Rare diseases: fight against oblivion

We are ending the month of February, and this means that the Rare Disease Day is approaching. Marfan syndrome, Williams syndrome, DiGeorge syndrome, Crohn’s disease, Fanconi anaemia, mucopolysaccharidosis, among many others make up the list of these diseases. Why are they called minority diseases or rare diseases?

WHAT ARE MINORITY DISEASES?

A minority disease is that which affects less than 1 in 2,000 people. Although individually they are rare, there are many diseases of this type (6,000-7,000), so there are many affected patients.

Although the definition of minority disease is what I have just said, in the pharmaceutical industry it is that disease in which it is not profitable to develop a drug due to the low number of patients, the limited information available, the poor diagnosis, the lack of clinical studies and the difficult location of patients. It is for this reason that the families themselves create their own foundations to obtain financing for the investigation of these diseases.

A few years ago these diseases were socially forgotten, but, fortunately, they are now socially transcendental and recognized.

As I said, there are around 7,000 minority diseases described and every year between 150 and 250 new ones are described, thanks to new technologies.

A large number of these diseases affects children, that is, they manifest themselves at an early age. It is necessary to know that most have a genetic basis, caused by mutations in specific genes such as cystic fibrosis or several muscular dystrophies. But there are also related to environmental factors, such as some types of anaemia due to lack of vitamins or due to medications. This is the case of malignant mesothelioma, a breast cancer, in which more than 90% of cases are due to asbestos exposure. However, there are still many without knowing their origin or data on their prevalence.

MINORITY DISEASES IN NUMBERS

The fact that these diseases affect few people and the ignorance of their symptoms by the public and professionals, it is estimated that the time that elapses between the appearances of the first symptoms until diagnosis is 5 years. In 1 of every 5 cases, more than 10 years may pass until the correct diagnosis is obtained. This means not receiving support or treatment or receiving inadequate treatment and worsening the disease.

Not all hospitals have the means to treat those affected, for this reason it is estimated that practically half of sufferers have had to travel and travel in the last 2 years out of their province because of their illness, either in look for a diagnosis or treatment.

Minor diseases represent a significant economic cost. The cost of diagnosis and treatment accounts for around 20% of the annual income of each affected family. This means an average of more than 350€ per family per month, a figure very representative of the high cost involved in the care of rare diseases. The expenses to cover in the majority of cases are related to the acquisition of medicines and other health products, medical treatment, technical aids and orthopaedics, adapted transport, personal assistance and adaptation to housing.

TREATMENT FOR MINORITY DISEASES

Only 1-2% of minority diseases currently have some type of treatment, therefore, much remains to be investigated.

There are 4 basic types of treatment for rare genetic diseases:

PHARMACOLOGICAL THERAPIES

It consists in the modification of a normal or pathological biochemical reaction by an external chemical agent.

The development of a drug is a very expensive process and difficult to quantify. Currently many millions have to be invested for a new drug to reach the patient.

But what is a medication? A medicine is a small organic molecule, which typically has to be:

• Specific to solve a molecular problem (ex: prevent an abnormal interaction between two proteins)
• Very active and very tuned for your target
• Very little toxic
• Distribute well throughout the body and reach the target tissue
• Cheap to produce or, at least, that can be synthesized in industrial quantities
• Stable
• New (patentable)
• It has to be commercialized

GENE THERAPY

Attempt to correct defective genes responsible for diseases in the somatic (non-sexual) line, either by:

• Loss of function: incorporate the normal gene (ex: phenylketonuria)
• Function gain: eliminate the responsible mutation, eliminating the protein (ex: Huntington)

Limitations:

• Only the reversible characteristics of a genetic disease can be corrected
• The size of the DNA to be incorporated in the patient’s genome
• Immune response against the viral vector (retroviruses, adenoviruses, adenoassociates)
• Inactivation of an essential gene that can cause a problem greater than the disease
• Directionally to appropriate target cells

CELLULAR THERAPY

Describes the process of introducing new cells into an affected tissue, with or without previous gene therapy. It is necessary to introduce many cells because the treatment is effective and, sometimes, these cells can go to unwanted tissues or have some types of abnormal growth.

