GENE THERAPYTHE STATE OF

THE ART

 Dr. Abdel Aziz El Bayoumi

Professor of Genetics

Dr. Khalid Al AliLecturer of Genetics

Department of Biological SciencesUniversity of Qatar,

Doha

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 AltwaijriDirector GeneralIslamic 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.

The process of transcription uses one strand only of DNA as a template by the help of RNA polymerase. A newly synthesized RNA strand is formed that is an exact copy of the DNA strand. By this way the information content of the DNA molecule is maintained during transcription into a mRNA molecule (Fig 5).

Most eukaryotic genes including man are interrupted by stretches of DNA that are not translated into proteins. These are known as intervening sequences or entrons, whereas the translated sequences are known as exons. When DNA is transcribed into RNA, the primary product contains both entrons and exons. Entrons are then spliced out and the exons are unitedtogether to form the mature mRNA that will be translated to the different amino acids. Other modification to the mRNA is carried out, that is adding at one end a cap made of 7 methyl

Figure 5 : The process of transcription and translatation

guanosine and the other end a tail composed of polyadenine. The capping and tailing of RNA is necessary to export the RNA from the nucleus (Fig 6).

Figure 6 : splicing out of intron from precursor to form the maure RNA

Translation occurs on the ribosomes in the cytoplasm. The mRNA functions as a template for the synthesis of protein. The process is complex, but it depends on the genetic information in the mature mRNA that determines the correct amino acid sequence in the polypeptide chain. The sequence of the bases present in the mRNA represent the genetic code. The code is triplets code i.e. every three-consequative bases represent a codon, which specifies the particular amino acid.

3.3 Recombinant DNA technology

Recent advances in molecular biology paved the road to more advances in manipulating the gene. The most important advances were the development and application of what is known as recombinant DNA technology. This refers to the creation of a new combination of DNA segments or DNA molecules that are not found together naturally. The recombinant DNA technology enhances the understanding of gene organization, structure and expression. It was possible, to increase the amount of DNA encoding a specific gene by a process known as gene cloning.

Recombinant DNA technology uses techniques derived from the biochemistry of the nucleic acids. Among these techniques are the isolation of pure populations of specific DNA segments, the way to know the exact sequence of bases in the DNA, cutting the DNA at specific positions and identifications of a specific DNA segments using radioactive isotopes and many other techniques.

3.3.1 Making Recombinant DNA

The basic procedures for constructing a recombinant DNA involves first isolating DNA in a pure form and cutting the DNA in small fragments then ligate it in a vector.

3.3.2 Restriction Enzymes

DNA fragments are generated by using enzymes called restriction endonuclease. These enzymes can recognize a specific site on DNA known as restriction sites. These sites are characterized by having a few numbers of bases arranged in palindrome arrangement on the DNA molecule i.e. it can be read from right to left in one DNA strand and from left to right on the opposite strand. There are a number of restriction enzymes each can act on a specific site. There are enzyme types that cut the restriction sites in a staggered way

producing complementary-single- stranded tails. The resulting single-stranded tails can anneal with complementary tails from other DNA fragments to form recombinant DNA molecules.

3.3.3 Vectors

The fragments produced by the action of the restriction enzymes, which represent a specific gene, have to be moved and introduced into a recipient cell. Vehicles known as vectors can achieve this. These vectors could be either plasmids, which are naturally occurring in bacteria or different types of viruses. The plasmid is a double–stranded DNA molecule that can replicate autonomously within the bacterial cells. They are present as a separate small circular molecule, and contain a number of restriction sites and specific antibiotic resistance genes. The presence of this resistant gene can help in detecting the success of recombinant DNA formation.

Other types of vectors are the viruses. The bacteriophage Lambda is widely used in recombinant DNA. Other types of viruses can also be used as a vector especially for gene therapy such as retroviruses, adenoviruses and adenoassociated viruses. When the fragments produced by the action of restriction enzymes are put together with a vector after treating both the fragments and vectors with the same restriction enzyme. The two types of DNA are joined together using ligase enzymes i.e. the vector and the DNA fragment forming the recombinant DNA.

3.3.4 Cloning DNA

The recombinant DNA molecule consisting of a vector carrying an inserted DNA segment is transferred to a host cell. Within this cell, the recombinant DNA molecule replicates, producing large number of identical copies known as clones. A number of cell types can be used as hosts for replication of cloned DNA;one of them is bacteria. Fig (7) shows the steps for the creation of recombinant DNA molecules and cloning it in bacteria using a plasmid vector.

However, to study gene expression and gene regulation of eukaryotic genes, it is more convenient to use eukaryotic hosts. The use of recombinant DNA has many applications in animal and medicine such as diagnosis of genetic diseases, construction of gene mapping. The most important is its use for gene transfer, which is very useful in gene therapy.

4. TYPES OF GENE THERAPY

There are different strategies for correcting an inherited disease by gene therapy, depending on the type of the cell and the way of introducing the therapy. Among these types are the following.

4.1 Somatic Gene Therapy

When selected genes are introduced to a somatic cell, this is known as somatic gene therapy. The gene defect is corrected only in the somatic cells of a person affected by the disease. In such case they cannot be transmitted to subsequent generations, and the changes are confined to the lifetime of the treated person. This is the type of gene therapy most commonly used in many laboratories. (Fig. 8).

4.2 Germline Gene Therapy

This involves manipulating and introducing the gene in the germ cells i.e. gametes, fertilized eggs or embryo before the germline is specialized. In such case the genetic changes will pass onto the next generation. Little or no research is currently being conducted in the germline (Gordon1,998). This is

due to many technical and ethical reasons.

 Figure 7 : Cloning a gene with a plasmid vector in bacteria 5. TARGET CELLS5.1 In Vivo Gene Therapy

In this type the genetic material is transferd to cells located within the recipient host. This is also termed the direct method of gene transfer. This type of gene therapy is suitable for genetic defects that affect blood cells. All blood cells are derived from stem cells in the bone marrow. These cells can be isolated, cultured in vitro and then reintroduced in vivo to the patient.

Figure  8 : Types of gene therapy

5.2 Ex-Vivo (In-Vitro) Gene Therapy

It involves the transfer of the genetic material to cells located outside the host. Following transfer of the genetic material to the cells, the cells are implanted back into the host. This type is also called the indirect method of gene transfer.

