ADA, Adenosine deaminase; Hb, hemoglobin; IV, intravenous; PEG, polyethylene glycol; SCID, severe combined immunodeficiency; WAS, Wiskott-Aldrich syndrome.

In this section, we outline the potential, methods, and probable limitations of gene transfer for the treatment of human genetic disease. The minimal requirements that must be met before the use of gene transfer can be considered for the treatment of a genetic disorder are presented in the Box.


Essential Requirements of Gene Therapy for an Inherited Disorder

Identity of the molecular defect

The identity of the affected gene must be known.

A functional copy of the gene

A complementary DNA (cDNA) clone of the gene or the gene itself must be available. If the gene or cDNA is too large for the current generation of vectors, a functional version of the gene from which nonessential components have been removed to reduce its size may suffice.

An appropriate vector

The most commonly used vectors at present are derived from the adeno-associated viruses (AAVs) or retroviruses, including lentivirus.

Knowledge of the pathophysiological mechanism

Knowledge of the pathophysiological mechanism of the disease must be sufficient to suggest that the gene transfer will ameliorate or correct the pathological process and prevent, slow, or reverse critical phenotypic abnormalities. Loss-of-function mutations require replacement with a functional gene; for diseases due to dominant negative alleles, inactivation of the mutant gene or its products will be necessary.

Favorable risk-to-benefit ratio

A substantial disease burden and a favorable risk-to-benefit ratio, in comparison with alternative therapies, must be present.

Appropriate regulatory components for the transferred gene

Tight regulation of the level of gene expression is relatively unimportant in some diseases and critical in others. In thalassemia, for example, overexpression of the transferred gene would cause a new imbalance of globin chains in red blood cells, whereas low levels of expression would be ineffective. In some enzymopathies, a few percent of normal expression may be therapeutic, and abnormally high levels of expression may have no adverse effect.

An appropriate target cell

Ideally, the target cell must have a long half-life or good replicative potential in vivo. It must also be accessible for direct introduction of the gene or, alternatively, it must be possible to deliver sufficient copies of the gene to it (e.g., through the bloodstream) to attain a therapeutic benefit. The feasibility of gene therapy is often enhanced if the target cell can be cultured in vitro to facilitate gene transfer into it; in this case, it must be possible to introduce a sufficient number of the recipient cells into the patient and have them functionally integrate into the relevant organ.

Strong evidence of efficacy and safety

Cultured cell and animal studies must indicate that the vector and gene construct are both effective and safe. The ideal precedent is to show that the gene therapy is effective, benign, and enduring in a large animal genetic model of the disease in question. At present, however, large animal models exist for only a few monogenic diseases. Genetically engineered or spontaneous mutant mouse models are much more widely available.

Regulatory approval

Protocol review and approval by an institutional review board are essential. In most countries, human gene therapy trials are also subject to oversight by a governmental agency.

General Considerations for Gene Therapy

In these instances, precisely where the transferred gene inserts into the genome of a cell would, in principle, generally not be important (see later discussion). If gene editing (see earlier discussion and Table 13-4) to treat inherited disease becomes possible, then correction of the defect in the mutant gene in its normal genomic context would be ideal and would alleviate concerns such as the activation of a nearby oncogene by the regulatory activity of a viral vector, or the inactivation of a tumor suppressor due to insertional mutagenesis by the vector. In some long-lived types of cells, stable, long-term expression may not require integration of the introduced gene into the host genome. For example, if the transferred gene is stabilized in the form of an episome (a stable nuclear but nonchromosomal DNA molecule, such as that formed by an adeno-associated viral vector, discussed later), and if the target cell is long-lived (e.g., T cells, neurons, myocytes, hepatocytes), then long-term expression can occur without integration.

Gene therapy may also be undertaken to inactivate the product of a dominant mutant allele whose abnormal product causes the disease. For example, vectors carrying siRNAs (see earlier section) could, in principle, be used to mediate the selective degradation of a mutant mRNA encoding a dominant negative proα1(I) collagen that causes osteogenesis imperfecta (see Chapter 12).

Gene Transfer Strategies


Figure 13-16 The two major strategies used to transfer a gene to a patient. For patients with a genetic disease, the most common approach is to construct a viral vector containing the human complementary DNA (cDNA) of interest and to introduce it directly into the patient or into cells cultured from the patient that are then returned to the patient. The viral components at the ends of the molecule are required for the integration of the vector into the host genome. In some instances, the gene of interest is placed in a plasmid, which is then used for the gene transfer.

The Target Cell

An important logistical consideration is the number of cells into which the gene must be introduced in order to have a significant therapeutic effect. To treat PKU, for example, the approximate number of liver cells into which the phenylalanine hydroxylase gene would have to be transferred is approximately 5% of the hepatocyte mass, or approximately 1010 cells, although this number could be much less if the level of expression of the transferred gene is higher than wild type. A much greater challenge is gene therapy for muscular dystrophies, for which the gene must be inserted into a significant fraction of the huge number of myocytes in the body in order to have therapeutic efficacy.

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Nov 27, 2016 | Posted by in GENERAL & FAMILY MEDICINE | Comments Off on Therapy

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