Gene therapy

Gene therapy using an adenovirus vector. In some cases, adenovirus will insert the new gene into a cell. If the treatment is successful, the new gene will produce a functional protein to treat a disease.

Gene therapy has been developed as a method of approach to the treatment of human diseases, based on the transfer of genetic material to the cells of an individual.

The purpose of this transfer of genetic material is to restore a cell function that was abolished or defective, to introduce a new function or to interfere with an existing function. Thus, the different gene therapy strategies are based on the combination of three key elements, the genetic material to be transferred, the transfer method and the cell type that will incorporate said genetic material.

Initially the focus was on the treatment of monogenic hereditary diseases, but subsequently most of the clinical trials (more than 400) have addressed the treatment of cancer.

Human gene therapy is feasible and can be useful, but the tools need to be perfected before it can become part of the standard therapeutic armamentarium.

Applications

• Genetic marking: The gene marking aims not to cure the patient, but to follow the cells, i.e. to check if the specific cells that have been marked are present at a certain site of the body. An example of this would be the delivery point for clinical trials, allowing, for example, that at times when a cancer patient (leukemia) and a self-transplant has been performed, you can know where the cells come from, whether they are transplanted cells or cells that have survived the treatment.

• Therapy of hereditary monogenic diseases: It is used in those diseases in which the administration of the deficit protein cannot be performed or is not efficient. The defective or absent gene is provided.

• Therapy of acquired diseases: Among these types of diseases the most prominent is cancer. Different strategies are used, such as inserting certain suicidal genes into tumor cells or inserting tumor antigens to enhance the immune response.

Types of gene therapy

• Somatic gene therapy: is done on the somatic cells of an individual, so the modifications that involve therapy only take place in that patient.

• Therapy in vivo: Cell transformation takes place within the patient who is given therapy. It consists in administering a gene to the patient through a vehicle (e.g. a virus), which should locate the cells to infect. The problem that this technique presents is that it is very difficult to get a vector to locate a single type of target cells.
• Therapy ex vivo: Cell transformation takes place from a biopsy of the patient's tissue and then the already transformed cells are transplanted. As it happens outside the patient's body, this type of therapy is much easier to perform and allows greater control of infected cells. This technique is almost completely reduced to hematopoietic cells because they are cultivable cells, thus constituting a transplantable material.

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

Procedure

Although many different approaches have been used, in most gene therapy studies, a copy of the working gene is inserted into the genome to make up for the defective one. If this copy is simply introduced into the host, it is called addition gene therapy. If we try, by means of homologous recombination, to eliminate the defective copy and replace it with the functional one, this is substitution therapy.

Currently, the most common type of vectors used are viruses, which can be genetically altered to stop being pathogenic and carry genes from other organisms. However, there are other types of vectors of non-viral origin that have also been used for this. Likewise, DNA can be introduced into the patient by physical (non-biological) methods such as electroporation, monoclonal antibodies, biobalistics... If we know the DNA sequence that is defective in the patient, we can remove it and introduce the genetic material to achieve the correct expression of the gene. Generally, a class of enzymes called restriction endonucleases are used. These enzymes are capable of recognizing certain genes based on the amino acid sequence, to later join that gene, cut it and, subsequently, introduce the correct genetic material to achieve gene expression. It is a process that may seem complex, but nothing is further from reality since we only have to know the wrong sequence and use a restriction endonuclease that recognizes it, cuts it, and introduces the correct gene sequence of interest.

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 carry out its function and, in theory, correct the defect that caused the disease.

Vectors in gene therapy

The great diversity of situations in which gene therapy could be applied makes it impossible to have a single suitable type of vector. However, the following characteristics can be defined for an "ideal vector" and then adapt them to specific situations:

• Make it reproducible.
• Make it stable.
• Allow the insertion of genetic material without size limit.
• To allow transduction both in cells in division and in those that are not proliferating.
• To enable the integration of the therapeutic gene into a specific site of the genome.
• To be integrated once per cell, to be able to control the dose.
• Recognise and act on specific cells.
• May the expression of the therapeutic gene be regulated.
• It lacks elements that induce an immune response.
• That can be completely characterized.
• Make it safe or your possible side effects are minimal.
• Make it easy to produce and store.
• May the whole process of your development have a reasonable cost.

The vectors will contain the elements that we want to introduce to the patient, which will not only be functional genes, but also elements necessary for their expression and regulation, such as promoters, enhancers or specific sequences that allow their control under certain conditions.