The advantages of such type of gene therapy are

- Cells from the patient will not be rejected by the immune system

- Transferring the genes into cells in -vitro is more efficient. In addition, using certain markers on the vectors, it may be possible to isolate the transfected cells.

The target cell to be used for ex-vivo gene therapy must satisfy some pre requisites among these they must be:

- Easily removed, and withstand the subsequent manipulation and reimplantation.

- Capable of dividing and passing their genetic material to their descendants

- The cells should be able to express a range of gene products

Only few cell types meet these requirements such as bone marrow-derived cells, and skin cells. (Aoki, et al 1997, Overturf , et al 1998 and Kerr, 1998).

5.3 The Bone Marrow Cells

The bone marrow cells contain the stem cells of all the hematopiotic series including red blood cells and all immunological cells such as T cells and B cells and macrophages. Some of these cells are long living and circulate throughout the body. The transplantation of bone marrow is now a routine clinical procedure but it is an expensive one.

5.4 Skin Cells

Two types of skin cells can be used for gene therapy Skin Fibroblasts which are easily infected by retrovirus and grow well in vitro, and it was shown to express proteins such as adenine deaminase (ADA an enzyme found in B cells) and factor IX (protein involved in blood clot formation). Their reintroduction under the skin or by skin graft is cheaper than bone marrow transplantation, although these fibroblasts do not circulate in the blood stream skin keratinocytes. The primary cells in the epidermis are also suitable cells for gene therapy. Cultured cells from sheets that are often used to regenerate skin in burn victims.

5.5 Liver cells or hypatocytes

Are also excellent choices for gene carriers for many defects that affect the liver, such as hemophilia hypercholesterolemia and antitrypsin defects.

5.6 Retinal Pigment Epithelial Cells

A preliminary report has described the use of cultured retinal pigment epithelial cells to provide gene therapy for retinal degeneration (Dunaief, et al 1995) Retroviral gene transfer into retinal pigment epithelial cells followed by transplantation into rata retina.

5.7 T-Cells

The T-cells of the individual are removed from the patient, then placed in tissue culture stimulated to proliferate, by treating them with the lymphokine, Interlekin 2(1L-2), then infected with the retroviral vector. Finally, they reinfected to the child. The infection has to be repeated because T-cells live for only 6-12 months in the blood.

5.8 Stem Cells

These are the precursors of all followed cell types e.g. blood stem cells removed from the umbilical cords of new born babies. The blood stem cells, produce by mitosis all the types of blood cells including T and B-lymphocytes. It also produces by mitosis more stem cells this ensuring and inexhaustible supply of cells. The blood stem cells are present in everyone’s bone marrow. Umbilical cord blood has a much higher proportion of stem cells in it.

6. THE TECHNOLOGY OF GENE THERAPY

The following basic steps represent the basic requirement protocols for gene therapy:

- Isolation of the gene to be used for gene therapy and cloned for the relevant genes related to the disease.

- Delivering and targeting the appropriate tissue

- Controlled expression of the therapeutic gene with regards to both gene transfer and gene expression.

7. DELIVERY SYSTEMS TO THE APPROPRIATE TISSUE

One major step for gene therapy relies upon methods of introducing the desired genes into the target cells. This can be achieved by using a vehicle for delivering these genes. The choice of a specific vehicle depends on a number of factors, among these factors are the following:

- It must be efficient in delivery to the target cell

- It must be delivered to the specific target cell

- Whether the vehicle affects the dividing or non-dividing target cell

- Whether the vehicle will stimulate the immune system

- The size of the DNA that can be carried

- The stability and longevity of the gene in the target cell.

- The expression of the introduced gene and its rate of expression

Two main types of delivery systems are currently used. These are viral and non-viral vectors.

7.1 Viral Vectors for Gene Therapy

The modification of viruses for the delivery of exogenous genes was first reported in 1968, using the tobacco mosaic virus. Then followed by using viruses, infecting mammalian cells and led to development of modified simian papiloma virus SV40. Recombinant DNA technology, now using vectors capable of transferring and expressing the genes in culture are in progress. The most commonly used viral vectors are Retrovirus, Adenovirus, Adeno associated virus and herpes viru

7.1.1 Retrovirus Vectors

Retrovirus is a virus that infects animal cells. Structurally, it is composed of a protein coat and genetic material made of RNA. It has a unique life cycle. The virus attaches itself to a specific cell surface receptor, then it enters the cell. Inside the cell, the RNA genome is copied by an enzyme reverse transcriptase to form DNA. This DNA is then integrated into the host genome randomly, to form a provirus encodes to form more copies of viral RNA and protein envelopes to form new viruses. The newly produced viruses leave the cell by budding off from the cell carrying the part of the outer membrane (Fig. 9)

Figure 9 : Retrovirus life cycle

The retroviral vectors were first described to be used for gene therapy by Wei et al (1981) and the first patient was treated for adenosine deaminase deficiency was carried out by Anderson et al 1990.

7.1.1.2 The use of retrovirus for gene therapy

Retroviruses have several advantages for introducing genes into human cells. Among these are:

- The envelope protein enables the virus to infect human cells.

- RNA copies of the human gene can be incorporated into the retroviral genome.

To use retrovirus, as a vehicle for gene therapy requires the removal of all the genes: gag, pol and env regions, and then replaced by the gene of interest. This means that the virus loses its ability to replicate but still has the capability of integration to the target cell genome. However, its limitation lies in that they only infect replicating cells. This could be an advantage for certain types of cancer therapies. Retroviruses are used for gene therapy in a number of diseases and acquire diseases such as HIV/ AIDS (Poeschla, 1996,Marcel and Grausz, 1997).

7.1.2 Adenovirus Vectors

Adenoviruses, generally infect a variety of tissues including hypatocytes (liver cells), muscle cells, cardiac myocytes, synoviocytes (joints tissues, primary epithelial cells, and neurons (nervous system and brain). Therefore they offer a wide variety of gene therapy applications. (Kotir 1994, and Schneider, et al 1998). The virus is not enveloped, the capsid consists of three types of protein, and some are important for receptor binding and cell internalization. Its genome is a double stranded DNA. When infection, it does not integrate with the host genome, but remains as episome in the target cells. Thus avoiding the dangers of uncontrolled integration.

