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Facts about gene therapy

Gene Therapy Therapeutic technique by which a functional gene is inserted into a patient’s cells to correct a genetic defect or to give the cells a new function.


Human gene therapy (TG) is the deliberate administration of genetic material to a human patient with the intention of correcting a specific genetic defect, that is, the insertion of genetic material into cells of an organism by replacing mutated alleles with genes with the desired fusion , in order to treat or prevent the development of a disease .

Basic Requirements for (TG)

  1. The gene must be isolated and available for transfer.
  2. There must be an effective method for cloning.
  3. The target tissue must be accessible for gene transfer.
  4. There should not be another effective therapy available.
  5. The therapy should not harm the patient.

Types of gene therapy

Somatic gene therapy: it is carried out on the somatic cells of an individual, so that the modifications implied by the therapy only take place in said patient.

  • In vivo therapy: the cellular transformation takes place within the patient to whom the therapy is administered. It consists of administering to the patient a gene through a vehicle (for example a virus), which must locate the cells to be infected. The problem with this technique is that it is very difficult to get a vector to locate a single type of target cells.
  • Ex vivo therapy: the cellular transformation is carried out from a biopsy of the patient’s tissue and then the transformed cells are transplanted. As it happens outside the patient’s body, this type of therapy is much easier to carry out and allows a greater control of the infected cells. This technique is almost completely reduced to hematopoietic cells because they are cultivable cells, thus constituting a transplantable material.

Germinal gene therapy: it would be performed on the germ cells of the patient, so that the changes generated by the therapeutic genes would be hereditary. For ethical and legal issues, this kind of gene therapy is not carried out today.


Although very different approaches have been used, in most gene therapy studies, a copy of the functional gene is inserted into the genome to compensate for the defective one. If this copy is simply introduced into the host, it is gene-addition therapy. If we try, by means of homologous recombination, to eliminate the defective copy and change it for the functional one, it is substitution therapy.

Currently, the most common type of vector used are viruses, which can be genetically altered to stop being pathogenic and carry genes from other organisms. The patient’s target cells are infected with the vector (in the case of a virus) or transformed with the DNA to be introduced. This DNA, once inside the host cell, is transcribed and translated into a functional protein, which will perform its function, and, in theory, to correct the defect that caused the disease.

Vectors in Gene Therapy


All viruses are capable of introducing their genetic material into the host cell as part of their replication cycle. Thanks to this, they can produce more copies of themselves, and infect other cells. Something common to most strategies with viruses is the need to use “packaging” cell lines or helper viruses, which carry the genes that we eliminate to our vectors and that allow infectionSome types of viruses insert their genes physically in the genome of the host, others pass through several cellular organelles in their cycle of infection and others replicate directly in the cytoplasm, so depending on the therapy to perform we may be interested in one or the other . Some examples are retroviruses, adenoviruses, adeno-associated viruses, herpes viruses and viral vectors.

  • Retrovirus: The genome of retroviruses is made up of single-stranded RNA, in which three clearly defined zones are distinguished: an intermediate one with structural genes, and two flankers with genes and regulatory structures. When a retrovirus infects a host cell, it introduces its RNA along with some enzymes that are found in the matrix, namely a protease, a reverse transcriptase and an integrase. The action of retrotranscriptase allows the synthesis of the genomic DNA of the virus from RNA. Next, the integrase introduces this DNA into the host’s genome. From this point, the virus can remain dormant or can activate replication massively. To use the retroviruses as viral vectors for gene therapy, the genes responsible for their replication were initially eliminated and these regions were replaced by the gene to be introduced followed by a marker gene.
  • Adenoviruses: Adenoviruses have a genome of double-stranded DNA, and do not integrate their genome when they infect the host cell, but the DNA molecule remains free in the cell nucleus and is transcribed independently. This supposes that the positional effect or insertion mutagenesis does not occur in these vectors, which does not mean that they do not have other disadvantages. In addition, due to the fact that in their natural cycle they are introduced into the nucleus of the cell, they can infect both dividing cells and quiescent cells.
  • Adeno-associated virus (AAV): AAVs are small viruses with a single-stranded DNA genome. They can be specifically integrated into chromosome 19 with a high probability. However, the recombinant AAV that is used as a vector and that does not contain any viral gene, only the therapeutic gene, is not integrated into the genome. Instead, the recombinant viral genome fuses its ends through the ITR (inverted terminal repeats), appearing recombination of the circular and episomal shape that is predicted to be the cause of long-term gene expression.

