Thymine. Adenine always pairs to Guanine on the “complementary strand” (the opposing strand of the double helix), while Cytosine always pairs to Thymine. It is the varying arrangement of these these 4 simple bases which gives DNA it’s final complexity, and which accounts for the innumberable possibilities– complexity is from the simple, repeated over and over!
The Restriction Endonucleases “cleave” the DNA at a particular site, such as when a Cytosine follows two consecutive Adenines which themselves follow a Guanine. There may be many such sites in a long DNA molecule, and so several Restriction Endonucleases which cut in different areas may be used to isolate a sequence of interest. Even after cutting the DNA up, we still must somehow tease out the piece of it we want. Since the pieces of DNA will have different lengths, they will also have different weights and we can separate them by using an electrical current to drag them along a “medium”, like filter paper or special “agarose gel”. The heavier pieces will move more slowly than the lighter ones, and so can distinguish them by weight (and thus by length). Once collected, we are now ready to put our piece of DNA, with it’s particular genes(s), into a receiving“host” cell. This is called“Gene Transfer”, and results in a new “combination” of genes of genes in the host. Thus the whole process is called “Recombitant DNA Technology”.
The initial uses for this new “Recombitant DNA Technology” was in putting genes that manufactured specific proteins or enzymes into bacteria, to make useful chemicals in large quantities. For instance, it takes many pig (“porcine”) pancreas’ to extract enough purified insulin for diabetics, but simple bacteria (like the E.Coli found in the human intestines) can be modified by insertion of the insulin-producing gene to become factories manufacturing insulin. These bacteria can be grown in large cultures (glass dishes), and the insulin they produce drained off, packaged, and sold to diabetic patients. It turns out to be a cheaper, safer way to make insulin, since there are no foreign pig proteins contaminating it (which can cause allergic reactions). Human Growth Hormone used to be extracted from thousands of dead people’s brain pituitary glands, and was very expensive. It’s a needed hormone for children who lack it, since they will be midgets without it. Recombitant DNA Technology allowed the gene that “codes” for the production of growth hormone to be isolated, cut out of the neighboring DNA, and transferred into bacteria. These bacteria can then produce great quantities of Growth Hormone at much less cost and logistical problems than collecting it from deceased people’s brains. A further use was developing “Oil Eating” bacteria to combat oil spills from tankers. Thus, the basic technology of “Gene Transfer”, at least into bacteria, is already well established and used every day .
The next logical step was using Recombitant DNA Technology in Human cells instead of just bacteria. The idea was to insert a gene to make a necessary enzyme in human cells that were missing them. The lack of an enzyme, caused by a flaw in or absence of necessary genes, is responsible for many “metabolic” diseases. While these diseases are mostly rare, they are often severe or fatal for those afflicted. Examples include Cystic Fibrosis and Adenine Deaminase Deficiency . If we could insert the proper genes into cells lacking them, the crucial enzymes would be produced, and these diseases cured. This was the first real attempt at primary “Gene Therapy”, but proved to be a lot harder than working with bacteria. For instance, it is more difficult to get the new genes into human cells, and to have them “integrate” into the right place in the human genome. With bacteria, we can bombard them with the new genes, see which bacteria take them up and separate them out of the bunch. Then we can induce them to divide to produce new bacteria with the desired gene, and grow them in vast quantities. Trying to put genes into Human Cells is tougher, since we can’t just “select out” the cells which have uptaken the new gene, or cause them to divide and grow more than they ordinarily would. Also, even after we get the new genes into the Human cells, there is no guarantee that they will migrate to the right place in the genome to function properly; they might just lie dormant and do nothing.
Furthermore, even when they do go to the right place, and start working, their action (called“expression” ) may be temporary. For reasons we are still trying to figure out, they may get turned off and quit working, defeating the whole therapy. Moreover, there are safety issues. it is possible that inserting the new gene into the diseased cells will actually turn on an Oncogene or turn off a Suppressor Gene, starting a cancer! Also, if we use “living” retroviruses to transfer genes, they may mutate into dangerous forms capable of “replication”, and actually start a new disease in the Human population! In general, however, this risk is small and the possible benefits of inserting functioning genes into gene-lacking cells cells are great– curing otherwise deadly diseases. The first success has already been seen with this therapeutic gene transfer, for the Adenine Deaminase Deficiency Syndrome.
When the Adenine Deaminase gene is successfully integrated into a diseased child’s genome, the disease is cured. Cystic Fibrosis cells have been “cured” in Clinical Trials, and future possibities include cure of all sorts of hereditary genetic diseases, from hemophilia and Tay Sachs disease to Muscular Dystrophy and Parkinson’s Disease.
The most complex use of Gene Therapy is going beyond replacing a non-functioning gene with a working one– it is actually turning on or off existing genes within Human Cells, or even fixing damaged ones. We are currently mapping the entire Human Genome, consisting of about 3 billion base pairs, to localize the genes for every physical human trait- – eye color, height, fingerprints, inborn diseases, etc. This enormous undertaking will give us a “road map” of all Human genes, enabling us to quickly identify missing, broken or duplicate ones in patients. When we can go into cells and modify the genes at will, we shall have incredible powers to cure disease– and“genetically engineer” ourselves. For example, we will be able to change a person’s height or eye color, cure Down’s Syndrome and maybe regrow amputated limbs or weak organs. We shall likely be able to increase our lifespan by turning off “aging genes”. We will be able to cure most every illness, including any cancer, by merely shutting off the “grow” message in sick cells, and encouraging the healthy cells to grow instead . The possibilities are limited only by our imagination. The usage of them, for good or evil, will surely test the basic nature of humankind.
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