7.1.2.1 Viral Genome

It is a double stranded DNA, about 36 kb in size. Each end is characterized by having 100 base sequence that is inverted repeats of the sequence present on the other end. Inside the genome there are early and late genes. The early regions include regions called E1A1 and E1B to E3. They can be expressed to form m-RNA molecules. The m-RNA is produced in an over lapping way, giving rise to proteins that are subunits of one another. The E1A region is transcribed first immediately after the entry of the virus to the nucleus. It is essential for viral DNA replication. The E3 region plays a role in pathogenesis.

To use the adenovirus as a vector in gene therapy, the early regions E1A and B must be deleted from the genome therefore preventing viral replication. Such vectors are capable of infecting a cell only once and no viral propagation occurs. Then followed by inserting the desired gene in place of the deleted region. This deletion can make room for inserting a foreign desired DNA gene, up to a size of about 6kb.

The adenoviral vector used in gene therapy has to be constructed to contain two parts, first the viral DNA vector and the second is a packaging cell line. The adenoviral DNA vector is a plasmid DNA that contains a portion of the viral genome after deleting E1A region and replaced by the gene of interest. This adenovirus vector can be produced using either in vitro ligation or homologous recombination. (Fig. 12).

Figure 12 : adenovirus vector

7.1.2.2 Life Cycle

The infection of the adenovirus is highly complex. It first binds to the cellular membrane receptors, then enter the cell by the process of endocytosis. Inside the cell, the virus is released from the endosome and the viral capsid disassembles during its movement towards the nuclear pore. The viral DNA enters the nucleus through the nuclear pore and remains in the nucleus as a separate episomal state. The DNA is then replicated by the help of the early proteins. The transcription then followed by the help of late genes to give rise to the capside protein.

7.1.2.3 Construction of vector

The vector is constructed by deleting the early E1A and E1B genes and substitutes them by the gene of interest. In some cases substitutions and deletions in the E3 and late regions have also been used. Substitution of E1 genes is important as the E1 proteins may have oncogenic and lytic properties.

7.1.2.4 Replication of Adenoviral vectors

Adenoviral replication depends on the presence of the E1A, ELB region of the viral genome, therefore these regions must be deleted from genome. Such vectors are capable of infecting a cell only once and no viral propagation occurs. This deletion can make room for inserting a foreign DNA, up to size of about 6kb. The process of substitution can be carried out by either recombination or by molecular biological techniques (Rosenfeld, et al 1990)

An alternative method, the foreign genes are inserted into the E3 region of an E1 deleted vector (Mittal et al 1993). It is also possible to make adenoviral vectors by recombination employing cells infected with adenovirus or adenoviral vectors and co-transfected with the foreign gene sequence in a plasmid carrying appropriate adenovirus DNA sequence. This forms recombinant adenoviral molecules with the foreign gene inserted in the vector.

The strategy for recombinant adenoviral construction involves that particular region of the genome EIA and EIB that are essential for replication are replaced by the desired

gene of interest. The adenoviral vector is composed of two parts, viral DNA vector and a packaging cell line. The adenoviral DNA vector is a plasmid DNA that contains a portion of the viral genome after deleting the E1A region, and replaced the gene of interest.

The adenoviral vector is produced using either in vitro ligation or homologous recombination. Isolating the wild type adenoviral DNA, then cut by the restriction enzyme ClaI can carry this out. The digested viral DNA is then ligated with the adenoviral DNA vector containing the gene of interest, which has been treated with the same enzyme ClaI. Both the adenoviral DNA vector and the viral DNA component are introduced into a packaging cell line. These DNA are recombined in the cell producing the vector. (Stratford- Perricaudet, et al 1992, Wolff, 1994,Rosenfeld, et al 1990 and Mettal, et al 1993).

7.1.2.5 Cell Targeting of Adenovirus

Adenoviruses are capable of infecting a broad range of cell types. It can infect both dividing and non-dividing cells. Adenoviral vectors have been successfully administrated to humans by aerosolization into the lungs. In animal models, successful transduction has been repeated following intramuscular injection (Stratford – Pericandet, 1992) and intravenously (Lemarchand, 1992)

7.1.2.6 Advantages and Disadvantages of Adenovirus

Its advantages of the adenoviral vectors are their efficiency in transduction and capable of inserting DNA of size up to 8kb, and infects both replicating and differentiated cells. Since it is not integrative with the host chromosome, therefore, it causes no mutagenic effect caused by the random integration into the host genome. The disadvantages include that the expression is transient since it is not integrative so that the therapy would need to be repeated. Adenovirus is a common pathogen to human and in vivo delivery it provokes a strong immune response. Recently adenovirus vehicles with deletions of other early genes such as E2, E3, E4 have been constructed to further reduce late gene expression which might be responsible for the immune response.

7.1.3 Adeno Associated Viruses AAV

This type requires coinfection with either adenovirus or herpes simplex virus (HSV). The genome is double stranded DNA molecule and contains a palindrome sequence at each end, referred to as inverted tandem repeats (ITR). These ITR are important for integration to a specific site on chromosome No. 19 in human. AAV have advantages over other virus system in that they are not naturally pathogenic, they are capable of infecting nondividing cells, they show longer lasting expression (up to 6 months in animal models) in addition to its specificity to integrate to specific sites on chromosome 19. They also lack of initiating and immune response. Unfortunately, they cannot incorporate genes larger than 5kb. Several trials for cystic fibrosis gene therapy have been carried out using this vector.

7.1.3.1 Construction of AAV

Adeno-associated vectors are prepared by replacing the capsid genes with the genes of interest. The vector consists of recombinant AAV plasmid DNA and AAV helper plasmid that encodes the AA capsid protein. It also requires either wild type adenovirus or HSV and cell line for viral propagation. Cells are first infected with the wild type adenovirus or HSV, and they are then recombined. Both the recombinant AAV vector plasmid DNA and the non-rescuable AAV helper plasmid are transfected into the cell. The cells produce mature recombinant AAV vectors and wild type adenovirus or HSV. The wild type adenovirus or HSV is then removed.

