Fundamentals of biotechnology
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Fundamentals of Biotechnology. Gene therapy. Gene therapy. The term gene therapy describes any procedure: intended to treat or alleviate disease by genetically modifying the cells of a patient.

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Fundamentals of Biotechnology

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Fundamentals of biotechnology

Fundamentals of Biotechnology

Gene therapy

Gene therapy

Gene therapy

  • The term gene therapy describes any procedure:

  • intended to treat or alleviate disease by genetically modifying the cells of a patient.

  • It encompasses many different strategies and the material transferred into patient cells may be

  • genes, gene segments or oligonucleotides.



  • The genetic material may be transferred directly into cells within a patient (in vivo gene therapy),

  • or cells may be removed from the patient and the genetic material inserted into them in vitro, prior to transplanting the modified cells back into the patient (ex vivo gene therapy).

Major disease classes include

Major disease classes include:

  • infectious diseases(as a result of infection by a virus or bacterial pathogen);

  • cancers(inappropriate continuation of cell division and cell proliferation as a result of activation of an oncogene or inactivation of a tumor suppressor gene or an apoptosis gene.

  • inherited disorders(genetic deficiency of an individual gene product or genetically determined inappropriate expression of a gene);

  • immune system disorders(includes allergies, inflammations and also autoimmune diseases, in which body cells are inappropriately destroyed by immune system cells).



  • Gene therapy has the potential to treat all of the above classes of disorder.

  • Depending on the basis of pathogenesis, different gene therapy strategies can be considered

General gene therapy strategies

General Gene therapy strategies

Gene augmentation therapy (GAT):

  • Disease caused by loss of function of a gene, introducing extra copies of the normal gene may increase the amount of normal gene product to a level where normal phenotype is restored.

  • Mostly use for autosomal receives disorders not for dominant one.

    Target killing of the specific cells:

  • Popular for cancer therapies. Genes are directed to the target cells and then expressed so as to cause cell killing.



Direct cell killing:

  • Possible if the introduced genes are expressed to produce a lethal toxin (suicidal genes), or a gene encoding a prodrug is inserted, conferring susceptibility to killing by a subsequent administered drug, selective lytic virus can be used.

  • Indirect cell killing uses immune stimulatory genes to provoke or enhance an immune response against the target cells.



Target inhibition of gene expression:

  • If disease cells display a novel gene product or inappropriate expression of a gene (in case of cancer in many infections)

  • Block gene expression at DNA, RNA or at protein level.

    Targeted mutation correction:

  • Gene targeting methods based on homologous recombination or at the RNA transcript level e.g. by using particular type of therapeutic ribozymes or therapeutic RNA Editing.

Subdivision of gene therapy

Subdivision of Gene Therapy

  • Classical gene therapy.The rationale of this type of approach is to deliver genes to appropriate target cells with the aim of obtaining optimal expression of the introduced genes.

  • Once inside the desired cells in the patient, the expressed genes are intended to do one of the following:

    • produce a product that the patient lacks;

    • kill diseased cells directly, e.g. by producing a toxin which kills the cells;

    • activate cells of the immune system so as to aid killing of diseased cells.



  • Nonclassical gene therapy. The idea here is to inhibit the expression of genes associated with the pathogenesis, or to correct a genetic defect and so restore normal gene expression.

    Current gene therapy is exclusively somatic gene therapy, the introduction of genes into somatic cells of an affected individual.

    The prospect of human germline gene therapy raises a number of ethical concerns, and is currently not sanctioned.

The technology of classical gene therapy

The technology of classical gene therapy

  • Genes can be inserted into the cells of patients by direct and indirect routes, and the inserted genes can integrate into the chromosomes or remain extrachromosomal



  • An essential component of classical gene therapy is that cloned genes have to be introduced and expressed in the cells of a patient in order to overcome the disease.

  • Practically, this usually involves targeting the cells of diseased tissues.

  • However, deliberate targeting of unaffected cells may be preferred in some approaches:



  • Immune system-mediated cell killing.In many gene therapies the target cells are healthy immune system cells, and the idea is to enhance immune responses to cancer cells or infectious agents.

  • Delivery of gene products from cells at a remote location.Genes may be targeted initially to one type of tissue while the gene products may be delivered to a remote location.

  • For example, the myonuclei in muscle fibers have the advantage of being very long lived.

  • Genetically engineered myoblasts therefore have the potential to ameliorate some nonmuscle diseases through long-term expression of exogenous genes which encode a product secreted into the blood stream.



  • Two major general approaches are used in the transfer of genes for gene therapy:

  • Transfer of genes into patient cells outside of the body (ex vivo)


  • Inside the body (in vivo).

In vitro

In vitro

  • This initially involves transfer of cloned genes into cells grown in culture.

  • Those cells which have been transformed successfully are selected, expanded by cell culture in vitro, then introduced into the patient.

  • To avoid immune system rejection of the introduced cells, autologous cells are normally used:

  • the cells are collected initially from the patient to be treated and grown in culture before being reintroduced into the same individual.



  • Clearly, this approach is only applicable to tissues that can be removed from the body,

  • altered genetically and returned to the patient where they will engraft and survive for a long period of time (e.g. cells of the hematopoietic system and skin cells).

  • Note that this type of gene therapy involves transplantation of autologous genetically modified cells and so can be considered a modified form of cell therapy.

In vivo

In vivo

  • Here the cloned genes are transferred directly into the tissues of the patient.

  • This may be the only possible option in tissues where individual cells cannot be cultured in vitro in sufficient numbers (e.g. brain cells) and/

  • or where cultured cells cannot be re-implanted efficiently in patients.

  • Liposomesand certain viral vectors are increasingly being employed for this purpose.



  • In the latter case, it is often convenient to implant vector-producing cells (VPCs), cultured cells which have been infected by the recombinantretrovirusin vitro:

  • in this case the VPCs transfer the gene to surrounding disease cells.

  • As there is no way of selecting and amplifying cells that have taken up and expressed the foreign gene,

  • the success of this approach is crucially dependent on the general efficiency of gene transfer and expression.

Cell therapy

Cell therapy

  • Unlike gene therapy cell therapy is a well established form of treating disorders

  • where patient can be treated with cells from different sources.

  • Often treatment with performed by transplanting cells from another individual

  • (allotransplantation, bone marrow transplantation is a useful example and is widely used to treat leukemia and various genetic disorders of blood cells.



  • Transplanted cells from an animal (xenotransplantation) e.g. parkinson’sdisease pig neurons.

  • The isolation of human pluripotent stem cells may provide a valuable source of cell transplantation and gene therapy.

Principles of gene transfer

Principles of gene transfer

  • Classical gene therapies normally require

  • efficient transfer of cloned genes into disease cells so that the introduced genes are expressed at suitably high levels.

  • an artificial minigene may be used: a cDNA sequence containing the complete coding DNA sequence is engineered to be flanked by appropriate regulatory sequences for ensuring high level expression, such as a powerful viral promoter.



  • Following gene transfer, the inserted genes may integrate into the chromosomes of the cell, or remain as extrachromosomal genetic elements (episomes).

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