We can distinguish two main categories of vectors used in gene therapy: viral and non-viral.

Viruses

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.

Some types of viruses physically insert their genes into the host genome, others pass through several cell organelles in their infection cycle and others replicate directly in the cytoplasm, so depending on the therapy to be performed, we may be interested One or another.

Common to most virus strategies is the need to use "packaging" or helper viruses, which carry the genes that we eliminate from our vectors and which allow infection.

Retroviruses

The genome of retroviruses is made up of single-stranded RNA, in which three clearly defined zones can be distinguished: an intermediate one with structural genes, and two flanking ones with genes and regulatory structures. When a retrovirus infects a host cell, it introduces its RNA along with some enzymes found in the matrix, namely a protease, reverse transcriptase, and integrase.

The action of reverse transcriptase allows the synthesis of the genomic DNA of the virus from the RNA. The integrase then introduces this DNA into the host genome. From this point on, the virus can lie dormant or it can activate replication en masse.

In order to use 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.

The LTR sequences remained from the viral genome; and the necessary elements to produce the vectors on a large scale and to transform the cells are provided from other vectors, either plasmids or in specific cell lines. In the case of using plasmid vectors, strategies such as cotransforming with several different plasmids that code for the retrovirus proteins, and that the transcription of their sequences is subject to eukaryotic promoters, can contribute to minimizing the risk of recombination generating recombinant viruses.

Currently, strategies such as the above are being sought to achieve greater security in the process. The addition of polyadenine tails to the transgene to prevent transcription of the second LTR sequence is an example of this.^{[citation needed]}

Retroviruses as vectors in gene therapy present a considerable drawback, and that is that the integrase enzyme can insert the genetic material into any area of the host genome, and can cause deleterious effects such as modification in the expression pattern (positional effect) or insertional mutagenesis of a wild-type gene.

Gene therapy trials using retroviral vectors to treat X-linked severe combined immunodeficiency (X-SCID) represent the most successful application of therapy to date. Thus, more than twenty patients have been treated in France and Great Britain, with a high rate of reconstitution of the immune system. However, similar trials were restricted in the United States when leukemia was reported in patients.^{[citation needed]} To date, four cases of French children and one British who developed leukemia as a result of insertional mutagenesis of retroviral vectors, and all but one of these children responded well to conventional leukemia treatment. ^{[citation needed]} Currently, gene therapy to treat SCID continues to be successful in the United States, Great Britain, Italy, and Japan.^{[citation required]}

Adenoviruses

Adenoviruses have a double-stranded DNA genome, 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 means that the positional effect or insertional mutagenesis does not occur in these vectors, which is not to say that they do not have other drawbacks. Furthermore, due to the fact that in their natural cycle they enter the cell nucleus, they can infect both dividing and quiescent cells.

Part of the E1 gene, basic for replication, was deleted from the first generation vectors, and from the 2nd generation vectors, other early genes in the virus cycle were deleted. In both cases, when an infection is carried out with a high concentration of virus, the expression of other genes is produced, causing a considerable immune response.

For this reason, the latest adenovirus-based vectors have been practically devoid of most of their genes, with the exception of the ITR regions (inverted repeat regions), and the area necessary for encapsidation.

Adeno Associated Virus (AAV)

AAVs are small viruses with a single-stranded DNA genome. They can specifically integrate 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, does not integrate into the genome. Instead, the recombinant viral genome fuses its ends via ITRs (inverted terminal repeats), resulting in recombination of the circular and episomal fashion 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 little, given the size of the virus. The production and infection process are also quite complex. However, since it is a non-pathogenic virus, in most of the treated patients there are no immune responses to eliminate the virus or the cells with which they have been treated.

Many trials with AAV are ongoing or in preparation, mainly in the treatment of muscles and ocular diseases, the two tissues where the virus appears to be particularly useful.^{[citation needed]} However, clinical trials are beginning, where AAV-based vectors are used to deliver the genes into the brain.^{[citation needed]} This is possible because AAV can infect cells that are not in a dividing state, such as neurons.

Herpes virus

Herpesviruses are DNA viruses capable of establishing latency in their host cells. They are genetically complex, but for use as vectors they have the advantage of being able to incorporate large exogenous DNA fragments (up to 30 kb). Furthermore, although their lytic cycle is carried out at the site of infection, they establish latency in neurons, which are implicated in numerous diseases of the nervous system, and are therefore targets of great interest.