The disadvantages of AAV-based systems lie mainly in the limitation of the size of recombinant DNA that we can use, which is very small given the size of the virus. Also the process of production and infection are quite complex. However, since it is a non-pathogenic virus, in the majority of treated patients, there are no immune responses to eliminate the virus or the cells with which they have been treated.

  • Herpes virus: Herpes viruses are DNA viruses capable of establishing latency in their host cells. They have the advantage of being able to incorporate large exogenous DNA fragments (up to about 30 kb). Furthermore, although their lytic cycle is performed at the site of infection, they establish latency in neurons, which are involved in numerous diseases of the nervous system, and are therefore of great interest.
  • Viral vectors: Viral vectors have natural populations of host cells that they infect efficiently. However, some cell types are not sensitive to infection by these viruses. The entry of the virus into the cell is mediated by proteins on its outer surface (which can be part of a capsid or a membrane). These proteins interact with cellular receptors that can induce structural changes in the virus and contribute to their entry into the cell by endocytosis .

Non-viral methods

  • Naked DNA: This method consists in the injection of naked DNA plasmids (that is, uncoated plasmids) which contain the desired genetic information and are able to enter the cells and restore the desired function. This is the simplest non-viral transfection method that exists, and although it has shown some positive results, the expression still remains very low in comparison with other methods, which has led to an investigation with more efficient methods of transformation, such as electroporation, sonication, or the use of biobalistics, which involves firing gold particles coated with DNA into the cell using high gas pressures.
  • Oligonucleotides: The use of synthetic oligonucleotides in gene therapy aims to inactivate the genes involved in the disease process.

Artificial chromosomes

The creation of stable human artificial chromosomes (HACs) is one of the possibilities that is currently being worked on as one of the ways of permanently introducing DNA into somatic cells for the treatment of diseases through the use of gene therapy. They have a high stability, in addition to allowing to introduce large amounts of genetic information.

  • Lipoplexes: The DNA vector can be covered by lipids forming an organized structure, such as a micelle or a liposome. When the organized structure forms a complex with the DNA then it is called lipoplex. There are three types of lipids: anionic, neutral or cationic, being the anionic and toxic neutrals the cationic ones are the most used. These, due to their positive charge, interact with DNA, which presents a negative charge, in such a way that it facilitates the encapsulation of DNA in liposomes. The use of cationic lipids improved the stability of the lipoplexes, in addition, as a result of their charge, they also interact with the cell membrane, allowing endocytosis as the main way in which the cells absorb lipoplexes. Once inside the cell the endosome must break and release the DNA load if this does not happen, it is eliminated. The lipoplexes have as a disadvantage a low efficiency in this stage so they require the help of other lipids as destabilizers of the endosome membrane.

Hybrid methods

Due to the deficiencies of many of the gene transfer systems, some hybrid methods have been developed that combine two or more techniques. Virosomes are an example, and they combine liposomes with the inactivated HIV virus or the influenza virus.

  • Dendrimers: A dendrimer is a very branched macromolecule with a spherical or variable shape. Its size is in the nanoscale and its surface can be functional in many ways and many of its properties derive from it. In particular, it is possible to construct a cationic dendrimer, that is, with a positive surface charge. In this way, it interacts with the nucleic acid, negatively charged, and form a complex that can enter by endocytosis in the cell.

Target cells

The target cells are selected according to the type of tissue in which the introduced gene must be expressed, and must also be cells with a long half-life. Likewise, it must be taken into account if the cell target is a dividing or quiescent cell. The ideal target cells would be the stem cells, since the insertion of a gene in them would produce a long-term effect. Due to the experience in bone marrow transplantation, one of the most studied cell targets is hematopoietic stem cells. Other cellular targets that have been worked with are: lymphocytes, respiratory epithelial cells, hepatocytes, dermal fibroblasts and muscle cells.

Application of gene therapy in humans

The most controversial and controversial application of transgene technology is that regarding human gene therapy, that is, the treatment and alleviation of human genetic diseases by the addition of exogenous wild genes to correct the defective function of the mutations. In humans, two basic types of gene therapy can be used: the somatic and the germinal.

The somatic gene therapy is so far the one that has dissimilar applications in a few diseases such as: Cystic fibrosis, Lysosomal diseases, hypercholesterolemia, malignant melanomas (cancer), Severe Combined Immune Deficiency, Duchenne Muscular Dystrophy, Beta Thalassemia, HIV / AIDS, among others.


  • Course of Introduction to Biotechnology. University for all. Applied Biotechnology Magazine
  • Durai S, Mani M, Kandavelou K, Wu J, Porteus MH, Chandrasegaran S (2005). “Zinc finger nucleases: custom-designed molecular scissors for genome engineering of plant and mammalian 

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