This article provides an overview on the principles of gene transfer.
Classical gene therapies normally require efficient transfer of cloned genes into disease cells so that the introduced genes are expressed at suitably high levels. In principle, there are numerous different physicochemical and biological methods that can be used to transfer exogenous genes into human cells.
The size of DNA fragments that can be transferred is in most cases comparatively very limited, and so often the transferred gene is not a conventional gene. Instead, an artificially designed minigene may be used: a cDNA sequence containing the complete coding DNA sequence is engineered to be flanked by appropriate regulatory sequences for ensuring high level expression, such as a powerful viral promoter. Following gene transfer, the inserted genes may integrate into the chromosomes of the cell, or remain as extra chromosomal genetic elements (episomes).
Genes Integrated into Chromosomes:
The advantage of integrating into a chromosome is that the gene can be perpetuated by chromosomal replication following cell division (Fig. 23.3). As progeny cells also contain the introduced genes, long-term stable expression may be obtained. As a result, gene therapy using this approach may provide the possibility of a cure for some disorders.
For example, in tissues composed of actively dividing cells, the key is to target the stem cells (a minority population of undifferentiated precursor cells which gives rise to the mature differentiated cells of the tissue). This is so because stem cells not only give rise to the mature tissue cells, but during this procedure they also renew themselves.
As a result, they are an immortal population of cells from which all other cells of the tissue are derived. High efficiency gene transfer into stem cells, and subsequent stable high level expression of a suitable introduced gene, can, therefore, provide the possibility of curing a genetic disorder.
Chromosomal integration has its disadvantages, however, because the insertion often occurs almost randomly: the location of the inserted genes can vary enormously from cell to cell. In many cases, inserted genes may not be expressed because of integration into a highly condensed heterochromatic region.
In some cases, the integration event can result in death of the host cell (for example, by insertion into a crucially important gene, thereby inactivating it). Such an event has consequences only for the single cell in which the integration occurred.
A greater concern is the possibility of cancer: an integration event in one of the many cells that are targeted could disturb the normal expression patterns of genes that control cell division or cell proliferation, for example by activating an oncogene or inactivating a tumor suppressor gene or a gene involved in apoptosis (programmed cell death).
Ex vivo gene therapy at least offers the opportunity for selecting cells where integration has been successful, amplifying them in cell culture and then checking the phenotypes for any obvious evidence of neoplastic transformation, prior to transferring the cells back into the patient.
Nonintegrated Genes:
Some gene transfer systems are designed to insert genes into cells where they remain as extra chromosomal elements and may be expressed at high levels (see Table 23.3). If the cells are actively dividing, the introduced gene may not segregate equally to daughter cells and so long-term expression may be a problem.
As a result, the possibility of a cure for a genetic disorder may be remote: repeated treatments involving gene transfer will be necessary. In some cases, however, there may be no need for stable long-term expression. For example, cancer gene therapies often involve transfer and expression of genes into cancer cells with a view to killing the cells. Once the malignancy has been eliminated, the therapeutic gene may no longer be needed.
Most gene therapy protocols have used mammalian viral vectors because of their high efficiency of gene transfer:
The method chosen for gene transfer depends on the nature of the target tissue and whether transfer is to cultured cells ex vivo or to the cells of the patient in Vivo. No one gene transfer system is ideal; each has its limitations and advantages. However, mammalian viral systems have been particularly attractive because of their high efficiency of gene transfer into human cells.
The major classes of recombinant virus vectors are based on certain retroviruses which are integrating but only infect actively dividing cells, and adenoviruses which infect a wide range of cell types but do not integrate efficiently.
In addition, increasing use is made of other systems such as adeno-associated viruses, while some viruses are particularly suitable for infecting specific tissues, as in the case of herpes simplex virus (HSV).
Retrovirus Vectors:
Retroviruses are RNA viruses which possess a reverse transcriptase function, enabling them to synthesize a complementary DNA form that can integrate into chromosomal DNA (see Fig. 23.4). They are very efficient at transferring DNA into cells, and integration of viral DNA occurs usually at a single chromosomal site.
The integrated DNA can be stably propagated, offering the possibility of a permanent cure for a disease. Simple injection of retroviral vectors is usually inappropriate for in vivo gene therapy because they can generally be killed by human complement.
Retroviruses can only be produced at relatively low titers and only infect actively dividing cells, thereby excluding their use in treating tissues composed of non-dividing cells (e.g.’ neurons, etc.). This same property is, however, beneficial to gene therapy for cancers of tissues that normally have non-dividing cells; the actively dividing cancer cells can be selectively infected and killed without major risk to the non-dividing cells of the normal tissue. Widely used murine retrovirus vectors can accommodate inserts up to 8 kb.
Adenovirus Vectors:
Adenoviruses are DNA viruses that produce infections of the upper respiratory tract and have a natural tropism for respiratory epithelium, the cornea and the gastro-intestinal tract Unlike retroviruses, which can only infect actively dividing cells, adenoviruses can infect a very wide variety of cell types.
Entry into cells occurs by receptor-mediated endocytosis (Fig. 23.4; see also below) and is efficient, but the inserted DNA does not appear to integrate and so expression of inserted genes can only be sustained over short periods. Adenovirus vectors can be produced at very high titers, typically accept insert sizes up to 7-8 kb and, because of their ability to infect many different types of cells, have found widespread applications, notably in vivo gene therapy strategies.
