GENE THERAPY
THE STATE OF THE ART
Dr. Abdel Aziz El Bayoumi
Professor of Genetics
Dr. Khalid Al Ali
Lecturer of Genetics
Department of Biological Sciences
University of Qatar, Doha
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FOREWORD
Scientific innovations have brought radical changes in our society and improved the quality of life. It has opened new vistas for development and offered tremendous opportunities for socio-economic development. In order to achieve any realistic progress in the present era, it is necessary to understand clearly these technological innovations and utilize them for sustainable development.
The Islamic Educational, Scientific and Cultural Organization (ISESCO), has accorded a high priority to disseminate scientific knowledge regarding the new and advanced technologies vital for the socio-economic development. In order to improve the teaching and research capacities of the institutes of higher learning in the member States, special importance is attached to the preparation and dissemination of state-of-the-art studies on various advanced scientific and technological fields. Application of Genetic engineering in various disciplines like agriculture, energy production, health, population control, environmental protection, and industrial sectors has opened new horizons for the benefit of mankind. In the medicine and biomedical field, Gene Therapy has shown great progress and has provided cure to millions of people suffering from genetic disorders and acquired diseases.
The present State-of-the-Art-Study on Gene Therapy defines the basic and molecular concepts of genetic disorders and explains in a simplified manner various applications of Gene Therapy for the cure of genetic diseases. Since the unguided application of Gene Therapy may pose a serious threat to ethical and Islamic values of our society, necessary explanations have been provided regarding sensitive applications in the light of the recent guidelines. ISESCO wishes to express its gratitude to Prof. Dr. Abdel Aziz El Bayoumin and Dr. Khalid Al Ali, Department of Biological Sciences, University of Qatar, Doha, State of Qatar for the efforts exerted in the preparation of the present Study.
ISESCO is pleased to present this Study to researchers and public at large, hoping that it will help to promote knowledge and research in genetic therapeutic techniques for the treatment of the human ailments.
May Allah bless our efforts in the service of the Muslim Ummah.
Dr. Abdulaziz Othman Altwaijri
Director General
Islamic Educational, Scientific and Cultural Organization (ISESCO)
ABSTRACT
Gene therapy uses the transfer of nucleic acid to prevent disease. It is now considered as a powerful therapeutic approach to a large number of genetically based and acquired human diseases such as cancer, acquired immunodeficiency syndrome (AIDS) and cystic fibrosis. The basic and molecular concepts of genetic diseases are discussed. The technology of gene therapy involves, vehicles for gene transfer and delivery, searching for the right target cells and the regulation of the gene expression i.e. getting the right gene into the right cells.
A number of vectors, viral and non-viral, are used for gene transfer. The viral vectors most commonly used are retroviruses, adenoviruses, adeno-associated viruses and herpes viruses. Each of these vectors has advantage and disadvantage. Each system for delivery has special features and the choice of vehicle is based upon a variety of factors including toxicity or immunogenecity of the viruses. The non-viral gene therapy exhibits safety aspects.
Gene transfer by the vectors applied in both ex-vivo and in-vivo way, which resulted in a promising success. A large number of genetic diseases were subjected to gene therapy. Focus in this review will be on general types of diseases such as the inherited diseases of the lung, the non-heritable diseases such as HIV and cancer. Many of these are now under thorough investigations in clinical trials.
In spite of the progress in gene therapy a number of major aspects need to have more basic research. These include improved types of vehicles, improved specificity of transfer or expression, generation of safer vectors, modulation of the host response against the virus, and the stability of gene expression.
Gene therapy raises a number of ethical issues especially on the germline gene therapy.