7.1.3.2 Herpes Simplex Virus HSV

This virus infects the nervous system. It is transmitted through direct contact and replicates in the skin or mucosal membrane before infecting the nervous cells. It shows both lytic and latent function. The lytic cycle, induces cell death after viral replication. The latent function keeps the virus in the host cell for a long time. (Wolf, J1994, Glorioso et al 1995)

The following must be taken into consideration for an efficient HSV vector that are suitable for gene therapy

1. The vector must be non-toxic for nerve cells as well as for cell lines

2. The desired gene must be expressed in sufficient level, during the latency condition of the vector.

A number of HSV vectors have been developed. They are constructed by deleting at least one of the essential early regulatory genes, making the virus less cytotoxic and still latent (Glorioso et al 1995)

7.2 Non Viral Systems for Gene Therapy

The viral systems show lack of specificity for the targeting to specific tissues. In addition that they can carry a DNA of limited size up to 7kb, and could be suppressed in certain cells. These facts together with the random integration to chromosome sites and an unwanted immune response to the virus, make the investigators to find other systems for delivery. These systems are using non-viral systems, which are also known as physical mechanisms of gene transfer. The non-viral vectors can be divided into two categories, those which are limited to in vitro applications, and those which have both in vitro and in vivo applications.

7.2.1 Non-Viral Vectors Used in In-Vivo Applications7.2.1.1 Calcium Phosphate

The technique involves, mixing plasmid DNA in a solution of calcium chloride, and then added to a phosphate- buffered solution. After 20 minutes a fine precipitate forms in the solution, and this solution is then added to the cells in culture. However, the number of cells that take the introduced genes and express the desired gene is usually quite limited and reaches around 10%, where in some cases its level is only less than 1%. Treating the transfected cells with glecerol can increase the efficiency. The DNA precipitates most probably enter the cell by endocytosis (Ledley, F.D. 1995). It is common to transfer plasmid DNA into a variety of cell cultures and packaging cell lines. The low level of transgene expression is a disadvantage of this technique, in spite of its minimal toxicity and inexpensive

7.2.1.2 Microinjection

This involves the direct injection of DNA or RNA to the nucleus using a glass pipette. This method avoids the degradation effect of the lysosomes, therefore efficient gene expression can expected from the surviving cells. This could be useful in infecting genes in the germ line, which has been used in animals to produce transgenic animals.

7.2.1.3 Electroporation

The DNA can be introduced to the cells by the application of high voltage to a mixture of DNA and cells in suspension. The DNA enters the cells through holes formed in the cellular membrane during the electrical pulse. Best results have been obtained from rapidly dividing cells. This technique is not efficient for mammalian cells, since these cells do not survive the high voltage nature of this procedure.

7.2.2 Non-Viral Techniques with Both In Vitro And In Vivo Applications7.2.2.1 Liposomes

The liposomes are spheres consisting of lipid molecules surrounding an aqueous interior. The liposomes, is a model where cellular membranes entrapping DNA. The most commonly used liposomes are the Cationic liposomes. Cationic liposomes consists of a positively charged lipid and a co-lipid, which interact with the negatively charged DNA molecules to form a stable complex.(Nabel et al 1993). A number of positively charged lipids are commercially available for example lipofectin. Lipofectin can interact with DNA spontaneously to form complexes that have a 100% loading efficiency. Lipofectin has been used to deliver linear DNA, plasmid DNA, and RNA to a variety of cells in culture. It was also shown that lipofectin could be used to deliver gene in vivo after injecting the tissues such as lung, and liver. The released DNA can be expressed extrachromosomally within the recipient cell. (Lasic, 1998)

The advantages of using this system in gene therapy are numerous such as:

1. The liposomes are stable and available commercially since they offer protection to DNA degradation.

2. Novel liposomes are easily prepared in bulk.

3. Naked DNA or RNA have high binding efficiency with the no limits to the size, they can carry a large pieces of DNA.

4. They can transfect most types of cells.

5. They have no immumogenicity

6. The recipient cells are not necessary replicating to uptake the DNA.

.2.2.2 Polycation Conjugation

This method overcomes the nonspecificity of the fusion of the liposomes. This method involves the conjugation of DNA and receptor binding molecules that leads its way to the target cell. It is then followed by internalization by the process of endocytosis (Perales et al 1994). One example is the use of cationic polylysine covalently linked to a ligand to cell surface receptor such as transferin, this is attached to the DNA of interest (Figure 13). The conjugate DNA complex binds to the appropriate receptor and in internalized by the receptor mediated endocytic pathway this transporting the therapeutic gene into the cell. The DNA moved to the nucleus where the gene is expressed. One of the problems is the inability of the transmitted DNA to escape from the endosome. However, the presence of adenovirus help active endosomal exits especially the presence of hexon protein with other capsid proteins and such DNA escape.

The cationic conjugates are formed by the attraction of the hydrophilic domain, which is positively charged (cationic) to both the negatively charged DNA and the negatively charged cell surface. These cationic lipids were very efficient at encapsulating DNA. The main advantage of this system of gene transfer, is that the lipoplexes are non-immunogenic so they are safer than virus based methods and also its ease of preparation. But the disadvantage is low efficiency of the transfer.

Figure 13 : Gene transfer to the target cell by endocytosis of polycation conjugation

7.2.2.3 Naked DNA Injection

The injection of naked DNA into tissues is currently used to deliver therapeutic genes. It was found that injection of DNA into muscle tissue of mice was taken up and expressed inside the muscle cells. However, this method is considered a crude way to introduce new genes, since the mechanism of uptake stability and expressions of DNA are still ill defined. Despite of this it is considered a way to target to particular tumor masses. A biolistic gun is used to fire DNA –coated tungsten or gold microparticles at cells. The force produced is able to get the DNA into the cells. Many cells are destroyed using this method. For example, some researches are being under consideration using genetic vaccination to give immune response to tumor antigens, such as carcinoembryonic antigens, pathogenic agents such as herpes simplex, rabies, hepatitis B and malaria and anti p53 for cancer therapy. (Cai et al 1995, Caplen, 1998).