Because they can infect virtually all human cells, cancer gene therapies involving cell-killing without causing toxicity to normal surrounding cells could be a problem. Another problem is that adenovirus vectors can induce significant inflammatory responses as happened when used to treat cystic fibrosis.
Herpes Simplex Virus Vectors:
HSV vectors are tropic for the central nervous system (CNS) and can establish lifelong latent infections in neurons. They are non-integrating and so long-term expression of transferred genes is not possible. Their major applications are expected to be in delivering genes into neurons for the treatment of neurological diseases, such as Parkinson’s disease, and for treating CNS tumors. They have a comparatively large insert size capacity (>20 kb).
Adeno-associated Virus Vectors:
Adenoassociated viruses (AAVs) are a group of small, single-stranded DNA viruses which cannot usually undergo productive infection without co-infection by a helper virus, such as an adenovirus or HSV. In the absence of co-infection by a helper virus, unmodifie human AAV integrates into chromosomal DNA, usually at a specific site on 19q13.3—qter. Subsequent super-infection with an adenovirus can activate the integrated virus DNA, resulting in progeny virions.
AAV vectors can only accommodate inserts up to 4.5 kb, but they have the advantage of providing the possibility of long-term gene expression with a high degree of safety; they integrate into chromosomal DNA but, because 96% of the parental AAV genome has been deleted—the AAV vectors lack any viral genes and recombinant AAV vectors only contain the gene of interest.
Concerns over the safety of recombinant viruses have prompted increasing interest in non-viral vector systems for gene therapy:
Increasingly concern has been expressed regarding the safety of viral vector systems. The recombinant viruses which are used for ex vivo gene therapy are designed to be disabled: typically some viral genes required for viral replication are deleted, and the therapeutic genes that are to be transferred are inserted in their place. The resulting replication-incompetent viruses are then intended to infect individual cells.
In the case of retrovirus vectors, chromosomal integration is still possible but they—like other replication-incompetent virus vectors—should not be able to undergo a productive infection in which they replicate, assemble new virions and infect new cells. However, there is the remote possibility that the introduced viruses can recombine with endogenous retroviruses, resulting in recombinant progeny that can undergo productive infection.
Additionally, adenoviruses are generally non-integrating and the repeated injections that may be required may provoke severe inflammatory responses to the recombinant adenoviruses as has happened recently in a gene therapy trial for cystic fibrosis. Increasingly, therefore, attention has been focused towards studying alternative methods of gene transfer.
Direct Injection/Particle Bombardment:
In some cases, DNA can be injected directly with a syringe and needle into a specific tissue, such as muscle. This approach has been considered, for example, in the case of DMD, where early studies investigated intramuscular injection of a dystrophin minigene into a mouse model, mdx .
An alternative direct injection approach uses particle bombardment techniques: DNA is coated on to metal pellets and fired from a special gun into cells. Successful gene transfer into a number of different tissues has been obtained using this approach. Such direct injection techniques are simple and comparatively safe.
However, there is poor efficiency of gene transfer, and a low level of stable integration of the injected DNA. The latter property is particularly disadvantageous in the case of proliferating cells, and would necessitate repeated injections, it may be less of a problem in tissues such as muscle which do not regularly proliferate, and in which the injected DNA may continue to be expressed for several months.
Receptor-mediated Endocytosis:
The DNA is coupled to a targeting molecule that can bind to a specific cell surface receptor, inducing endocytosis and transfer of the DNA into cells. Coupling is normally achieved by covalently linking polylysine to the receptor molecule and then arranging for (reversible) binding of the negatively charged DNA to the positively charged polylysine component.
For example, hepatocytes are distinguished by the presence on the cell surface of asialoglycoprotein receptors which clear asialoglycoproteins from the serum. Coupling of DNA to an asialoglycoprotein via a polycation such as polylysine can target the transfer of exogenous DNA into liver cells.
The complexes can be infused into the liver either via the biliary tract or vascular bed, whereupon they are taken up by hepatocytes. A more general approach utilizes the transferrin receptor which is expressed in many cell types, but is relatively enriched in proliferating cells and hemopoietic cells (see Fig. 23.5). Gene transfer efficiency may be high but the method is not designed to allow integration of the transferred genes.
A further problem has been that the protein-DNA complexes are not particularly stable in serum. Additionally, the DNA conjugates may be entrapped in endosomes and degraded in lysosomes, unless previously co-transferred with, or physically linked to, an adenovirus molecule (see Fig. 23.5 )
Liposomes:
Liposomes are spherical vesicles composed of synthetic lipid bilayers which mimic the structure of biological membranes. The DNA to be transferred is packaged in vitro within the liposomes and used directly for transferring the DNA to a .suitable target tissue in vivo (Fig. 23.6).
The lipid coating allows the DNA to survive in vivo, bind to cells and be endocytosed into the cells. Liposomes have become popular vehicles for gene transfer in vivo gene therapy because of the safety concerns when using recombinant viruses. However, the efficiency of gene transfer is low, and the introduced DNA is not designed to integrate into chromosomal DNA, so expression of the inserted genes is transient.