1. INTRODUCTION
There are at least 4000 human diseases, which could be of genetic origin. Nuclear genes (one or more genes) or mitochondrial disorders or chromosomal imbalance causes some of these diseases. For example, about 5% of live born babies suffer from a significant medical disorder, most of them have a genetic component. A number of these disorders can be cured by supplementing, the target deficient cells with external drug, that replaces the product of the expression of the gene. However, the rate of such treatment is low by using the traditional mode of treatment. But, recently, a new way of treating many of these diseases has immerged in the scientific literature, called gene therapy. This involves, treating the genetic disease by introducing the nondefective gene into the patient, replacing or adding a new gene in order to create a more favourable phenotype. The first successful gene treatment was reported in 1990 by Anderson, of that of the fatal genetic disease, severe combined immunodeficiency (SCID). This disease destroys much of the immune system, particularly the white blood cells (T cells) due to the absence of the enzyme adenosine deaminase. The gene responsible for the enzyme adenosine deaminase is a recessive one, preventing the formation of this enzyme. Without the enzyme, the body fails to break down chemicals produced during normal
Recent advances in molecular biology and DNA technology explained the role of any specific gene product in causing the disease. Isolation, identification of any gene followed by the determination of its DNA sequences has enriched our knowledge on how the gene functions. It is now well understood that the gene product is a type of protein. If the gene is defective, this will lead to the lack of such protein causing the disease. An international effort was launched to identify every single human gene, with a project known as the human genome project This project is expected to finish approximately in year 2005. (Hawley, and Mori 1999)
Recent advances in research on gene therapy covers curing diseases such as cystic fibrosis (Wagnerand and Gardener1997, Welsh. and Oestedgoard 1998), liver diseases (Davern and Scharschmidt 1998, Wa et al 1998) and cell anemia. It is now used in a wide range of other diseases including cancer (Dachs et al 1997, Ficazzola and Tanejn 1998), cardiovascular diseases (Snowden and Grave 1998), arthritis and neurodegenerative disorders (Kaplit et al 1998) and acquired diseases such as HIV AIDS (La Frace and Mang 1997)
2. BASIC GENETICS OF HUMAN DISEASES
Gregor Mendel founded the basic rules of genetics in 1865. He started his experiments using the plant Pisum sativum. But later it was found that these rules applied also on animals including man. Mendelian rules explain how the traits are inherited throughout the different generations. He established that a pair of genetic factors now known as alleles controls each character. These factors segregate during gamete formation, and reunite during embryo formation. It is now known that these factors are located on rod-like structures, which are the chromosomes. Each individual is characterized by having a constant chromosome number in the somatic cell, which is known as the diploid number (2n). Gametes, which are produced by the meiotic cell division, contain half the chromosome number, which is the haploid number (n). For example in man his diploid number is 2n = 46 chromosome, while his haploid number is n = 23. The sexually reproduced individuals contain special chromosomes that determine the type of sex, these are the sex chromosomes, and the rest of the chromosomes are known as autosomes. In female, the two sex chromosomes are similar and designated as XX, while in male they are different and designated as XY. Normally the Y-chromosomes do not carry genes.
The physical position of a gene on a chromosome is the locus. Each gene has two forms that are the alleles located at the same locus on both of the homologous pair of chromosomes. When there are two identical alleles at a locus a person is known as homozygous, when the two alleles are different, the person is heterozygous. The genetic constitution is the genotype. The expression of the genotype is the phenotype. The two alleles are present in two forms, one is known as the dominant allele and the other is known as the recessive allele. Any individual could have only a pair of these alleles. The dominant allele masks the expression of the recessive allele, when present in the heterozygous condition.
The genes located on the autosomes are autosomal while the genes located on the sex chromosomes as sex-linked genes. Because females have two X-chromosomes and male have only one the inheritance of characteristics that are X-linked is different from that of chromosomes determined by the other 22 autosome pairs. Therefore, Mendelian inheritance shows one of three patterns, autosomal dominant, autosomal recessive or sex X-linked (which may be dominant or recessive)
2.1 Autosomal Dominant Inheritance
Human disorders inherited as autosomal dominants could be either homozygous or heterozygous. In case of the heterozygous, there will be a 50% chance that the child of an affected parent will be affected (Fig.1-a).
However, the clinical expression of disorder will be the same in both homozygous and heterozygous. Affected individuals for example are Huntington’s disease, myotic dystrophy and neurofibromatosis.