8. TARGETING AND GENE EXPRESSION

One of the most important aspects of gene therapy is the ability to target a specific gene to a specific cell type and also regulating its expression in

quantities needed for the therapy. This could be achieved by either making use of cell properties such as its rate of cell division. So it involves altering the gene transfer vector, so that it only delivers its gene to the dividing cells. Retroviral vectors are examples for this type of targeting, where they can only infect the rapidly dividing cells. This strategy is suitable in targeting tumour cells. (Salmon and Guinzburg, 1993,and Dachs. et al 1997),

Other strategy depends on controlling the target gene expression, at the level of transcription. It focuses on restricting the transgene expression in target cells through the regulatory mechanism of the genetic elements such as cell specific promoter and or/ enhancer elements. Retroviral vectors that contain the tetracycline inducible system have been developed by Paulus et al (1996). In this system, the gene can be switched on and off by administering the tetracycline to the cell. (Figure 14).

Another example for cell targeting is by activating the expression of certain gene using steroid receptors. In the presence of a certain steroid, it is bounded to a cognate ligands attaches itself to the specific target cell, enters and moves towards the nucleus. They can then selectively induce the transcription of the specific gene. For example, a drug known as antiprogestin activates a recombinant steroid receptor that in turn binds specifically to the promoter region of the therapeutic gene. This gene switch does not affect other cellular pathways. In vivo experiment showed that when antiprogestin is administered orally, it activates the gene to be switched on. (Kasahar, et al 1994), increased the efficiency of transfection by incorporating adenoviruses or other endosome-lysing agents into molecular conjugate vector composition (Figure 14). Similarly, adenovirus was coupled with the transferrin-polylysine/DNA complexes that enhance the receptor-mediated gene delivery and expression of transfected genes (Wagner, et al 1992).

Figure 14 : Regulation of gene expression by the action of drugs on the promoter

Other system of targeting is the use of liposomes that were prepared to incorporate specific proteins onto their surface and combined with DNA. This type of liposomes has the ability to recognize a specific cell population to deliver genes to these cells. Molecules such as monoclonal antibodies, carbohydrate ligands and protein ligands are used to be incorporated with the lysosomes to

target the DNA to specific cell types.

9. GENERATION OF MOUSE MODELS FOR GENE THERAPY

Normally, gene therapy like any other administration of a new drug has to be tested in animal model, before clinical application on human. Mice are good candidates for administering and studying the effect of new drugs. The mice have to be prepared in way to generate genetically manipulated mice, for example that carry or lack a specific gene. These mice provide tools to analyze the function of new genes in a whole organism environment and provide a model of a human disease.

The introduction of any gene to be incorporated within the genome, form what is known as transgenic mice. Therefore, it is possible to introduce specific gene together with promoter region, which will be expressed in specific tissue and forming the final protein product.

Normally the gene of interest is cloned and constructed then injected into the pronuclei of fertilized mouse eggs, leading to the integration of this DNA into the mouse genome. The injected eggs are transplanted into female. Expression of the gene and any resultant phenotypic changes can then be studied. (Figure 15) Transegenic mice are normally used in gene therapy to show the expression targeting types and longevity of the gene expression (Tsukui, 1996).

10. CANDIDATES OF INHERITED DISEASES OF THE LUNG

There are a large number of inherited human diseases. However, not all are subject for gene therapy. The ideal candidate should satisfy the following:

1. It must be caused by a single gene defect.

2. It affects only one cell type which is ready for removal or replacement

3. The gene responsible for the disease must be well identified and cloned.

4. The regulated expression of the gene is not required to correct the defect.

5. The disease represents a significant cause of mortality and no effective conventional treatment is available.

Figure 15 : Production of transgenic mice as a model for gene therapy

Table 1 represents some common inherited diseases that could be candidate for gene therapy. Most of these diseases are controlled by single gene. There are however other diseases that are controlled by more than one gene. These are so far very difficult for gene therapy

Table 1 Some common inherited diseases that could be candidate for gene

therapy

Disease Defective Gene Sickle cell disease Beta chain of hemoglobin

Cystic fibrosis ATP-binding cassette (ABC) family protein

Huntington’s disease Identified;function unknown Duchenne’s muscular dystrophy Dystrophin

Phenylketouria mental retardation Phenylaianine hydrroxylaseLesch-Nyhan syndrome Hypoxanthineguanine – phosphoribosy

ltransferaseGaucher’s disease GlucocerebrosidaseTay-Sach’s disease Alpha chain of lysosomal

hexosaminidase AGalactosemia Galactose accumulation

Maple syrup urine disease Amino acids and ketoacid accumulationAdenosine deaminase (ADA) deficiency Adenosine deaminase

Thalassemias Beta chain of hemoglobinAlphal-antitrypsin deficiency Alphal- antitrypsin

X-linked chronic granulomatous disease (CGD) Cytochrome b558 gp91-phox

Autosomal CGD P47-phoxFamilial hypercholesteremia Receptor for low-density lipoprotein

Ornithine transcarbamylase deficiency Ormithine transcarbamylaseModified from, Maulik, S. and S. Potel 1997.

10.1 Gene Therapy for Inherited Diseases of Lung

Gene therapy was originally developed as an approach for treatment of classic genetic diseases. The transfer of a normal copy of a single defective gene would compensate for the damaged gene and revert the disease. Recently it was used to treat other disease such as the lung diseases. There are three genetically controlled diseases of the lung due to a mutation at a single gene. These diseases are cystic fibrosis (CF), a1-antitrypsin deficiency (µ 1-ATD) and surfactant protein B (SF-B) deficiency.

10.1.1 Vector System for Gene Delivery

Gene delivery for the lung involves delivery via the airway that has direct access to lung epithelia. Alternatively, gene delivery could also be via the pulmonary circulation that permits vascular diseases.

Viral and non-viral vectors are used for gene therapy of lung diseases

10.1.1.1 Viral Vectors

i. Retroviral Vectors

Most of the viral vectors be employed for gene therapy were delivered from retroviruses. The retrovirus life cycle as mentioned before includes converting RNA viral genome into double-stranded DNA, followed by randomly integrates into the host chromosomal DNA. A major disadvantage of these vectors is that they are very labile in the presence of serum

complement, which restrict their use to ex vivo protocols. In addition, they have little effects on non-dividing cells. Also is produced only in relatively low titers.

ii. Adenoviral Vectors

Recombinant adenoviral vectors have been widely employed as vehicles for gene delivery to the lung, because of its attraction to the respiratory tract. Adenovirus has high efficient gene transfer to a range of cell types in vivo including both dividing and non-dividing. In addition, they can be concentrated to high titers. One problem for the use of adenoviral vectors for gene therapy is that host cellular immune responses result in transient expression of the delivered therapeutic gene that requires re-administration of the same vector. A second generation of this vector has now been engineered to minimize the expression of viral antigens. Another disadvantage is that the adenoviral genome is not integrated into the host genome so that expression of the therapeutic gene is only transient.

iii. Adeno-Associated Virus

It is non-pathogenic parvovirus with a single stranded DNA genome (Muzyczka, 1992). It is tropic to the airway epithelium. Its main advantage in gene therapy for the lung diseases is that its ability to integrate with the host genome and affects a variety of cell types including both dividing and non-dividing. However, its disadvantages are that it is produced in low titer and it can accommodate small size of DNA, which is limited to 4.5 kb.