The herpesvirus vectors implemented have used two main strategies:

• The homologue recombination between the complete virus genome and the content in a plasmid that carried the transgen into the area that encodes for non-essential genes in terms of replication and infection.

• The use of vectors with virus replication origins as well as corresponding packing sequences, and their introduction into well-coinfected cellular stretchers with wild viruses or carriers of the other genes involved in encapsidation and replication, to allow the formation of recombinant viral particles with which to perform the treatment.

However, the use of vectors based on HSV (herpes simplex virus) can only be carried out in patients who have not been previously infected by it, since they may present immunity.

Protein "pseudotyping" of viral vectors

The viral vectors described above have natural populations of host cells that they efficiently infect. 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 cell receptors that can induce structural changes in the virus and contribute to its entry into the cell by endocytosis.

In either case, entry into host cells requires a favorable interaction between a virus surface protein and a cell surface protein. Depending on the purpose of a given gene therapy, the range of cells susceptible to infection by a vector could be limited or expanded. For this reason, vectors known as "pseudotyped" have been developed, in which the viral envelope of wild-type proteins has been replaced by peptides from other viruses, or by chimeric proteins, which consist of parts of the viral protein. necessary for its incorporation into the virion, as well as the sequences that are supposed to interact with specific receptors of cellular proteins.

For example, the most popular retroviral vector for use in gene therapy trials has been the simian immunodeficiency virus coated with the G-protein envelope of vesicular stomatitis virus. This vector is known as VSV and can infect almost all cells, thanks to the G protein with which this vector is coated.^{[citation needed]}

There have been many attempts to limit the tropism (ability to infect many cells) of viral vectors. This advance could allow for the routine administration of a relatively small amount of the vector. Most attempts have used chimeric envelope proteins, which included antibody fragments.

Non-viral methods

These methods have certain advantages over viral methods, such as large-scale production facilities and low immunogenicity. Previously, low levels of transfection and gene expression kept non-viral methods in a less advantageous position; however, recent advances in vector technology have produced molecules and transfection techniques with efficiencies similar to those of viruses.

Complex DNA

This is the simplest method of non-viral transfection. It consists of the localized application of, for example, a plasmid with naked DNA. Several of these assays gave successful results. However, expression has been very low compared to other transformation methods. In addition to plasmid assays, assays with PCR products have been performed, with similar or greater success. This achievement, however, does not exceed other methods, which has led to research with more efficient transformation methods, such as electroporation, sonication, or the use of bioballistics, which consists of shooting gold particles coated with DNA into cells using high gas pressures.

Oligonucleotides

The use of synthetic oligonucleotides in gene therapy aims to inactivate the genes involved in the disease process.

There are several strategies for treatment with oligonucleotides

One strategy, "antisense" it uses oligonucleotides with the sequence complementary to the mRNA of the target gene, which activates a gene silencing mechanism. It can also be used to alter the transcription of the defective gene, for example by modifying its intron and exon editing pattern.

Use is also made of small RNAi molecules to activate a mechanism of gene silencing similar to that of antisense therapy

Another possibility is to use oligodeoxynucleotides as a decoy for the factors that are required in the activation of the transcription of the target genes. The transcription factors bind to the decoys instead of the promoter of the defective gene, thereby reducing expression of the target genes. In addition, single-stranded DNA oligonucleotides have been used to direct a single base change within the sequence of a mutant gene.

Like naked DNA methods, they require transformation techniques to enter the cell.

Artificial Chromosomes

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

Lipoplexes and polyplexes

Vector DNA can be covered by lipids forming an organized structure, such as a micelle or liposome. When the organized structure forms a complex with DNA then it is called a lipoplexe.

There are three types of lipids: anionic, neutral, or cationic. Initially, anionic and neutral lipids were used in the construction of lipoplexes for synthetic vectors. However, these are relatively toxic, incompatible with body fluids, and have the potential to adapt to remain in a specific tissue. Furthermore, they are complex and time consuming to produce, so attention turned to the cationic versions. These, due to their positive charge, interact with DNA, which has a negative charge, in such a way that it facilitates the encapsulation of DNA in liposomes. Later, it was found that the use of cationic lipids improved the stability of the lipoplexes. Furthermore, as a result of their charge, cationic liposomes also interact with the cell membrane, and endocytosis is believed to be the main route by which cells take up lipoplexes.

