Molecular Biotechnology: Principles and Applications - Glick, B. R., Pasternak, J. J. 2002

Molecular Biotechnology of Microbiological Systems
Gene Therapy

For a long time, medical genetics focused on a single challenge: identifying the GENETIC BASIS OF inherited human diseases. Diagnostic tests were developed to detect various such conditions in newborns or fetuses, enabling Genetic Counseling for at-risk families. This helped prepare prospective parents for the possibility of a child inheriting the disorder. Furthermore, it was occasionally possible to mitigate the negative consequences of genetic defects through drug therapy, Blood transfusions, or specialized diets.

The normal functioning of the Organism is ensured by the coordinated activity of numerous interconnected genes, and a mutation in even a single Gene can lead to diverse consequences. For instance, a mutation altering The activity of a specific enzyme can cause the accumulation of a toxic substrate or a deficiency in a compound essential for normal cellular function, whereas a mutation in a structural protein gene can lead to severe cellular, tissue, or organ-level dysfunctions. Moreover, a mutation in a gene expressed in one tissue can profoundly impact an entirely different tissue, resulting in a wide array of symptoms. For example, a mutation in the gene encoding the hepatic enzyme phenylalanine hydroxylase, which blocks The conversion of phenylalanine to Tyrosine, severely affects The Nervous system. In an individual homozygous for the defective gene, this enzyme is either not produced at all or synthesized in extremely low amounts; this leads to elevated levels of endogenous phenylalanine in the blood, defective myelination of axons in the Central Nervous System, and, consequently, severe mental retardation. This congenital disorder, known as phenylketonuria, occurs in Caucasians with a frequency of 1 in 10,000 newborns. Although each tissue in the body expresses a distinct subset of the total gene Complement, some Mutations cause systemic diseases affecting virtually all Organs and Tissues: Muscles, eyes, Liver, bones, Kidneys, Heart, nervous system, Skin, Brain, Stomach, intestines, and the hematopoietic system. The ultimate goal of medical genetics research is to develop treatments for all inherited diseases. Table 21.1 lists the products of several genes, briefly describes the symptoms of the disorders caused by mutations in these genes, and outlines their treatments. Inherited disorders have complex clinical manifestations, and their Treatment is largely symptomatic. Some Metabolic Disorders are managed by prescribing specialized diets, which help reduce the levels of toxic substances in the body whose accumulation is driven by mutations in specific genes. For instance, in phenylketonuria—detected in newborns via specialized biochemical blood screening—a phenylalanine-free diet is prescribed. To alleviate the symptoms of genetic DISEASES ASSOCIATED WITH specific protein defects, functional, non-immunogenic forms of the protein are administered intravenously. Such "replacement" therapy is used, for example, to treat hemophilia, severe combined immunodeficiency (SCID), and Gaucher disease. Occasionally, Bone Marrow or other organ transplantation is performed to compensate for lost Functions. Unfortunately, intensive treatment for many Hereditary diseases is often initiated only when the patient is in critical condition, yielding only marginal life extension. Because Genetic Disorders are frequently systemic and progressively debilitate the organism, developing effective therapies is a formidable challenge. Current treatments are generally inefficient, with very few patients surviving to old age or having children. In most cases, therapies must be administered repeatedly, making them exceptionally costly and prolonged. Consequently, The Development of novel therapeutic approaches is of paramount importance.

Class="center">Table 21.1. Current treatments for selected monogenic human diseases

Once the Molecular Basis of Bacterial Transformation (gene transfer between strains) was established, scientists hoped that a similar mechanism—introducing normal genes into defective somatic Cells—could be used to treat inherited human diseases. The Prospects for somatic gene correction became more realistic in the 1980s, by which time Methods for isolating genes had been developed, eukaryotic expression vectors created, and gene transfer experiments in mice rendered routine. In 1990, the first attempt at Gene Therapy was performed on two young girls with SCID (Table 21.1).

The approach was as follows: cloned adenosine deaminase (ADA) cDNA was introduced into lymphocytes harvested from each patient. The modified, ADA-synthesizing cells were cultured and periodically infused into the girls over a two-year period. Four years after the treatment began, both patients exhibited ADA Gene Expression and an alleviation of SCID symptoms. However, the precise cause of the improvement remained somewhat ambiguous: whether it resulted from replacement therapy (intravenous administration of a stabilized form of ADA, polyethylene glycol-ADA) or actual gene therapy. One thing was certain: this trial demonstrated the safety of gene therapy. Similar results were observed in other SCID patients who concurrently received both treatments. Research was subsequently expanded to a larger cohort of patients.

Under US legislation, before a new drug can be approved for clinical use, it must undergo four strictly defined phases of testing.

1. Preclinical testing, which includes extensive in vitro experiments and studies on laboratory animals.

2. Phase I clinical trials, conducted on a small number (6 to 10) of patients, primarily aimed at evaluating drug safety.

3. Phase II clinical trials, conducted on a larger patient cohort to assess therapeutic efficacy.

4. Phase III clinical trials, involving a large cohort of subjects and comprehensive Analysis of the drug's safety and efficacy, utilizing data gathered from previous stages.

Before clinical testing can begin, the trial protocol must be reviewed and approved by the appropriate regulatory authorities. Between 1990 and 1992, over ten Phase I gene therapy protocols were approved, and by 1997, more than 200 gene therapy protocols targeting various malignancies, hemophilia, AIDS, cystic fibrosis, hypercholesterolemia, and AMYOTROPHIC LATERAL SCLEROSIS, among others, had been approved (Table 21.2). Prior to initiating Phase I clinical trials, several crucial factors must be considered: the proposed study must aim to treat a definitively diagnosed disease, comply with established bioethical and experimental guidelines, and entail minimal risk to the patient. Because gene therapy is a novel field and the disorders targeted are highly diverse, numerous approaches are currently under evaluation. At present, all gene therapy research focuses on correcting Genetic Defects in somatic cells rather than germline (reproductive) cells. This is driven by ethical, technical, and safety considerations, as DNA introduced into human Germ Cells would be inherited by subsequent generations.

Table 21.2. Selected diseases targeted by gene therapy clinical trials since 1990

In the broadest sense, human somatic gene therapy refers to the correction of a specific inherited disorder through the Introduction of a functional, expressing gene into target cells. However, this simple definition conceals a host of challenges. For instance, how does one access the cells intended for correction? How should the therapeutic gene be delivered? What proportion of target cells must receive the gene for the disease to recede? Is precise transcriptional control of the inserted gene necessary for efficacy? Might overexpression of the introduced gene trigger adverse side effects? Will the modified cells persist indefinitely, or will repeated administrations be required?

Although somatic gene therapy is still in its infancy, Answers have already been found for several questions regarding specific inherited conditions. Novel approaches to somatic gene therapy continue to emerge, broadly divided into two main categories: in vivo and Ex Vivo Gene therapy. Furthermore, specialized nucleic acid-based drugs are being developed, including antisense oligonucleotides, engineered ribozymes, and oligonucleotides designed to correct Gene Mutations in vivo.