Molecular Genetic & Treatment of Diseases | Human Genetics

Let us make an in-depth study of the molecular genetic based approaches for the treatment of diseases.

Principles of Molecular Genetic-Based Approaches to the Treatment of Diseases:

Once a human disease gene has been characterized, molecular genetic tools can be used to dissect gene function and explore the biological processes involved in the normal and pathogenic states. The resulting information can be used to design novel therapies using conventional drug-based approaches.

In addi­tion, molecular genetic technologies have recently provided a variety of novel therapeutic approaches that depend on the ability to clone individual types of gene, transfer them into recipient cells and express them; the ability to re-design proteins; and the ability to inhibit specifically the expression of a predetermined (characterized) gene in vivo.

Novel molecular genetic-based therapeutic approaches can be categorized into two broad groups, depending on whether the therapeutic agent is a gene product/vaccine or genetic material.

Recombinant pharmaceuticals and vaccines can be provided by genetic engi­neering. Possible approaches include:

Expression cloning of normal gene products – Cloning of (usually) human genes and expression in suitable expression systems to make large amounts of a medically valuable gene product;

Genetically engineered antibodies – antibody genes can be manipulated to form novel antibodies, including partially or fully humanized antibodies, for use as therapeutic agents;

Genetically engineered vaccines – novel cancer vaccines and vaccines against infec­tious agents.

Gene Therapy:

Gene therapy is the genetic modifica­tion of the cells of a patient in order to combat disease. This very broad definition includes many different possible approaches, and can involve transfer of cloned human genes, double-stranded human gene seg­ments, genes from other genomes, oligonu­cleotides and various artificial genes, such as antisense genes.

Such gene transfer can result in genetic modification of the cells of the patient. In most cases’; the gene therapy is designed to lead to genetic modification of disease cells, but some approaches are delib­erately; designed to target non disease cells, notably immune system cells, and constitute a form of vaccination.

Cloned Human Genes can be Used as a Source of Medically Important Products:

Once a human gene has been cloned, large amounts of the purified product can be obtained by using a suitable system for expres­sion cloning. This often involves expressing the desired gene in bacterial cells, which have the advantage that they can be cultured easily in large volumes, and large amounts of prod­uct can be obtained.

Even if long-standing alternative treatments are available, the gene cloning—expression approach may be pre­ferred because it minimizes safety risks. For example, diabetes sufferers traditionally have been treated with insulin prepared from cows or pigs. Because of species differences in the amino acid sequence of the product, however, animal products are potentially immunogenic and may produce unwanted side-effects in highly immunoreactive individuals.

The administration of biochemically puri­fied human products may also be hazardous. Recently, many hemophiliacs have developed acquired immune deficiency syndrome (AIDS) as a result of treatment with factor VIII which was purified from the serum of unscreened human donors. Growth hormone deficiency was treated in the past by purified human growth hormone.

However, some patients developed Creutzfeldt-Jakob disease (a rare neurological disorder which is a human coun­terpart of scrapie in sheep and of ‘mad cow disease’ because the hormone was extracted from large numbers of pooled cadaver pituitaries.

Cloning of a medically important human gene and expression in suitable expression- cloning systems is, therefore, attractive. If bio­chemical purification of the product from a human or animal source is difficult or impossi­ble, it provides the only product-based thera­peutic route.

Even if biochemical purification of the desired product from animals or humans is possible, large quantities of the human product can be produced by expres­sion-cloning without the attendant hazards of the type described above. Recombinant human insulin was first marketed in 1982 and, subsequently, a number of other cloned human gene products of medical interest have been produced commercially (see Table 23.1). Treatment with the products of cloned genes may also pose risks, however.

For example, patients who completely lack a normal pro­duct may mount a vigorous immune response to the administered pharmaceutical product as in the case of some patients with severe hemophilia A who have been treated with recombinant factor VIII.

Expression cloning often involves the use of microorganisms, but this approach may not always be suitable. For example, expres­sion of a human gene in a bacterial cell can give a product that shows differences from the normal human product: the polypeptide may have the same sequence of amino acids but patterns of glycosylation, etc., produced by post-translational processing may be dif­ferent.

This may mean that the gene product is not particularly stable in a human environ­ment, or it may provoke an immune response, or its biological function may be less effective than desired. Alternative expres­sion systems have been utilized, and increas­ing attention has been paid to constructing transgenic livestock whose post-transla­tional processing systems are more similar to analogous human systems. For example, a cloned human gene can be fused to a sheep gene specifying a milk protein and then inserted into the genome of the sheep germ line.

The resultant transgenic sheep can secrete large quantities of the fusion protein in its milk. The design of the fusion gene nor­mally permits the ability to cleave the secret­ed fusion protein using a specific protease in order to generate the human protein residue which can be purified easily.

Antibody Engineering has Permitted the Construction of Humanized Antibodies and Fully Human Antibodies:

Antibodies are natural therapeutic agents which are produced by B lymphocytes. In each B-cell precursor, a cell-specific rearrangement of antibody gene components occurs. Additional diversity is provided by other mechanisms, including frequent somatic muta­tion events.

As a result, each individual has a population of B cells which collectively ensures a huge repertoire of different antibo­dies as a defense system against a diverse array of foreign antigens. The antibody may be thought of as an adaptor molecule: it con­tains binding sites for foreign antigen at the variable (V) end, and for effector molecules at the constant (C) end. Binding of an antibody may by itself be sufficient to neutralize some toxins and viruses, but it is more common for the antibody to trigger the complement system and cell-mediated killing.

