Gene therapy is a novel therapeutic branch of modern medicine. Its emergence is a direct consequence of the revolution heralded by the introduction of recombinant DNA methodology in the 1970s. Gene therapy is still highly experimental, but has the potential to become an important treatment regimen. In principle, it allows the transfer of genetic information into patient tissues and organs. Consequently, diseased genes can be eliminated or their normal functions rescued. Furthermore, the procedure allows the addition of new functions to cells, such as the production of immune system mediator proteins that help to combat cancer and other diseases.
Gene therapy uses genetic engineering the introduction or elimination of specific genes by using molecular biology techniques to physically manipulate genetic material to alter or supplement the function of an abnormal gene by providing a copy of a normal gene, to directly repair such a gene, or to provide a gene that adds new functions or regulates the activity of other genes. Cell-based gene therapy approach will direct a patient’s own cells to produce and deliver a therapeutic agent. Clinical efforts to apply genetic engineering technology to the treatment of human diseases date to 1989. Initially, gene therapy clinical trials focused on cancer, infectious diseases, or disorders in which only a single gene is abnormal, such as cystic fibrosis. Increasingly however, efforts are being directed toward complex, chronic diseases that involve more than one gene.
Gene therapy researchers have employed two major strategies for delivering therapeutic transgenes into human recipients. Gene therapy can be performed either by direct transfer of genes into the patient or by using living cells as vehicles to transport the genes of interest. Both modes have certain advantages and disadvantages.
The first is to “directly” infuse the gene into a person. Direct gene transfer is particularly attractive because of its relative simplicity. Genes are delivered directly into a patient’s tissues or bloodstream by packaging into liposomes (spherical vessels composed of the molecules that form the membranes of cells) or other biological microparticles. Alternately, the genes are packaged into genetically-engineered viruses, such as retroviruses or adenoviruses. Because of biosafety concerns, the viruses are typically altered so that they are not toxic or infectious (that is, they are replication incompetent). These basic tools of gene therapists have been extensively optimized over the past 10 years. However, their biggest strength simplicity is simultaneously their biggest weakness. In many cases, direct gene transfer does not allow very sophisticated control over the therapeutic gene. This is because the transferred gene either randomly integrates into the patient’s chromosomes or persists unintegrated for a relatively short period of time in the targeted tissue. Additionally, the targeted organ or tissue is not always easily accessible for direct application of the therapeutic gene.
The second strategy involves the use of living cells to deliver therapeutic transgenes into the body. This procedure is relatively complex in comparison to direct gene transfer, and can be divided into three major steps. In the first step, cells from the patient or other sources are isolated and propagated in the laboratory. Second, the therapeutic gene is introduced into these cells, applying methods similar to those used in direct gene transfer. Finally, the genetically-modified cells are returned to the patient.
The use of cells as gene transfer vehicles has certain advantages. In the laboratory dish (in vitro), cells can be manipulated much more precisely than in the body (in vivo). Some of the cell types that continue to divide under laboratory conditions may be expanded significantly before reintroduction into the patient. Moreover, some cell types are able to localize to particular regions of the human body, such as hematopoietic (blood-forming) stem cells, which return to the bone marrow. This “homing” phenomenon may be useful for applying the therapeutic gene with regional specificity. Investigators can genetically engineer, or “program,” the cells’ level and rate of production of the therapeutic agent. Cells can be programmed to steadily churn out a given amount of the therapeutic product. In some cases, it is desirable to program the cells to make large amounts of the therapeutic agent so that the chances that sufficient quantities are secreted and reach the diseased tissue in the patient are high. In other cases, it may be desirable to program the cells to produce the therapeutic agent in a regulated fashion. In this case, the therapeutic transgene would be active only in response to certain signals, such as drugs administered to the patient to turn the therapeutic transgene on and off.
A major disadvantage, however, is the additional biological complexity brought into systems by living cells. Isolation of a specific cell type requires not only extensive knowledge of biological markers, but also insight into the requirements for that cell type to stay alive in vitro and continue to divide. Unfortunately, specific biological markers are not known for many cell types, and the majority of normal human cells cannot be maintained for long periods of time in vitro without acquiring deleterious mutations.