Endosomes are formed as a result of endocytosis. However, if the genes cannot be released into the cytoplasm by disruption of the endosome membrane, the liposomes and the contained DNA will be destroyed. The efficiency of this "endosomal escape" in the case of liposomes constituted only by cationic lipids it is low. However, when "helper lipids" (usually electroneutral lipids, such as DOPE) are added, the efficacy is much greater. Furthermore, certain lipids (fusogenic lipids) have the ability to destabilize the endosome membrane. The use of certain chemical compounds, such as chloroquine, allows exogenous DNA to escape from the lysosome, although they must be used with caution, since it is toxic and must be used in small doses so as not to affect the transfection target cell.

However, cationic lipids have dose-dependent toxic effects, which limits the amount that can be used and therefore the therapy itself.

The most common use of lipoplexes is gene transfer into cancer cells, where the delivered genes activate tumor suppressor genes in the cell and decrease the activity of oncogenes.

Recent studies have shown that lipoplexes are useful in the epithelial cells of the respiratory system, so they can be used for the genetic treatment of respiratory diseases such as cystic fibrosis.

DNA polymer complexes are called polyplexes and most consist of cationic polymers, regulated by ionic interactions.

A major difference between the methods of action of polyplexes and lipoplexes is that some polyplexes cannot release their loaded DNA into the cytoplasm, so they require countertransfection with agents that contribute to endosome lysing. There are other elements that form polyplexes, such as chitosan or polyethylamine, which are capable of being released from the endosome.

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 one example, and combine liposomes with inactivated HIV virus or influenza virus.

Dendrimers

A dendrimer is a highly branched macromolecule with a spherical or variable shape. Its surface can be functional in many ways and many of its properties derive from it. In addition, its size —at the nano scale— allows its use in biomedicine.

In particular, it is possible to build a cationic dendrimer, that is, one with a positive surface charge. In this way, it interacts with the nucleic acid, which is negatively charged, and forms a complex that can enter the cell by endocytosis. This is useful in gene therapy, to introduce foreign genes.

Production costs are high, but techniques are being developed to make it cheaper, since it is a technique with very low toxicity, and its main disadvantage is at a productive level.

Comparative table of main viral vectors

Target cells

The target cells are selected based on the type of tissue in which the introduced gene must be expressed, and they must also be cells with a long half-life, since it does not make sense to transform cells that are going to die after a few days. Likewise, it must be taken into account whether the cell target is a dividing or quiescent cell, because certain viral vectors, such as retroviruses, only infect dividing cells.

Based on these considerations, the ideal target cells would be stem cells, since inserting a gene into them would produce a long-term effect. Due to the experience in bone marrow transplantation, one of the most worked cell targets are hematopoietic stem cells. Gene therapy in these cells is technically possible and it is a very suitable tissue for ex vivo transfer. Other cellular targets that have been worked on are:

• Lymphocytes: They are medium-life cells and easy access (they are found in peripheral blood). They constitute a target for ex vivo therapies of melanomas and immunodeficiencies.
• Respiratory epithelium: they are very slow division cells and in them it is not possible to transfer ex vivo, but its transformation through adenovirus and lipoplexes.
• Hepatocytes: their transformation into possible both ex vivo (it is feasible to cultivate cells and transplant them by portal circulation) and in vivo (specific hepatocyte protein receptors are being developed).
• Dermal fibroblasts: they are easily accessible and cultivated cells, and can be transformed both ex vivo and in vivo, but they usually have transient effects.
• Muscle cells: they can be transformed by in vivo DNA injection and also by adenovirus, but with a very limited success in the latter case.

Main events in the development of gene therapy

2002 and earlier

Gene therapy appeared in the 1970s to try to treat and alleviate diseases of a genetic nature and the first tests with viruses were given, which failed. Years later, in the 1980s, attempts were made to treat thalassemia using betaglobin. In this case, it was successful in animal models, although it could not be used in humans.

In 1990, W. French Anderson proposed the use of bone marrow cells treated with a retroviral vector that carries a correct copy of the gene that codes for the enzyme adenosine deaminase, which is mutated. It is a disease that is part of the group of severe combined immunodeficiencies (SCID). He performed the ex-vivo transformation with the patient's T cells, which were then reintroduced into his body. Five years later, they published the results of the therapy, which contributed to the scientific community and society considering the possibilities of this technique.