SURGERY

For example in congenital heart defects.

RARE DISEASE DAY

For rare diseases to cease to be, Rare Disease Day is celebrated on the last day of February, with the aim of raising awareness and awareness among the public about rare diseases; as well as showing the impact on patients’ lives and reinforcing their importance as a priority in public health.

It was established in 2008 because, according to the European Organization for Rare Diseases (EURORDIS), the treatment of many rare diseases is insufficient, as well as in social networks to support people with minority diseases and their families. In addition, while there were already many days devoted to people suffering from individual diseases (such as AIDS, cancer, etc.) before there was not a day to represent people suffering from minority diseases. It was chosen on 29^th February because it is a “rare” day. But it is celebrated on the last day of February in years that are not leap years.

Then I leave the promotional video for the Rare Disease Day 2015:

Video 1. Rare Disease Day 2015 Official Video (Source: YouTube)

REFERENCES

• FEDER
• EURORDIS
• Fundación Mehuer
• Rare Disease Day

What is gene therapy?

In the last years we have heard discuss gene therapy and its potential. However, do we know what gene therapy is? In this article, I want to make known this promising tool that can cure some diseases that therapies with conventional drugs cannot it. I discuss approaches of gene therapy and their key aspects, where we find animal models.

INTRODUCTION

A clinical trial is an experimental study realized in patients and healthy subjects with the goal to evaluate the efficiency and/or security of one or various therapeutics procedures and, also, to know the effects produced in the human organism.

Since the first human trial in 1990, gene therapy has generated great expectations in society. After over 20 years, there are a lot of gene therapy protocols have reached the clinical stage.

Before applying gene therapy in humans it is necessary to do preclinical studies; these are in vitro or in vivo investigations before moving to clinical trials with humans. The aim of these is protect humans of toxic effects that the studied drug may have.

An important element in preclinical studies are animal models. First, tests are made with small animals like mice. If they are successful, then tests are made with larger animals, like dogs. Finally, if these studies give good results then they are passed to higher animals: primates or humans.

WHAT IS GENE THERAPY?

Gene therapy represents a promising tool to cure some of those diseases that conventional drug therapies cannot. This therapy consists in the transfer of genetic material into cells or tissues to prevent or cure a disease.

Initially gene therapy was established to treat patients with hereditary diseases caused by single gene defects, but now, at present, many gene therapy efforts are also focused on curing polygenic or non-inherited diseases with high prevalence (Video 1).

Video 1. Explanation about what gene therapy is (Source: YouTube)

APPROACHES IN GENE THERAPY

There are two types of approaches in gene therapy (Figure 1):

• In vivo gene therapy: introduce a therapeutic gene into a vector which then is administered directly to the patient. The vector will transfer the gene of interest in the target tissue to produce the therapeutic protein.
• Ex vivo gene therapy: transfer the vector carrying the therapeutic gene into cultured cells from the patient. After, these genetically engineered cells are reintroduced to the patients where they now express the therapeutic protein.

Figure 1. Differences between the two types of approaches in gene therapy (Source: CliniGene – Gene Therapy European Network)

KEY ASPECTS OF GENE THERAPY

When designing a gene therapy approach there are some key aspects to be considered:

1/ THERAPEUTIC GENE

The gene of interest is that which is introduced into the body to counteract the disease. For the one hand, for the diseases are caused by the lost or dysfunction of a single protein, the gene to be transferred is more identifiable, being that only a correct copy of the gene whose dysfunction causes the diseases will be introduced. For the other hand, for the diseases whose origin is more complex the choice of the therapeutic gene may be more difficult and will have to make several studies and know well the disease.

2/ VECTOR

Vehicle by which the gene of interest is transported to the target cells. The perfect vector should be able to transduce target cells without activating an immune response either against itself or the therapeutic gene. But there aren’t a universal vector to treat any disease.

