The major reason for using stem cells as vehicles in cell-based gene therapies is that they are a self-renewing population of cells and thus may reduce or eliminate the need for repeated administrations of the gene therapy. Stem cells can be classified as embryonic or adult, depending on their tissue of origin. The role of adult stem cells is to sustain an established repertoire of mature cell types in essentially steady-state numbers over the lifetime of the organism. Although adult tissues with a high turnover rate, such as blood, skin, and intestinal epithelium, are maintained by tissue-specific stem cells, the stem cells themselves rarely divide. However, in certain situations, such as during tissue repair after injury or following transplantation, stem cell divisions may become more frequent. The prototypic example of adult stem cells, the hematopoietic stem cell, has already been demonstrated to be of utility in gene therapy.
To date, about 40 percent of the more than 450 gene therapy clinical trials conducted in the United States have been cell-based. Of these, approximately 30 percent have used human stem cells specifically hematopoietic stem cells as the means for delivering transgenes into patients. Of the stem cell-based gene therapy trials that have had a therapeutic goal, approximately one-third have focused on cancers (e.g., ovarian, brain, breast, myeloma, leukemia, and lymphoma), one-third on human immunodeficiency virus disease (HIV-1), and one-third on so-called single-gene diseases (e.g., Gaucher’s disease, severe combined immune deficiency (SCID), Fanconi anemia, Fabry disease, and leukocyte adherence deficiency).
Hematopoietic stem cells (HSC) have been a delivery cell of choice for several reasons. First, although small in number, they are readily removed from the body via the circulating blood or bone marrow of adults or the umbilical cord blood of newborn infants. In addition, they are easily identified and manipulated in the laboratory and can be returned to patients relatively easily by injection. The ability of hematopoietic stem cells to give rise to many different types of blood cells means that once the genetically engineered HSCs differentiate, the therapeutic transgene will reside in cells such as T and B lymphocytes, natural killer cells, monocytes, macrophages, granulocytes, eosinophils, basophils, and megakaryocytes. The clinical applications of hematopoietic stem cell-based gene therapies are thus also diverse, extending to blood and bone marrow disorders, and immune system disorders. In addition, hematopoietic stem cells “home,” or migrate, to a number of different spots in the body primarily the bone marrow, but also the liver, spleen, and lymph nodes. These may be strategic locations for localized delivery of therapeutic agents for disorders unrelated to the blood system, such as liver diseases and metabolic disorders such as Gaucher’s disease.
The only type of human stem cell used in gene therapy trials so far is the hematopoietic stem cell. However, several other types of stem cells are being studied as gene-delivery-vehicle candidates. They include muscle-forming stem cells known as myoblasts, bone-forming stem cells called osteoblasts, and neural stem cells. Myoblasts appear to be good candidates for use in gene therapy because of an unusual and advantageous biological property: when injected into muscle, they fuse with nearby muscle fibers and become an integral part of the muscle tissue. Moreover, since muscle tissue is generally well supplied with nerves and blood, the therapeutic agents produced by the transgene are also accessible to nerves and the circulatory system. Thus, myoblasts may not only be useful for treating muscle disorders such as muscular dystrophy, but also possibly nonmuscle disorders such as neurodegenerative diseases, inherited hormone deficiencies, hemophilia, and cancers. Several promising animal studies of myoblast-mediated gene therapy have been reported.
In a series of experiments in rodents, a team of investigators has been testing neural stem cells as vehicles for cell-based gene therapy for brain tumors known as gliomas. The genetically modified human neural stem cells can produce a protein cytosine deaminase that converts a nontoxic precursor drug into an active form that kills cancer cells. The animal studies also revealed that neural stem cells were able to quickly and accurately “find” glioma cells, regardless of whether the stem cells were implanted directly into the tumors, implanted far from the tumors (but still within the brain), or injected into circulating blood outside the brain.