However, support for the therapy was questioned when some children treated for SCID developed leukemia. Clinical trials were temporarily halted in 2002, due to the shock of the case of Jesse Gelsinger, the first publicly recognized person as deceased from gene therapy. His death was due to the use of the adenoviral vector for the transduction of the gene necessary to treat his disease, which caused an excessive immune response, with multiple organ failure and brain death. There is a large bibliography on the subject, and the report issued by the FDA pointing out the conflict of interest of some of the doctors involved in the case, as well as the failures in the procedure, is noteworthy. In 2002, four ongoing gene therapy trials were halted when a leukemia-like disease developed in a treated child. Subsequently, following a review of procedures, ongoing projects were resumed.

2003

A team of researchers at the University of California, Los Angeles inserted genes into the brain using liposomes coated with a polymer called polyethylene glycol (PEG). Gene transfer into this organ is a significant achievement because viral vectors they are too large to cross the blood-brain barrier. This method has the potential for the treatment of Parkinson's disease.

Also in that year, RNA interference was proposed to treat Huntington's disease.

2006

NIH scientists successfully treat metastatic melanoma in two patients, using T cells to attack cancer cells. This study constitutes the first demonstration that gene therapy can be an effective treatment against cancer.

In March 2006, an international group of scientists announced the successful use of gene therapy for the treatment of two adult patients infected with a disease that affects myeloid cells. The study, published in Nature Medicine, is a pioneer in showing that gene therapy can cure diseases of the myeloid system.

In May 2006, a team of scientists led by Dr. Luigi Naldini and Dr. Brian Brown from the San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET) in Milan, reported the development of a form of prevent the immune system from being able to reject the entry of genes. Dr. Naldini's researchers observed that the natural function of microRNAs could be used to selectively turn off therapeutic genes in cells of the immune system. This work has important implications for the treatment of hemophilia and other genetic diseases.

In November of the same year, Preston Nix of the University of Pennsylvania reported on VRX496, an immunotherapy for the treatment of HIV that uses a lentiviral vector to transport antisense DNA against the HIV envelope. It was the first lentiviral vector therapy approved by the FDA for clinical trials. Phase I/II data is now available.

2007

On May 1, 2007, Moorfields Eye Hospital and College London's Institute of Ophthalmology, one year later Children's Hospital of Philadelphia announced the first gene therapy trial for inherited retinal disease. The first operation (in England) was carried out on a 23-year-old British male, Robert Johnson, earlier this year. While in Philadelphia Corey Haas was the first child to obtain this type of therapy. Leber's congenital amaurosis is a hereditary disease that causes blindness due to mutations in the RPE65 gene. The Moorfields/UCL results were published in the New England Journal of Medicine. Subretinal transfection by recombinant adeno-associated virus carrying the RPE65 gene was investigated, and positive results were found. Patients showed increased vision, and there were no apparent side effects. Clinical trials of this therapy are in phase II.

One of the stages to be carried out is the determination of the molecular type that concerns each disease (or http://es.wikipedia.org/wiki/Distrofias_de_la_retina).

2008

Researchers at the University of Michigan in Ann Arbor (United States) developed a gene therapy that slows and restores gums in the face of advancing periodontal disease, the leading cause of tooth loss in adults. Researchers discovered a way to help certain cells by using an inactivated virus to make more of a protein called the TNF receptor. This factor is found in low amounts in patients with periodontitis. The administered protein makes it possible to decrease excessive levels of TNF, a compound that worsens inflammatory bone destruction in patients suffering from arthritis, joint deterioration and periodontitis. The results of the work showed that between 60 and 80 percent of periodontal tissues were spared from destruction when using gene therapy.^{[citation needed]}

2009

In September 2009, it was reported in Nature that researchers at the University of Washington and the University of Florida were able to give squirrel monkeys trichromatic vision using gene therapy.

In November of that same year, the journal Science published encouraging results on the use of gene therapy in a very serious brain disease, adrenoleukodystrophy, using a retroviral vector for treatment.

2012

On November 2, the European Commission authorized Glybera, a German company (Amsterdam), to launch a treatment for a rare genetic disorder —lipoprotein lipase (LPL) deficiency.