10.1.1.2 Non-Viral Vectors

A number of non-vectors have been developed including injection of naked DNA, complexing DNA with macromolecules in order to facilitate cellular entry. liposomes, have been also used to introduce the DNA to the cell via cell membrane fusion (Lee.and Huang,1997)

Other non-viral vectors were also used such as Cationic lipids complex with anionic plasmid DNA. At the moment strategies to improve liposomes have focused on developing new lipids or ligands to permit targeting to specific cell types. (Lasic, 1998). Molecular conjugate vectors have been also developed to deliver genes to the target cells via the receptor-mediated endocytosis pathway (Cristiano and Curiel1996)

10.2 Gene Therapy for Cystic Fibrosis

Cystic fibrosis is an autosomal recessive disease, located on chromosome number 7. It is responsible for the cystic fibrosis transmembrane conductance regulator (CFTR). This is an CAMP-medicated chloride channel protein, that regulates ion channels (Riordan,et al1989 and Rommens,etal1989). The cause of mortality of CF, is a pulmonary disease, due to the accumulation of viscous mucus in the lung Smith et al 1996). This facilitates chronic bacterial infection, airway inflammation and premature death at around 29 years of age.

Current treatment of cystic fibrosis showed no improvement in survival rate. Therefore gene therapy could be a potential way to correct the biochemical abnormality. Since the lung is the primary target for CF gene therapy. It involves the introduction of the wild type (normal) CFTR gene into the airway epithelial cells. The introduction of the normal CFTR gene in cultured CF airway epithelial cell lead to restoration of normal chloride ion transport (Rich et al 1990,Wagner, and Gardener1997). However, the results showed that only 6 to 10% of CF airway epithelial cells can restore normal chloride transport properties, (Johnson,et al 1995). Another experiments carried out using adenoviral vectors to human CF bronchial xenografts, showed also only 5% of the cells completely restored chloride transport. These previous studies have provided the way for delivering

the CFTR gene to intact airways first in animal models, followed by human clinical trials, based on nasal or and lung-directed gene transfer.

CF gene therapy at the moment uses Adenoviruses, Adeno-associated viruses and liposomes to achieve gene transfer via the airway epithelia resulting in at least partial correction of the chloride transport defect. However, there are still several problems with these systems, which needs to be overcome. Initial human clinical trials showed that the efficiency of gene transfer using viral vectors to uninjured airway epithelia by the present vectors is low. It was found that the inefficiency of gene transfer using the adenovirus vectors to fully differentiated epithelial cells is due to the paucity of the cellular receptors required for internalization of the virus (Goldman, and Su, 1996)

Another problem in gene therapy using viral vectors is its safety since a number of adenoviral particles cause direct viral toxicity. Further problem with the use of adenoviral vectors for CF gene therapy is that host cellular and humoral immune response result in transient expression of the delivered gene (Wilson ,1995)

11. GENE THERAPY FOR NON-HERITABLE DISEASES

Gene therapy is now extended to be used for non-heritable disorders, including the acquired diseases such as acquired immunodeficiency syndrome AIDS, and cancer diseases. The following represent an overview of the nature and gene therapy treatment for these types

11.1 Gene Therapy for HIV/ AIDS

It is well known that HIV virus is the causative agent for AIDS, This disease is now widely spread in different parts of the world. The search for a cure is one of the great challenges to human health. There are however a number of ways to manage the HIV disease, but little success towards the virus itself. Gene therapy showed a promising potential for long term treatment of HIV infection (Gilboa, 1994 Ho et al 1995, Wer et al 1995)

Current HIV gene therapy trials are mainly ex-vivo techniques. Hematopoietic cells (mature T cells, or Hematopoietic stem / progenitor cells) are isolated from the patient and utilized as the cellular vehicle of the therapeutic genes. The therapeutic gene is introduced into target cells using recombinant viral vectors. The transduced cells (those cells that take up and express the therapeutic transgene) are then reinfused intravenously into the number of transduced cells before the reinfusion.

11.1.1 HIV Life Cycle

HIV is a member of lentivirus family of retroviruses. RNA viruses that integrate into host chromosome through a double-stranded DNA intermediate provirus. Therefore AIDS is essentially an acquired genetic disease capable of suppressing the immune function (Poeschla, 1996)

All retroviruses contain gog, pol, and env genes. HIV contains numerous other genes including tat, rev, nef, vpu, vif, and vpr which are translated from differentially messenger RNA (mRNA) transcripts. Some of the regulatory functions for these genes are defined, while others are still obscure. (Figure 16)

Figure 16 : HIV genome

Full understanding of the biologic function of these genes and HIV – host interactions facilitate the design for gene therapy treatments for the AIDS. The complexity of HIV-host interactions depends on the functional integration of HIV into the fundamental cells of the immune system. The cellular receptors used for HIV binding and entry are essential receptors for immune function. HIV utilizes replication and gene expression. For example transcriptional factors that regulate activation of T cells can bind to the viral promoter regions within HIV LTR and regulate virus gene expression. Also, regulatory gene products from HIV, such as tat, can bind to host gene – promoter regions and induce expression of regulatory factors that are important for the immune system. HIV integrates into the host genomes and also functionally integrates into the complex regulatory machinery of the immune system cells (Brother, 1996).

11.1.2 Cellular Vehicles for HIV Gene Therapy

The cellular vehicles for gene therapy are hematopoietic stem cells, because these cells give rise to all of the hematopoietic cell lineages and have the capacity of self-renewal. Therefore provide long term protection.