2.1/ VIRAL VECTORS

These type of vectors derives from viruses, but this is not a problem because much or all of the viral genes are replaced by the therapeutic gene. This means that the viral vectors do not cause pathogenic disease because the gene was deleted.

2.2/ NON-VIRAL VECTORS

These type of vectors does not derive from viruses, but the therapeutic gene is part of a plasmid.

3/ TARGET CELLS

Any cell that has a specific receptor for an antigen or antibody, or hormone or drug… The therapeutic gene must be directed to target cells in specific tissues.

4/ ROUTES OF ADMINISTRATION

The therapeutic gene must be administered through the most appropriate route. The type of route depends, as like as vector, the target tissue, the organ to manipulate or the disease to be treated.

5/ ANIMAL MODELS

Are used to find out what happens in a living organism. They are mainly used in research to achieve progress of scientific knowledge, as many basic cellular processes are the same in all animals and can understand what happens to the body when it has a defect; as models for the study of a disease, because humans and animals share many diseases and how to respond to the immune system; to develop and test potential methods of treatment, being an essential part of applying biological research to real medical problems and allowing the identification of new targets for the intervention of the disease; and, finally, to protect the safety of people, animals and environment, researchers have measured the effects of beneficial and harmful compound on an organism, identifying possible problems and determine the dose administration.

Gene therapy represents a promising tool to cure some of those diseases that conventional drug therapies cannot. The availability of animal models is key to preclinical phases because it allows thorough evaluation of safety and efficacy of gene therapy protocols prior to any human clinical trials.

In the near future, gene therapy will be an effective alternative to pharmacological efforts, and enable treatment of many diseases that are refractory or not suitable for pharmacologic treatment alone. Thus, gene therapy is a therapeutic tool that gives us virtually unlimited possibilities to develop better and more effective therapies for previously incurable diseases.

REFERENCES

• Ramos Muntada M. “Animal Models for Gene Therapy of Glycogenosis” Final Degree Project. UAB, Genetics Bachelor’s Degree 2015
• Bosch F, Roca C, Anguela X and Ruzo A. “Gene Therapy, a new tool to cure human diseases” CliniGene-NoE. CBATEG-UAB 2011, CliniGene – Gene Therapy European Network
• Gene Therapy Technology Explained
• Australian Clinical Trials
• Main picture: Genetic Literacy Project

How is genetic engineering done in plants?

For years, by crossing, scientists have achieved plants with a desired characteristic after many generations. Biotechnology accelerates this process and allows to catch only the desired genes from a plant, achieving the expected results in only one generation. Genetic engineering allows us to do all this. In this article I will explain what it is and how does it work.

WHAT IS GENETIC ENGINEERING?

Genetic engineering is a branch of biotechnology that consists in modifying hereditary characteristics of an organism by altering its genetic material. Usually it is used to get that certain microorganisms, such as bacteria or viruses, increase the synthesis of compounds, form new compounds or adapt to different environment.

It is a safer and more efficient tool for improving species than traditional methods (crossing) as it eliminates much of the randomness. On the other hand, modern biotechnology also becomes a new technology that has the power to modify the attributes of living organisms by introducing genetic material prepared in vitro.

It could be defined as the set of methodologies to transfer genes from one organism to another and express them (to produce proteins for which these genes encode) in different organisms of the original organism. DNA which combines fragments of different organisms is called recombinant DNA. Consequently, genetic engineering’s techniques are called recombinant DNA techniques.

Currently there are more plant organisms genetically modified than animal organisms. For this reason I will explain genetic engineering based on plants.

GENETIC ENGINEERING vs. TRADITIONAL METHODS

This methodology has 3 key advantages compared with traditional methods of genetic improvement based on hybridization:

• The genes could come from any specie (for example a bacteria’s gene can be incorporated in soy‘s genome).
• At genetically improved plant you may introduce a single new gene preserving the remaining genes from the original plant to their offspring.
• This modification process delays less the deadlines than improvement by crossbreeding.