Percentages of Gene Therapy Trials Today

Diseases and gene therapy

There are numerous diseases that are the object of gene therapy, the most characteristic of which are those discussed below:

ADA

The first FDA-approved clinical protocol for the use of gene therapy was that used in the treatment of adenosine deaminase (ADA) deficiency, which causes a disorder of immunity, in 1990. In these patients, no he has been able to withdraw the exogenous enzymatic treatment necessary for his survival, if not only reduce it by half, and persistence in gene expression has been detected even after four years of starting the protocol. Although the complete cure of the patients has not been achieved (which would consist of withdrawing all the exogenous enzymatic contribution) this constitutes an unprecedented event in the therapeutic history. In 2009, a new experiment was carried out in which hematopoietic cells were extracted from the bone marrow for the introduction of the ADA gene ex vivo using a modified retrovirus (GIADA). The modified cells are reintroduced into the patient. The results of this experiment were successful because none of the patients developed leukemia (as had happened with the use of retroviruses). In addition, all patients developed correct expression of the ADA gene during the years of follow-up and achieved an increase in blood cells. Thus, 8 of the nine patients do not need exogenous enzymatic treatment to complement the gene therapy.

Cancer

Cancer treatment to date has involved destroying cancer cells with chemotherapeutic agents, radiation, or surgery. However, gene therapy is another strategy that in some cases has reduced the size of solid tumors by a significant percentage. The main methods used by gene therapy in cancer are:

1. Increased antitumoral cellular immune response (immunogenic therapy). It is based on the ability of the immune system to attack cancer. To do this, antigens are introduced into tumor cells allowing immune cells to recognize tumor cells. Thus, tumor cells can be transformed with CD80 protein, antigen-representative cell membrane glycoprotein that binds to T lymphocytes by boosting the immune response.
2. Introduction of drug-activating genes within tumor cells or suicide gene therapy. It consists of the selective introduction of genes in tumor cells and not others, which encode for susceptibility to drugs that otherwise would not be toxic. This leads to the insertion of enzymes; such as HSV-tk [Herpex simplex thymidine kinase virus]) and desaminese cytosine, which are harmless enzymes for mammal cells and convert prodrogues (vg ganciclovir and 5-fluorocytosine) into cytotoxic metabolites that destroy tumor cells in proliferation.
3. Standardization of the cell cycle. It consists of the inactivation of mutated oncogenes, such as ras, or in the reexpression of antioncogenes or inactive tumor suppressants such as p53. Clinical trials have been conducted in which retrovirus tumor cells express p53 are injected. The problem is that large amounts of viruses are needed to treat widespread tumors and retrovirus presents a low rate of infection efficiency.
4. Manipulation of bone marrow cells. It is mainly used in the gene therapy of hematological disorders, and is to transfer hematopoietic parent cells to chemoprotection or chemosensitization genes, among others. This is the case of the MDR1 gene studied in breast cancer that, transplanted into precursor cells of T and NKs lymphocytes (positive CD34 cells), makes the transfected cells more resistant to high doses of chemotherapy.
5. Use of ribozymes and anti-sense or "antisense" technology. Ribozymes are RNA with catalytic activity that would act by increasing the degradation of newly translated RNA, decreasing undesired specific proteins, which is sometimes associated with tumor alterations. Antisensing technology refers to RNA oligonucleotides that do not have catalytic activity, but are complementary to a gene sequence and which can act by blocking the processing of RNA, preventing the transport of mRNA or blocking the beginning of the translation.
6. CAR-T Therapy. This type of therapy is recent and consists of the removal of the patient's T lymphocytes to treat, the genetic modification of the same and, the subsequent introduction of the T lymphocytes modified in the patient's body. In this way, modified T lymphocytes are able to attack malignant B lymphocytes. In some types of leukemias or lymphomas it may occur that Lymphocytes B become malignant or tumoral due to the expression of a surface antigen known as CD19. The T Lymphocytes are unable to destroy these B malignant lymphocytes because they do not recognize them. After extracting and modifying the patient's T lymphocytes by means of this novel technique, modified T lymphocytes will be able to recognize malignant B lymphocytes and thus destroy them by slowing the progress of the tumor process. Hospitals such as Gregorio Marañón and 12 October in Madrid or Sant Joan de Déu in Barcelona, have begun to implement this type of therapy.