2.2 Autosomal Recessive Inheritance
The symptoms of the character only appear when the individual is having homozygous genotype. Both sexes are affected. Heterozygous of a single gene is normal and is considered as a carrier. Most individuals who have the disease arise from mating between two heterozygotes. In such cases there is a 25% chance of normal, 50% chance heterozygote and 25% chance of affected outcome (Fig-1-b). Most of the recessive conditions are quite rare. The frequency of heterozygotes is always much greater e.g. cystic fibrosis, phenylketonurea, sickle cell anemia, b-thalassaemia.
2.3 X-Linked Inheritance
The genes located on the X-chromosome are known as sex-linked genes. X-linked inheritance differs from autosomal in that females have two X-chromosomes where as males have one X-chromosome. X-linked inheritance is usually recessive. Mating between normal father and a carrier mother has an outcome with 25% chance of each an affected son or normal son, a carrier daughter or a normal daughter. In such case, the father contributes only normal X-chromosomes to his daughters and Y-chromosomes to his sons. However, if the father is affected all his daughters will be carriers and all his sons will be normal (Fig 2). Examples of X-linked disorders are fragile X-syndrome, duchenne muscular dystrophy & hemophilia A & B.
2.4 Multifactorial Disorders
There are other disorders that are controlled by more than single gene are called multifactorial. The final phenotype appears as a result of the gene interaction and the environment. Examples of these disorders are cleft lip palate, congenital dislocation of hip, diabetes, Hypertension, Multiple sclerosis and schizophrenia.
3. MOLECULAR BASIS OF HUMAN GENETIC DISEASES
It is now well established that the chromosomes are made of protein and deoxyribonucleic acid (DNA). Experiments demonstrated that DNA is the genetic material for all organisms except some types of viruses that use another type of nucleic acid, which is ribonucleic acid (RNA).
3.1 Structure and Function of DNA
From chemical analysis it was shown that DNA is composed of units known as nucleotides. Each nucleotide consists of pentose sugar deoxyribose, phosphate group and nitrogenous bases. There are four types of nitrogenous bases, Adenine (A), Guanine (G), Cytosine (C) and Thymine (T). Watson and Crick proposed the double helix model for the DNA molecule, with two chains interwined running in opposite directions (Fig 3).
Figure 3 : Structure of DNA double helix
Each DNA strand is composed of nucleotides arranged in a random fashion. The two strands opposite to each are connected together by the hydrogen bonds between the opposite nitrogenous bases. The opposite pairs are arranged in a specific way, where the guanine always bonding with cytosine and adenine bonds with thymine. Thus the two strands are complementary to each other.
One of the important characteristics for the genetic material is that it forms copies of itself, so that it can be transmitted to the daughter cells. The double helix structure of DNA suggests a model for replication of genetic material. This involves unwinding the double helix and each strand acts as a template to form a complementary strand depending upon the specificity of pairing between the nitrogenous bases, where A pairs with T and G pairs with C. Therefore, two daughter molecules are produced. Each daughter double helix molecule contains one parental strand and one newly synthesized strand (Fig 4).
Figure 4 : DNA Replication
3.2 Gene Expression
The sequence of bases in a DNA molecule represents the information content of a gene, which is known as the genetic code. These bases are arranged in a random fashion. The type, number and sequence of these four bases determine the type of the gene. Any change of these bases will lead to a change in gene function, i.e. forming a mutation. The total sum of DNA molecules found within every cell of the human body is known as the human genome. Nearly all the genome is found in the nucleus, although, mitochondria also contain essential genetic information.
This DNA contains all the information needed for the development of the zygote to form the adult. However, only about 3% of the total DNA of the human genome represent the functional genes. The rest of the DNA is non-functional or has a functional role in regulating and promoting gene expression.
It is now established that the gene functions through its control to the synthesis of protein. It involves two basic steps, the first is the synthesis of different types of RNA from DNA, by a process known as transcription. The information in the RNA can then be translated to a polypeptide chain by the process of translation.
Different types of RNA polymerase catalyze the process of transcription. Three types of RNA are formed namely the messenger RNA (mRNA) that contains the genetic code for the functional genes, transfer RNA (tRNA), that carries the amino acids to the site of protein synthesis and ribosomal RNA (tRNA), that combine with proteins to form the ribosomes. All types of RNA polymerase require the action of transcription factors to bind DNA and promote transcription.