Basic approach to gene therapy

The basic approach to gene therapy is outlined in Figure 14.1. The desired gene must usually be packaged into a vector system capable of delivering it safely inside the intended recipient cells. A variety of vectors can be used to effect gene transfer. These include both viruses (particularly retroviruses and adenoviruses) and non-viral carriers, such as plasmid-containing liposomes/ lipoplexes (Table 14.2 and Figure 14.2). Each such vector has its own unique set of advantages and disadvantages, as discussed subsequently in this chapter.

Nucleus

Vector

Gene to be transferred

Nucleus

(a)

Intracellular effect

Extracellular effect

Figure 14.1 Simplified schematic representation of the basis of gene therapy. The genetic material to be transferred is first usually packaged into some form of vector that serves to deliver the nucleic acid to the target cell. (a) Entry of the therapeutic nucleic acid, often still associated with its vector, into the cell cytoplasm. (b) Transfer of the nucleic acid into the nucleus of the recipient cell. This is often, though not always, followed by integration of the foreign genetic material into the cellular DNA. (c) The foreign gene (whether integrated or not) is expressed, resulting in the synthesis of the desired protein product. Regulatory elements of the nucleic acid transferred may be designed to ensure the protein product is retained within the cell, or is exported from the cell, as is necessary. Refer to the text for further details

Once assimilated by the cell, the exogenous nucleic acid must now travel/be delivered to the nucleus. In some cases, the mechanism by which this transfer occurs is understood, at least in part (e.g. in the case of retroviral vectors). In other cases (e.g. use of liposome vectors or naked DNA), this process is less well understood. At a practical level, gene therapy protocols may entail one of three different strategies (Figure 14.3).

Table 14.2 Vector systems used to deliver genes into mammalian cellsa

Viral-based vector systems Non-viral-based vector systems

Retroviruses Nucleic-acid-containing liposomes

Adenoviruses Molecular conjugates

Adeno-associated virus Direct injection of naked DNA

Herpes virus CaPO4 precipitation

Polio virus Electroporation

Vaccinia virus Particle acceleration aPrior to 2003 the majority of clinical trials undertaken utilized retroviral vector systems, although adenoviral-based systems have now come to the fore. Non-viral systems have generally been employed least often, although some, e.g. nucleic-acid-containing liposomes, may be used more extensively in the future. Some of the methods tested, e.g. calcium phosphate precipitation, electroporation and particle acceleration, are unlikely to be employed to any great extent in gene therapy protocols.

Figure 14.2 Vectors used thus far in gene therapy trials. 'Others' are mainly viral-based and include the use of pox, vaccinia and adeno-associated viruses, as well as herpes simplex virus. Data adapted from www.wiley. co.uk/genemed/clinical

Target cells for gene therapy

Vector

Target cells for gene theraphy

Figure 14.2 Vectors used thus far in gene therapy trials. 'Others' are mainly viral-based and include the use of pox, vaccinia and adeno-associated viruses, as well as herpes simplex virus. Data adapted from www.wiley. co.uk/genemed/clinical

Figure 14.3 The various practical approaches that may be pursued when undertaking gene therapy. (a) In vitro gene therapy entails removal of target cells from the body followed by their incubation with nucleic acid-containing vector. After the vector delivers the nucleic acid into the human cells, they are placed back in the body. (b) In situ gene therapy entails direct injection of the vector immediately adjacent to the body target cells. (c) In vivo gene therapy involves intravenous administration of the vector. The vector has been designed such that it will only recognize and bind the intended target cells. In this way, the nucleic acid is delivered exclusively to those cells. Refer to text for further details

Figure 14.3 The various practical approaches that may be pursued when undertaking gene therapy. (a) In vitro gene therapy entails removal of target cells from the body followed by their incubation with nucleic acid-containing vector. After the vector delivers the nucleic acid into the human cells, they are placed back in the body. (b) In situ gene therapy entails direct injection of the vector immediately adjacent to the body target cells. (c) In vivo gene therapy involves intravenous administration of the vector. The vector has been designed such that it will only recognize and bind the intended target cells. In this way, the nucleic acid is delivered exclusively to those cells. Refer to text for further details

The in vitro approach entails initial removal of the target cells from the body. These are then cultured in vitro and incubated with vector containing the nucleic acid to be delivered. The genetically altered cells are then reintroduced into the patient's body. This approach represents the most commonly adopted protocol to date. In order to be successful, however, the target cells must be relatively easy to remove from the body, and reintroduce into the body. Such in vitro approaches have successfully been undertaken utilizing various body cell types, including blood cells, stem cells, epithelial cells, muscle cells and hepatocytes.

A second approach involves direct injection/administration of the nucleic-acid-containing vector to the target cell, in situ in the body. Examples of this approach have included the direct injection of vectors into a tumour mass, as well as aerosol administration of vectors (e.g. containing the cystic fibrosis gene) to respiratory tract epithelial cells.

Although less complicated than the in vitro approach, direct in situ injection of vector into the immediate vicinity of target cells is not always feasible. This would be true, for example, if the target cells are not localized to one specific area of the body (e.g. blood cells). An alternative (in vivo) approach entails the development of vectors capable of recognizing and binding only to specific, predefined cell types. Such vectors could then be administered easily by, for example, i.v. injection. Through appropriate biospecific interactions, they would only deliver their nucleic acid payload to the specified target cells. The simplicity and specificity of this approach renders it the method of choice. However, thus far, no such vector systems have been developed for routine therapeutic use. Intensive efforts to develop these are underway, and a number of different strategies are being pursued. For example, the inclusion of an antibody on the vector surface, which specifically binds a surface-antigen uniquely associated with the target cell, would allow selective delivery. Another approach entails engineering the vector to display a specific hormone that would bind only to cells displaying the hormone receptor. The feasibility of this approach has been demonstrated using retroviral vectors engineered to display EPO on their surface.

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