The target cells for HIV –1 is CD4+ cells of the immune system including T cells and macrophages. HIV action is associated with progressive depletion of CD4+ T cells. A number of the HIV gene therapy clinical trials have focused on ex vivo treatment of CD4+Tcells, making them resistant to HIV infection, then infused into the patient.

The most commonly used vector delivery systems are both viral and non viral systems.

11.1.3 Gene Therapy Strategies For HIV / AIDS

A number of gene therapy strategies for HIV infection include: Gene transfer based immunotherapies vaccines and direct antiviral transgene.

11.1.4 Gene Therapy Based Immunotherapy

It was shown that immune response to HIV is initiated soon after infection. Therefore, gene therapy was developed based on stimulating the immune response against HIV by directly injecting a modified retrovirus expressing the HIV proteins env (envelope) and rev (a regulatory viral protein) into HIV- infected patients. The increase of the amounts of such env and rev proteins will raise the immune response of the individuals who are infected (Rock et al 1994). Clinical trials were carried out (Yu, et al 1994). It remains to be seen whether this will benefit the patient. One criticism of this approach is the high mutation rates of the virus, that some mutants could escape immune response.

11.1.5 Direct Antiviral transgene

The other approach to gene therapy for AIDS is to transduce cells with transgenes that are directly antiviral. This involves obtaining an HIV mutant of a specific gene essential for viral replication, therefore inhibiting the function of the wild-type gene products. One of these mutants is the HIV regulatory protein rev that interferes with the function of the normal rev molecules. The rev is essential for HIV replication. Therefore, the mutant produced will inhibit HIV replication (Liu , et al 1994).

A non-viral delivery system such as injecting DNA gold-coated particles for some mutants of the rev gene has also been explored (Woffendin, 1994). This approach has the advantage of the retroviral vector systems i.e. causing the disease and it only infects the dividing cells.

11.1.5.1 Interferon

Human interferon – alpha was found to be an inhibitor of retrovirus replication. Su et al (1995) demonstrated that expression of interferon-alpha inhibits HIV production, using a retroviral delivery system containing the interferon-alpha gene to the human T - cell line.

11.1.5.2 RNA Strategies

RNA gene therapy approaches using the RNA for viral regulatory proteins includes the ribozymes and antisense DNA / RNA. These RNA mimics a viral binding site for the regulatory protein and captures it so that it is not available for viral replication.

The antisense RNA encodes untranslated RNA molecules, that binds with the sense strand RNA’s transcribed from the HIV genome. The formation of antisense –sense duplexes blocks translation and stimulates degradation of the unstable RNA complex (Welch, 1996).

The ribozyme, which is a molecule of RNA that has enzymatic activity against specific target RNA sequences. Ribozyme binds to complementary sequences on target RNA transcript, which cleaves the substrate RNA in a catalytic manner. In the case of RNA viruses such as HIV, ribozyme can cleave incoming viral genomic RNA prior to integration and can disrupt mRNA translation and progeny RNA genomes that ready for packaging to produce new virions (Yu, et al 1993 and Welch1996).

11.2 Gene Therapy of Cancer11.2.1 Genetics of Cancer

Cell differentiation in eukaryote cell depends on the regulation of the cell division cycle. The cycle is controlled by a number of molecular factors. Healthy cells grow, when there is a balance of stimulatory and inhibitory signals obtained from outside the cell that favor the cell proliferation. A cancerous cell does not respond to the usual signals and reproduces continuously forming more malignant cells. Cancer results from the uncontrolled proliferation of cells and from the ability of such cells to migrate to other sites (metastasis). There are many different causes of cancer, such as spontaneous genetic changes either by gene mutations or chromosome mutations. The other are induced types, caused either by exposure to mutagens, radiation or cancer inducing viruses. Most cancers are considered of genetic disorders; i.e. they are caused by changes in DNA. Cancer results from genetic mutations that cause a loss in the ability of the cell to respond to regulatory signals, so the cell loses control on cell division. Mutant forms of gene involved in the regulation of the cell cycle are the cancer-causing genes. The cell cycle is regulated at two sites, with two types of gene products these are, cyclins and kinases.

Recently, a number of genes related to the onset of cancer are identified. There are three classes of genes that have been shown to be mutated frequently in cancer. These are oncogenes, tumour suppressor genes and mutator genes. The products of oncogenes normally stimulate cell proliferation. Mutant oncogenes either are more active or become active inappropriately. The products of tumour suppressor genes normally inhibit cell proliferation. Mutant tumour suppressor genes have lost their inhibitory function.

Oncogenes are genes that normally function to maintain cell division, these genes must be mutated or inactivated to stop cell division. If these genes become permanently switched on, cell division occurs in an uncontrolled way. Proto-oncogenes normally function to promote cell division. These genes can be on or off. When they are on, they promote cell division. To stop cell division these genes must be inactivated. If these genes are permanently switched on then uncontrolled cell division occur, this leads to tumour formation. Therefore the mutant form of proto-oncogenes are known as oncogenes.

Tumour suppressor genes normally act to suppress cell division. If mutation occurs in these genes, cancer will appear.

Tumor viruses induce other types of cancer. Both DNA & RNA tumour viruses are known. DNA tumour viruses transform cells to the cancerous state through the action of a gene or genes that are essential parts of the viral genome. All RNA tumour viruses are retroviruses. These retroviruses contain cancer-inducing genes termed oncogenes. Both normal cells and non viral-induced cancer cells contain DNA sequences that are related to the viral oncogenes. It is found that the tumour causing retroviruses have picked up normal cellular genes called proto-oncogenes, while losing some of its own genetic information (Klug and Cummings 1997).

11.2.2 Gene Therapy Strategies for Cancer

Traditional cancer treatment is either radiation or chemotherapy. These treatments depend on the action of these agents on destroying the rapidly dividing cells or by preventing cells from entering cell cycle and therefore stopping the cells from dividing. Recently, attention was made to use modern technology for cancer treatment. Among these technologies is gene therapy. (Vile, 1996 and Roth & Cristiano 1997) that specifically targets cancer cells.

For gene therapy of cancer to be successful, the gene must target specifically the malignant cells, while leaving the normal tissue. This means the development of selective gene delivery with highly specific gene expression and specific gene product activity and possibly specific drug activation.

