Techniques of Gene Therapy Ž 11

Techniques of gene therapy

Gene therapy involves isolating a gene, putting Once the gene that causes a disease has been it into cells where it will be used, and ensuring identified, the corresponding normal gene must that the inserted gene functions in the new cells be isolated, unless it is already available because in a way that does not harm the patient. it has been studied for some other purpose. Using an abnormal gene to find its normal counterpart Genes are copied and passed on is usually done by exploiting the extensive simi- by DNA replication larity between the sequences of the normal and defective genes; they rarely differ greatly in over- Genetic information is transmitted from one cell all sequence (although the functional results are to its progeny by duplication, or replication, of quite different, or there would be no disease). its DNA. When a cell divides, it copies its DNA After the normal gene has been identified and and distributes a copy to each of two offspring isolated, then it must be copied. The process of cells. A new therapeutic gene introduced into a making multiple copies of a single gene is called cell in the laboratory can thus be reproduced 7 cloning. Cloning involves combining the gene of through the process of cell division when the cell interest with DNA sequences that allow it to be is placed into a patient and proliferates. copied in lower organisms—usually bacteria or Many breakthroughs in molecular genetics have yeasts. The DNA containing the gene of interest come from discoveries about how DNA replicates, is then inserted into bacteria or yeast (or, more how it can be specifically cut and reassembled, recently, into some types of mammalian cells and how to re-introduce the altered DNA back growing in culture). The DNA is copied as the cells into cells in such a way that its expression, or proliferate. The numerous copies of DNA are then translation into protein, can be controlled (Jud- purified from other cell components, and the gene son, 1980). Many of the techniques for splicing of interest can be cut away from unwanted DNA and controlling the expression of genes were first sequences, One now has millions or billions of discovered between 1970 and 1974, using some copies of a single gene. of the same techniques that led to the develop- These copies are then combined with DNA that ment of recombinant DNA (Watson, 1984). is suitable for insertion into human cells. Isolation and cloning of the Insertion into human cells normal gene The DNA that contains the normal gene can be The usual first step in approaching gene ther- administered to human cells in several ways: apy is identification of the abnormal gene. (This using viruses, physically injecting it, treating the step can be skipped when the corresponding nor- DNA chemically so that cells take it up, treating mal genes are already available, as was the case the cells so that they are induced to take in the for sickle cell disease.) Once the abnormal gene DNA, or by fusing the cells with membranes that has been found, then copies of the corresponding normal gene must be isolated and copied. There T’r)r details of cloning, see the ‘1’echnica] Notes. (honing a gene are several ways to identify abnormal genes. should not be umfused with cloning an organism, ‘1’}le term “clon- These involve analysis of patterns of inheritance ing” refers to reproduction mrithout mating: in the case of a g[>nc or DNA sequence, this merely means making copies of the relrwant of a disease, study of the metabolism of patients stretch of DNA. Cloning a whole organism, in contrast, in~’ol~ws co- who have the disease, and analysis of the genes p)ring all of a cell’s DNA so that a completely new organism that of those who have the disease, Identification of shares all its genes with the original is produced. The techniques tor (loning grnes are completely’ different frum those for cloning the gene that causes a particular disease requires organisms. Cloning an indliidual human Jt’ouki not help in thr pre- hundreds of experiments, luck, and extensive re- ! ent ion of genet i(’ disease, and is not d irerth’ related to the (lues - sort to recombinant DNA technology. tions raised b~r human gent’ therap~ 12 Human Gene Therapy—Background Paper contain the DNA. In the distant future, designed alternative that is ethically unacceptable for viruses or genetic elements may be used to trans- human experiments) (Anderson, 1984). fer genes to specifically targeted human cells. At present, however, more primitive methods are used. CHEMICAL AND PHYSICAL METHODS Some early experiments in gene transfer em- VIRUSES ployed mixing DNA with chemicals and subse- Viruses are small packages of genetic informa- quently applying the DNA to a large number of tion in the form of DNA or RNA that enter cells cells. Most cells would pick up the DNA, and some and either insert their information into that of would insert it into their own DNA, and, in some the infected cell or duplicate themselves using the cases, express it. The usual chemical treatment cell’s biochemical machinery. Viruses are usually employed calcium phosphate with relatively large covered with a coat of protein or membrane, but amounts of the desired DNA. The most common their most distinguishing characteristic is the physical method involved “electroporation”) in genetic information that they contain. Some which electrical treatment of the cells induced up- viruses promise to be practical for gene transfer take of DNA and other constituents from the because they are relatively simple and controlla- fluids bathing the cells. ble, and contain sequences that permit insertion Chemical and physical treatments have the of genes into the host’s DNA. Modified viruses are advantage of not requiring a vector to cause in- the most likely candidates for gene therapy in the sertion, but have two major disadvantages. First, long run, because they are highly efficient, can the DNA is only stably incorporated into a small affect many cells, and are relatively easy to manip- proportion of cells, usually only one in ten thou- ulate in the laboratory (Rawls, 1984). sand to one in a million. (This small proportion Several scientists are developing viruses that nevertheless usually represents hundreds or would not injure cells, would not propagate un- thousands of times more cells than could be di- controllably, and would enter only target cells rectly injected.) This feature requires that cells (Anderson, 1984). Such viruses have been suc- that take up and incorporate the desired DNA cessfully used to insert new genes into blood- must somehow be separated from cells that do forming cells of mice with relatively high effi- not, and there must be a very large number of ciency (A. D. Miller, 1984; Williams, 1984). At cells to treat in the first place. Second, the DNA some point, scientists may be able to design a usually inserts at random into the cell’s genome, virus that could be used for cloning as well as de- and often in multiple copies. DATA insertion fol- livery, saving yet more steps. lowing chemical and physical insertion methods is thus relatively uncontrolled and unpredictable MICROINJECTION (Anderson, 1984). Microinjection of DNA involves putting the DNA one wants to insert into a solution that can be MEMBRANE FUSION pushed directly into individual cells through ex- The final way to get DNA into cells involves put- tremely small needles made of glass. The tech- ting it inside of membranes that can then be fused nique is highly reliable, in that a high proportion with the outer membrane of target cells, allow- of cells that receive genes express them (Capec- ing the contents to spill into the cells. The mem- chi, 1981), but limited by the number of cells that brane sacs, called liposomes, can be made of ar- can be directly injected. Investigators can inject tificially constructed lipid mixtures or derived hundreds or thousands of cells, at most, for a from specially treated cells such as red blood cells given experiment, compared to billions that can or bacteria. The advantage of cell fusion is that be treated using viruses or chemical treatments. it is relatively simple, and large numbers of cells Microinjection has been the method of choice for can be treated. It is, like chemical treatment, experiments involving gene transfer in mice, be- unreliable and nonspecific at delivery. The tech- cause of its reliability, but its applicability to nique might prove useful in the future, however, humans is questionable because it is not com- if membranes are constructed that target specific pletely reliable, and often results in cell death (an cells with highly reliable delivery.