Wiskott-Aldrich Syndrome (WAS)

Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disease characterized by eczema, thrombocytopenia, recurrent infections, immunodeficiency as well as a high tendency to lymphomas and autoimmune diseases. There is also a milder version of this disease known as X-linked thrombocytopenia or XLT characterized by congenital microthrombocytopenia with small platelets. Both diseases are caused by mutations in the WAS gene that codes for a multidomain protein that is only expressed in hematopoietic cells, WASP. Therefore, the majority of those with this syndrome suffer an early death due to infection, hemorrhage, cancer, or severe autoimmune anemia. Currently, effective treatments have been performed in patients with Wiskott-Aldrich syndrome by transplantation of bone marrow or umbilical cord blood from an HLA-identical or matched donor.

In 2010, a study was published showing significant improvements in two children diagnosed with the disease. The therapy consisted of extracting the hematopoietic stem cells and transferring them back after integrating the WAS gene into the genome. After gene therapy, they detected significant levels of the WASP protein in the different cells of the patients' immune system. The result was that the patients had several significant improvements: one of them fully recovered from autoimmune anemia and the other patient reduced the eczema he suffered from.

Beta Thalassemia

Beta-thalassemia is a genetic disorder with mutations in the β-globulin gene that reduce or block the production of this protein. Patients with this disease suffer from severe anemia and require blood transfusions throughout their lives. Gene therapy aims to heal bone marrow stem cells by transferring the normal β-globin or β-globin gene into hematopoietic stem cells (HSCs) to permanently produce normal red blood cells. To carry it out, it is intended to use lentivirus because several studies show the correction of β-thalassemia in animal models. The goals of gene therapy with this disease are: to optimize gene transfer, the introduction of a large number of genetically modified HSCs, and to minimize the negative consequences that can result from the random integration of vectors into the genome.

Problems of gene therapy and its applications

A very important concept underlying some aspects of the safety of gene therapy is that of the Weismann barrier. It refers to the fact that hereditary information only goes from germ cells to somatic cells, and not the other way around.

Gen therapy in germ cells is much more controversial than in somatic cells, but even so, if the Weismann barrier were permeable to some exchange of information, as some authors point out, even somatic cell therapy could have ethical problems and security that previously would not have been considered. These types of aspects to take into account are included in the Universal Declaration on the Human Genome.

The nature of gene therapy itself and its vectors means that on many occasions patients must repeat the therapy from time to time because it is not stable and its expression is temporary.

The immune response of the organism to a foreign agent such as a virus or an exogenous DNA sequence. Furthermore, this response is reinforced in the successive applications of the same agent.

Problems related to viral vectors. They could be contaminated by both chemical substances and viruses capable of causing disease. They also imply risks of immune response.

Multigenic disorders: they represent a great challenge for this type of therapy, since these are diseases whose origin lies in mutations in several genes, and applying the treatment would encounter the classic difficulties of therapy multiplied by the number of genes to be treated.

Possibility of inducing a tumor by mutagenesis. This can occur if the DNA is integrated into a tumor suppressor gene, for example. This has been the case in clinical trials for X-linked SCID, in which 3 of 20 patients developed leukemia.

Gene therapy in other animals

The first example of mammalian gene therapy was the correction of deficient growth hormone production in mice. The recessive little (lit) mutation produces dwarf mice. Although they have an apparently normal growth hormone gene, they do not produce mRNA from this gene.

The first step in the correction of the defect consisted of the injection of five thousand copies of a linear DNA fragment carrying the structural region of the rat growth hormone gene fused to the promoter of the mouse metallothionein gene, in eggs lit. The normal function of metallothionein is the detoxification of heavy metals, so the regulatory region responds to the presence of heavy metals in the animal. The injected eggs were implanted in females. 1% of the offspring mice were found to be transgenic, and they grew larger.

Similar technology has been developed to generate transgenic varieties of Pacific salmon with a rapid growth rate, and the results have been spectacular. Salmon eggs were microinjected with a plasmid carrying the gene for growth hormone regulated by the metallothionein promoter and a small portion of the resulting fish were transgenic, weighing eleven times more than non-transgenic.

Gene therapy in popular culture

In television series such as Dark Angel, the topic of gene therapy is mentioned as one of the practices carried out on transgenic children and their mothers. Also in the Alias series, molecular gene therapy appears as an explanation for two identical individuals.

It is a fundamental element in the plot of video games like Bioshock or Metal Gear Solid, and plays an important role in the plot of movies like Die Another Day , by James Bond or I am Legend by Will Smith, The Bourne Legacy, among many others.