Gene transfer is a novel area of therapeutics in which the active agent is a nucleic acid rather than a protein or small molecule. Because delivery of naked DNA or RNA to a cell is an inefficient process, most gene transfer is carried out using a vector, or gene delivery vehicle. These vehicles have generally been engineered from viruses by deleting some or all of the viral genome and replacing it with the therapeutic gene of interest under the control of a suitable promoter (Table 68–1). Gene transfer strategies can be described in terms of three essential elements: (1) a vector; (2) a gene to be delivered, sometimes called the transgene; and (3) a relevant target cell to which the DNA or RNA is delivered. The series of steps in which the donated DNA enters the target cell and expresses the transgene is referred to as transduction. Gene delivery can take place in vivo, in which the vector is directly injected into the patient or, in the case of hematopoietic and some other target cells, ex vivo, with removal of the target cells from the patient, followed by return of the modified autologous cells after gene transfer in the laboratory. The latter approach offers opportunities to integrate gene transfer techniques with cellular therapies (Chap. 67).
Table 68–1 Characteristics of Gene Delivery Vehicles
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Table 68–1 Characteristics of Gene Delivery Vehicles
|Features||Retroviral||Lentiviral||Adenoviral||AAV||Human Foamy Virus||HSV-1||SV-40||Alphaviruses|
|Cell division requirement||Yes||G1 phase||No||No||No||No||No||No|
|Packaging limitation||8 kb||8 kb||8–30 kb||5 kb||8.5 kb||40–150 kb||5 kb||5 kb|
|Immune responses to vector||Few||Few||Extensive||Few||Few||Few in recombinant virus||Few||Few|
|Main advantages||Persistent gene transfer in dividing cells||Persistent gene transfer in transduced tissues||Highly effective in transducing various tissues||Elicits few inflammatory responses, nonpathogenic||Persistent gene expression in both dividing and nondividing cells||Large packaging capacity with persistent gene transfer||Wide host cell range; lack of immunogenicity||Limited immune responses against the vector|
|Main disadvantages||Theoretical risk of insertional mutagenesis (occurred in three cases)||Might induce oncogenesis in some cases||Viral capsid elicits strong immune responses||Limited packaging capacity||In need of a stable packaging system||Residual cytotoxicity with neuron specificity||Limited packaging capacity||Transduced gene expression is transient|
Gene transfer technology is still under development, although licensing applications for gene therapy products have been filed. Gene therapy is one of the most complex therapeutic modalities yet attempted, and each new disease represents a therapeutic problem for which dosing, safety, and efficacy must be defined. Nonetheless, gene transfer remains one of the most powerful concepts in modern molecular medicine and has the potential to address a host of diseases for which there are currently no cures or, in some cases, no available treatment. More than 5000 subjects have been enrolled in gene transfer studies, and serious adverse events have been rare. Gene therapies are being developed for a wide variety of disease entities (Fig. 68-1).
Indications in gene therapy clinical trials. The bar graph classifies clinical gene transfer studies by disease. A majority of trials have addressed cancer, with monogenic disorders and cardiovascular diseases the next largest categories. (Adapted from J Gene Med. New Jersey, Wiley, 2009.)
Gene Transfer for Genetic Disease
Gene transfer strategies for genetic disease generally involve gene addition therapy. This approach most commonly involves transfer of the missing gene to a physiologically relevant target cell. However, other strategies are possible, including supplying a gene that achieves a similar biologic effect through an alternative pathway (e.g., factor VIIa for hemophilia A), supplying an antisense oligonucleotide to splice out a mutant exon if the sequence is not critical to the function of the protein (as has been done with the dystrophin gene in Duchenne's muscular dystrophy), or downregulating a harmful response through an siRNA. Two distinct strategies are used to achieve long-term gene expression: one is to transduce stem cells with an integrating vector, so that all progeny cells will carry the donated gene; the other is to transduce long-lived cells such as skeletal muscle or neural cells. In the case of long-lived cells, integration into the target cell genome is unnecessary. Instead, because the cells are nondividing, the donated DNA can be stabilized in an episomal form, avoiding problems related to integration and insertional mutagenesis.
Immunodeficiency Disorders: Proof of Principle
Early attempts to effect gene replacement into hematopoietic stem cells (HSCs) were stymied by the relatively low transduction efficiency of retroviral vectors, which require dividing target cells for integration. Because HSCs are normally quiescent, they are a formidable transduction target. However, identification of cytokines that induced cell division without promoting differentiation of stem cells, along with technical improvements in the isolation and transduction of HSCs, led to modest but real gains in transduction efficiency.
The first convincing therapeutic effect from gene transfer occurred with X-linked severe combined immunodeficiency disease (SCID), which results from mutations in the gene (IL2RG) encoding the γc subunit of cytokine receptors required for normal development of T and NK cells (Chap. 316). Affected infants present in the first few months of life with overwhelming infections and/or failure to thrive. In this disorder, it was recognized that the transduced cells, even if few in number, would have a proliferative advantage compared to the nontransduced cells, which lack receptors for the ...