There are cell-based gene therapy system under investigation involves the use of osteoblasts, or boneforming stem cells. In a preliminary study examining a gene therapy approach to bone repair and regeneration, researchers genetically engineered osteoblasts to produce a bone growth factor.
Another adult bone marrow-derived stem cell type with potential use as a vehicle for gene transfer is the mesenchymal stem cell, which has the ability to form cartilage, bone, adipose (fat) tissue, and marrow stroma (the bone marrow microenvironment). Recently, a related stem cell type, the multipotent adult progenitor cell, has been isolated from bone marrow that can differentiate into multiple lineages, including neurons, hepatocytes (liver cells), endothelial cells and other cell types. Other adult stem cells have been identified, such as those in the central nervous system and heart, but these are less well characterized and not as easily accessible.
limitations of adult stem cell mediated gene therapy
Most of the cell-based gene therapies attempted so far have used viral vehicles to introduce the transgene into the hematopoietic stem cell. One way to accomplish this is to insert the therapeutic transgene into the one of the chromosomes of the stem cell. Retroviruses are able to do this, and for this reason, they are often used as the vehicle for infecting the stem cell and introducing the therapeutic transgene into the chromosomal DNA. However, mouse retroviruses are only efficient at infecting cells that are actively dividing. Unfortunately, hematopoietic stem cells are quiescent and seldom divide. The percentage of stem cells that actually receive the therapeutic transgene has usually been too low to attain a therapeutic effect. Because of this problem, investigators have been exploring the use of viral vehicles that can infect nondividing cells, such as lentiviruses (e.g., HIV) or adeno-associated viruses. This approach has not been entirely successful, however, because of problems relating to the fact that the cells themselves are not in an active state.
The major drawback of these methods is that the therapeutic gene frequently integrates more or less randomly into the chromosomes of the target cell. In principle, this is dangerous, because the gene therapy vector can potentially modify the activity of neighboring genes (positively or negatively) in close proximity to the insertion site or even inactivate host genes by integrating into them. These phenomena are referred to as “insertional mutagenesis”; In extreme cases, such as in the X-linked SCID gene therapy trials, these mutations contribute to the malignant transformation of the targeted cells, ultimately resulting in cancer.
One approach to improving the introduction of trans-genes into hematopoietic stem cells has been to stimulate the cells to divide so that the viral vehicles can infect them and insert the therapeutic transgene. however, this manipulation can change other important properties of the hematopoietic stem cells, such as plasticity, self-renewal, and the ability to survive and grow when introduced into the patient. This possibility might be overcome with the use of embryonic stem cells if they require less manipulation.
In some cases such as a treatment of a chronic disease achieving continued production of the therapeutic transgene over the life of the patient will be very important. Generally, however, gene therapies using hematopoietic stem cells have encountered a phenomenon known as “gene silencing,” where, over time, the therapeutic transgene gets “turned off” due to cellular mechanisms that alter the structure of the area of the chromosome where the therapeutic gene has been inserted.
Persistence of the cell containing the therapeutic transgene is equally important for ensuring continued availability of the therapeutic agent. The optimal cells for cell-mediated gene transfer would be cells that will persist for “the rest of the patient’s life; they can proliferate and they would make the missing protein constantly and forever”. Persistence, or longevity, of the cells can come about in two ways: a long life span for an individual cell, or a self-renewal process whereby a short-lived cell undergoes successive cell divisions while maintaining the therapeutic transgene. Ideally, then, the genetically modified cell for use in cell-based gene therapy should be able to self-renew (in a controlled manner so tumors are not formed) so that the therapeutic agent is available on a long-term basis. This is one of the reasons why stem cells are used, but adult stem cells seem to be much more limited in the number of times they can divide compared with embryonic stem cells. Another major limitation of using adult stem cells is that it is relatively difficult to maintain the stem cell state during ex vivo manipulations. Under current suboptimal conditions, adult stem cells tend to lose their stem cell properties and become more specialized, giving rise to mature cell types through a process termed “differentiation”.