With this way you can modify properties of plants more broadly, more accurate and faster.

In traditional crossing it generates a hybrid which combines randomly genes of both parental organisms, including the gene of interest encoding the desired trait. In contrast, biotechnology techniques only pass one or few genes which encode a specific trait known. The new plant has all the original genes of the plant and an introduced gene accurately and directed (Figure 1).

Figure 1. (A) Traditional method where, by crossing, a new variety is obtained. This carries the gene of interest (red), but also another genes randomly. (B) With genetic engineering we obtain a new variety of commercial plant with the gene of interest (red) of any other species (Source: Mireia Ramos, All You Need is Biology)

METHODOLOGY OF GENETIC ENGINEERING

Obtaining a transgenic organism through genetic engineering techniques involves the participation of an organism who gives the gene of interest and a receptor organism who will express the desired quality. The steps and the process techniques are:

0/ DECIDE THE AIM: MAKE KNOCK IN OR KNOCK OUT

KNOCK OUT:

This technique is to remove the expression of a gene, replacing it with a mutated version of itself, this being a non-functional copy. It allows the gene is not expressed.

KNOCK IN:

It is the opposite of the knock out process. A gene is replaced by a modified version of itself, which produces a variation in the resulting function of it.

In medicine, the knock in technique has been used as a strategy to replace or mutate genes that cause diseases such as Huntington’s chorea, in order to create a successful therapy.

1/ DOUBLE CHECK THAT THERE IS A GENE CODING FOR THE CHARACTERISTIC OF INTEREST

Firstly, you have to check the characteristic of interest comes from a gene, as this will be easier to transfer to a living organism that does not.

2/ CLONING THE GENE OF INTEREST

It is a complex process, but outline the steps are the following:

• Extract DNA
• Find a gene among the genes of this DNA
• Sequence it
• Build the recombinant vector

The DNA of interest is inserted into a plasmid, a circular DNA molecule with autonomous replication. The plasmids of bacterial origin are the most used (Video 1).

Video 1. “Clonación de un gen en un plásmido vector”. Explaining the use of plasmids as a vector in the process of cloning (Source: YouTube)

The development of these techniques was possible by the discovery of restriction enzymes. These enzymes recognize specific sequences and cut the DNA by these points. The generated ends can be sealed with ligase enzyme and to obtain a new DNA molecule, it called recombinant DNA (Figure 2).

Figure 2. (1) Plasmid’s DNA (2) DNA from another living organism (3a, 3b) The restriction enzyme cuts DNA (4) The restriction enzyme recognizes AATT sequence and cuts between A and T nucleotides (5) The two DNAs are contacted with the purpose of forming recombinant molecules (6) A ligase enzyme joins the DNA ends (Source: GeoPaloma)

3/ CHARACTERISE GENE OF INTEREST

If we know the gene sequence we can compare this sequence with known gene sequence through bioinformatics, provided to determine which gene looks and assign a possible function. So when we have predicted the function of cloned gene we confirm it in vivo, usually transferring it to a model organism.

4/ MODIFY GENE OF INTEREST

We can add (promoter, introns…) or mutate sequences inside the encoding region.

5/ TRANSFORMATION OF A LIVING ORGANISM WITH GENE OF INTEREST

When we have finished the gene building with the desired gene and the promoter, the recombinant DNA is inserted into the cells of the living organism that we want to modify.

6/ CHARACTERIZATION GMO

When we already have the GMO (Genetically Modified Organism) it is analysed from the molecular and biological point. In the molecular analysis it must demonstrate if you have one or more copies of the transgene or how and what tissues the gene is expressed. In the biological analysis it looks if it achieves the objective for which it was designed.

REFERENCES

• Por qué biotecnología
• Cultivos Transgénicos: Introducción y Guía a Recursos
• Agricultura Transgénica
• Main picture: FarmacoSalud