Artificially produced therapeutic anti­bodies are designed to be mono-specific (i.e. they will recognize a single type of antigenic site) and can be employed specifically to recognize particular disease-associated anti­gens, leading to killing of the disease cells.

Notable targets for such therapy are cancers, especially lymphomas and leukemia’s, infec­tious disease (using antibodies raised against antigens of the relevant pathogen) and auto­immune disorders (antibody recognition of inappropriately expressed host cell antigens). A favorite way of producing immortal mono-­specific antibodies is to fuse individual anti­body-producing B lymphocytes from an immu­nized mouse or rat with cells derived from an immortal mouse B-Lymphocyte tumor.

From the fusion products—a heterogeneous mixture of hybrid cells, those hybrids that have both the ability to make a particular antibody and the ability to multiply indefinitely in culture are selected. Such hybridomas are propagated as individual clones, each of which can provide a permanent and stable source of a single type of monoclonal antibody.

Until recently, the therapeutic antibody approach was not straightforward. It has proved very difficult to make human mono­clonal antibodies (mAbs) using hybridoma technology. Although rodent mAbs can be created against human pathogens and cells, they have limited clinical utility: the rodent mAbs have a short half-life in human serum; only some of the different classes can trigger human effector functions; and they can elicit an unwanted immune response in patients (human anti-mouse antibodies).

Once immunoglobulin genes had been cloned, how­ever, the possibility of designing artificial com­binations of immunoglobulin gene segments arose (antibody engineering). The process was assisted by the use of different exons to encode different domains of an antibody molecule: domain swapping could be done easily at the DNA level by artificial exon shuf­fling between different antibody genes. The resulting recombinant antibody genes could then be expressed to give chimeric antibodies

Humanized Antibodies:

One immediate goal of antibody enginee­ring was the production of humanized anti­bodies, that is rodent-human recombinant antibodies Humanizing of rodent antibodies allows, in principle, access to a large pool of well- characterized rodent mAbs for therapy, inclu­ding those with specificities against human antigens that are difficult to elicit from a human immune response.

Early versions con­tained the variable domains of a rodent anti­body attached to the constant domains of a human antibody: the immunogenicity of the rodent mAb is reduced, while allowing the effector functions to be selected for the thera­peutic application. A further stage of human­izing antibodies is possible.

The essential anti- gen-binding site is a subset of the variable region characterized by hyper variable sequences, the complementarity-determining regions (CDRs). Accordingly, second genera­tion humanized antibodies were CDR-grafted antibodies: the hyper variable antigen-binding loops of the rodent antibody were built into a human antibody.

Chimeric V/C antibodies and CDR-engrafted antibodies have been con­structed against a wide range of microbial pathogens and against human cell surface markers, including tumor cell antigens and, in some cases, their use has already been demonstrated in the clinic (see Table 23.2).

Fully Human Antibodies:

Two approaches have been taken towards the construction of fully human antibodies. One approach—phage display technology bypass­es hybridoma technology, and even immuniza­tion. Instead, antibodies are made in vitro by mimicking the selection strategies of the immune system, a procedure which should facil­itate the construction of human antibodies of therapeutic value, as well as research potential.

A second recent approach has involved using transgenic mice .This strategy involves transferring yeast artificial chromosomes containing large segments of the human heavy and kappa light chain immunoglobulin loci into mouse embryonic stem cells, and subsequent production of trans­genic mice.

The resulting mice produce a diverse repertoire of human heavy and light chain immunoglobulin’s and, upon immuniza­tion with tetanus toxin, can be used to derive antigen-specific fully human monoclonal anti­bodies.

By breeding the mice to mice that are engineered by gene targeting to be deficient in mouse immunoglobulin production, a mouse strain was obtained in which high levels of anti­bodies were produced, mostly with both human heavy and light chains. Such strains should per­mit the development of fully human monoclon­al antibodies with therapeutic potential.

Genetically Engineered Vaccines have Great Therapeutic Promise:

Recombinant DNA technology is also being applied to the construction of novel vaccines.

Several different strategies are being used:

(a) Direct DNA injection. Direct injection of a piece of influenza virus DNA (which was conserved between several different strains) into muscle cells in mice has result­ed in a potent antibody response against influenza. If this approach works as effec­tively in humans, the therapeutic potential is very considerable.

(b) Genetic modification of antigen. This can be achieved, for example, by fusion with a cytokine gene to increase anti­genicity.

(c) Genetic modification of microorgan­isms.

This can involve two approaches:

(i) Genetically disabling an organism (e.g. by removing genes required for pathogenesis or survival). This is a genetic method of attenuation so that a live vaccine can be used without undue risk.

(ii) Inserting an exogenous gene that will be expressed in bacteria or parasites.

One promising application is the use of genetically modified bacille Calmette-Guerin (BCG) as a vehicle for immunization. BCG is a live attenuated tubercle bacillus which is the most widely used vaccine in the world: it is inherently immunostimulatory, and has a very low incidence of serious complications. Recombinant BCG strains have been deve­loped using expression vectors containing BCG regulatory sequences coupled to genes encoding foreign antigens.

Such strains can elicit long-lasting antibody and cell-mediated immune responses to foreign antigens in mice, and hold the promise of allowing simultaneous expression of multiple protective antigens of different pathogens.

Recombinant organisms of this type could be used to carry genes not only for infectious agents, but also for tumors, providing cancer vaccines, and, theoretically, auto antigens. Note that some gene therapy approaches, such as adoptive immunotherapy, are effec­tively forms of genetically engineered vacci­nation.