The patient’s immune system response can be another significant challenge in gene therapy. Donor stem cells may be recognized as nonself by the patient’s immune system and be rejected.
Genetic Manipulation of Stem Cells
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. All of these techniques have been applied to various stem cells, including human embryonic stem cells. However, the destiny of the introduced DNA is relatively poorly controlled using these procedures. In most cells, the DNA disappears after days or weeks, and in rare cases, integrates randomly into host chromosomal DNA.
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 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. 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 the past, gene silencing and positional effects were a particular problem in mouse hematopoietic stem cells. These problems led to the optimization of retroviral and lentiviral vector systems by the addition of genetic control elements (referred to as chromatin domain insulators and scaffold/matrix attachment regions) into the vectors, resulting in more robust expression in differentiating cell systems, including human embryonic stem cells. 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. Recently, episomal systems have been applied to embryonic stem cells.
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, and has been used extensively with mouse embryonic stem cells to investigate gene function and create mouse models of human diseases. This technique results in the replacement of normal genomic DNA with recombinant DNA containing genetic modifications (the therapeutic gene). Gene targeting by homologous recombination has recently been applied to human embryonic stem cells.
The Potential of Human Embryonic Stem Cells for Gene Therapy
To date, only nonembryonic human stem cells have been used in cell-based gene therapy studies. The inherent limitations of these stem cells have prompted scientists to ponder and explore whether human embryonic stem cells might overcome the current barriers to the clinical success of cell-based gene therapies. Embryonic stem cells are capable of unlimited self-renewal. Even after months and years of growth in the laboratory, they retain the ability to form any cell type in the body.
Since establishing human ES cells in 1998, scientists have developed genetic manipulation techniques to determine the function of particular genes, to direct the differentiation of human ES cells towards specific cell types, or to tag an ES cell derivative with a certain marker gene. Human embryonic stem cells could be genetically manipulated to introduce the therapeutic gene. This gene may either be active or awaiting later activation, once the modified embryonic stem cell has differentiated into the desired cell type. Recently published reports establish the feasibility of such an approach.
Another method to test the function of a gene is to use RNA interference (RNAi) to decrease the expression of a gene of interest. In RNAi, small pieces of double-stranded RNA (siRNA; small interfering RNA) are either chemically synthesized and introduced directly into cells, or expressed from DNA vectors. Once inside the cells, the siRNA can lead to the degradation of the messenger RNA (mRNA), which contains the exact sequence as that of the siRNA. mRNA is the product of DNA transcription and normally can be translated into proteins. RNAi can work efficiently in somatic cells, and there has been some progress in applying this technology to human ES cells.
Several approaches have been developed to introduce genetic elements randomly into the human ES cell genome, including electroporation, transfection by lipid-based reagents, and lentiviral vectors. However, homologous recombination (Gene targeting technique), a method in which a specific gene inside the ES cells is modified with an artificially introduced DNA molecule, is an even more precise method of genetic engineering that can modify a gene in a defined way at a specific locus. While this technology is routinely used in mouse ES cells, it has recently been successfully developed in human ES cells, thus opening new doors for using ES cells as vehicles for gene therapy and for creating in vitro models of human genetic disorders such as Lesch-Nyhan disease. Gene targeting by homologous recombination has recently been applied to human embryonic stem cells. This is important for studying gene functions in vitro for lineage selection and marking. For therapeutic applications in transplantation medicine, the controlled modification of specific genes should be useful for purifying specific embryonic stem cell-derived, differentiated cell types from a mixed population, altering the antigenicity of embryonic stem cell derivatives, and adding defined markers that allow the identification of transplanted cells. Additionally, since the therapeutic gene can now be introduced into defined regions of the human genome, better controlled expression of the therapeutic gene should be possible. This also significantly reduces the risk of insertional mutagenesis.