The therapeutic gene needs to be introduced into the cell type used for therapy. Genes may be introduced into cells by transfection or transduction. Transfection utilizes chemical or physical methods to introduce new genes into cells. Usually, small molecules, such as liposomes, as well as other cationic-lipid based particles are employed to facilitate the entry of DNA encoding the gene of interest into the cells. Brief electric shocks are additionally used to facilitate DNA entry into living cells.
Transduction utilizes viral vectors for DNA transfer. Viruses, by nature, introduce DNA or RNA into cells very efficiently. Engineered viruses can be used to introduce almost any genetic information into cells. However, there are usually limitations in the size of the introduced gene. Additionally, some viruses (particularly retroviruses) only infect dividing cells effectively, whereas others (lentiviruses) do not require actively dividing cells. In most cases, the genetic information carried by the viral vector is stably integrated into the host cell genome.
An important parameter that must be carefully monitored is the random integration into the host genome, since this process can induce mutations that lead to malignant transformation or serious gene dysfunction. However, several copies of the therapeutic gene may also be integrated into the genome, helping to bypass positional effects and gene silencing. Positional effects are caused by certain areas within the genome and directly influence the activity of the introduced gene. Gene silencing refers to the phenomenon whereby over time, most artificially introduced active genes are turned off by the host cell, a mechanism that is not currently well understood. In these cases, integration of several copies may help to achieve stable gene expression, since a subset of the introduced genes may integrate into favorable sites. In some gene transfer systems, the foreign transgene does not integrate at a high rate and remains separate from the host genomic DNA, a status denoted “episomal”. Specific proteins stabilizing these episomal DNA molecules have been identified as well as viruses (adenovirus) that persist stably for some time in an episomal condition.
An elegant way to circumvent positional effects and gene silencing is to introduce the gene of interest specifically into a defined region of the genome by the gene targeting technique. The gene targeting technique takes advantage of a cellular DNA repair process known as homologous recombination. Homologous recombination provides a precise mechanism for defined modifications of genomes in living cells. Recombinant DNA is introduced by transfection into the cell, where it recombines with the homologous part of the cell genome.This technique results in the replacement of normal genomic DNA with recombinant DNA containing genetic modifications (the therapeutic gene).
Homologous recombination is a very rare event in cells, and thus a powerful selection strategy is necessary to identify the cells in which it occurs. Usually, the introduced construct has an additional gene coding for antibiotic resistance (referred to as a selectable marker), allowing cells that have incorporated the recombinant DNA to be positively selected in culture. However, antibiotic resistance only reveals that the cells have taken up recombinant DNA and incorporated it somewhere in the genome. To select for cells in which homologous recombination has occurred, the end of the recombination construct often includes the thymidine kinase gene from the herpes simplex virus. Cells that randomly incorporate recombinant DNA usually retain the entire DNA construct, including the herpes virus thymidine kinase gene. In cells that display homologous recombination between the recombinant construct and cellular DNA, an exchange of homologous DNA sequences is involved, and the non-homologous thymidine kinase gene at the end of the construct is eliminated. Cells expressing the thymidine kinase gene are killed by the antiviral drug ganciclovir in a process known as negative selection. Therefore, those cells undergoing homologous recombination are unique in that they are resistant to both the antibiotic and ganciclovir, allowing effective selection.
Scientists have recently demonstrated that certain adult cell types can be “reprogrammed” into other cell types in vivo using a well-controlled process of genetic modification. This strategy may offer a way to reprogram available cells into other cell types that have been lost or damaged due to disease. For example, one recent experiment shows how pancreatic beta cells, the insulin-producing cells that are lost or damaged in diabetes, could possibly be created by reprogramming other pancreatic cells. By “re-starting” expression of three critical beta-cell genes in differentiated adult pancreatic exocrine cells, researchers were able to create beta cell-like cells that can secrete insulin. The reprogrammed cells were similar to beta cells in appearance, size, and shape; expressed genes characteristic of beta cells; and were able to partially restore blood sugar regulation in mice whose own beta cells had been chemically destroyed. While not transdifferentiation by definition, this method for reprogramming adult cells may be used as a model for directly reprogramming other adult cell types.
In addition to reprogramming cells to become a specific cell type, it is now possible to reprogram adult somatic cells to become like embryonic stem cells (induced pluripotent stem cells, iPSCs) through the introduction of embryonic genes. Thus, a source of cells can be generated that are specific to the donor, thereby avoiding issues of histocompatibility, if such cells were to be used for tissue regeneration.