There are a number of approaches to gene therapy of cancer. These include, non-specific immune stimulation, selective enhancement of toxicity to cancer cells, general tumour suppressions and specific tumour suppression genes.

However, there are two principal obstacles that limit the advances in gene therapy of cancer. The first, is the development of the appropriate delivery system that must be reliable, safe and effective to introduce the genetic material into the target cells or tissues. The second problem is the understanding of the molecular basis of cancer, in order to determine where single changes might allow effective anticancer therapy.

Progress is underway to develop a number of vectors used for gene delivery. Viral vectors, such as retrovirus, adenovirus, adenoassociated virus are used (Robbins, et al 1998, Reynolds, et al 1999). Also non-viral methods such as using liposomes for injection are also used (Lasic,. 1998).

The following represent some of the approaches used for gene therapy of cancer.

11.2.2.1 Pro-drug Activation Vectors

One idea is to deliver a gene to a target cell whose product will activate a prodrug, which then kills the manipulated cell but does not affect the normal cells. Several experimental models are now in progress. These models use viral vector that carries a gene, its product would activate the non-toxic drug to be toxic only in the cancerous cell. For example the thymidine kinase gene, carried by a retrovirus, injected to the brain tumour to deliver the Thymidine kinase (Fig .17). The patient is then treated with the drug ganciclovir. This drug is an analogue of guanosine. It could be phosphorylated by thymidine kinase, which can be incorporated into DNA. Such incorporation inhibits DNA replication and kills the cell (Dachs, et al 1997).

Figure 17 : prodrug activation

Other example is a viral vector carries the gene for enzyme cytosine deaminase. This enzyme activates the non-toxic pro-drug 5-flurorcysteine to the toxic chemotherapeutic drug 5-fluorouracil, that kills the cancerous cells as a result of its interference with m-RNA metabolism (Licht , et al 1997) .

11.2.2.2 Immunotherapy

Another way for cancer gene therapy is to protect blood hoemopoietic stem cells from the effect of chemotherapy. Generally, resistant lines arise due to the production of high level of multiple drug resistance (MDR) proteins, which pump toxic drugs out of the cell. Meanwhile the hoemopoitic stem cells naturally produce very low levels of MDR and are killed by low concentrations of toxic drugs. This resistance could be overcome by increasing the initial concentration of drugs to kill the cancer cells before resistant lines arise. Gene therapy approach was carried out to increase the expression of MDR in hoemopoietic cells. Recombinant retrovirus can be used on hoemopoietic stem cells (Roth,. and Cristiano, 1997).

A second way for the immune system to fight cancer is to increase its sensitivity to cancer cells. The immune system in man can recognize the cancer cells and kill them but in well-established cancer, the system fails to do this. A method was developed to stimulate the production of cytokine that stimulate cell-mediated immunity. The latest version of this treatment is to produce the cytokines in the cancer cells. This stimulates the immune system, but in the vicinity of the tumour.

T lymphocytes play an important role in the host’s immune response to cancer. Modern cancer immunotherapies are designed to induce T cell reactivity against tumour antigens. To stimulate the immunoresponse investigators used cytokine genes. They developed a vaccine consisting of tumour cells transduced with cytokine genes. Therefore it is now possible to transfer cytokine gene directly into tumours in vivo (Tuting, et al 1997, Jaffee.& Pardoll. 1997).

A number of cancers have mutations in tumour suppressor genes such as p53. The p53 gene encodes a nuclear protein that acts as a transcriptional factor. Normally p53 is a tumour suppressor gene. Mutation of the gene would cause cancer. It was found that when normal genes are introduced into cell lines from tumours carrying these mutations, the tumour characteristics disappear (Caplen, 1998, Clayman, et al 1998 and Roth, et al 1998).

Large number of clinical trials using gene therapy for different types of cancer are under progress, in eye cancer, retinoblastoma (Aldred, 1999 and Hayashi, et al 1999), breast cancers (Wicha, 1998), prostate cancer (Ficazzola and Taneja 1998).

12. ETHICS OF GENE THERAPY12.1 Ethical Analysis

Moslem scholars and scientists have many potential ethical tasks, from providing information to suggesting principles for regional and international legal recognition. The International Bioethic committee (IBC) in its ethics evaluative role must suggest ethical principles that are grounded in universal ideas even as it takes account of the happy diversity of cultural and religious traditions that co-exist in the world.

The principles and materials explained in the holly book of Quran and Hadeith yield a rich supply of guidance for evaluating what should be done in discrete bioethics cases. As result Muslim scholars and scientists are encouraged to discuss the recent advances in the genetic fields among which is gene therapy.

12.2 Application to Somatic Cell Gene Therapy

So far as we know there has been no committee anywhere that has recommended outright prohibition of all somatic cell gene therapy. The 1982 Report “ Splicing life “ (United States presidential commission for the study of Ethical problem in medicine and biomedical and Behavioral research) laid the groundwork for world-wide recognition that the ethical problems presented by such therapies are not fundamentally different from those presented by other research techniques.

The principal problems in somatic cell gene therapy involve adequate supervision both of the safety of research practices in the laboratory, and of the decision to initiate human trials and the methods for securing full information from them.

From any ethical standpoint we know about, the central responsibility is maximize the gains and minimize the risks, and ultimately decide whether or not the benefits realistically to be gained make legitimate asking others to run the risks involved (Ivanov,1993).

12.3 Somatic Cell Therapies and the Problem of Enhancement Engineering:

Recent gene therapy discussions insist that somatic cell procedures should be revised for “ serious disease”. This is due to the highly experimental nature of procedures and the lack of sufficient experience for determination of the incidence and seriousness of side effects.

That accompanies various types of cellular alterations.

What is a serious disease? Despite the diversity of religion, cultures and world views, we believe the international community would reach virtual unanimity on a long list of serious diseases, and that list would include current targets of genet therapy protocols such as Cystic Fibrosis, ADA deficiency, Fanconi Anemia, Cancer and AIDS. Moreover, some kind of protocols that enhance human performance, for example by putting in genes that protect an individual against serious disease, must be conceptualized as protocols aimed at that serious diseases, and not objected to as efforts to improve mere behavioral traits.

Finally, should somatic cell gene therapy procedures that are designed to enhance performance in someway or another be regarded categorically as violations of international human rights principles or other religious or moral values, if so which one and why ?

We think that there is no need to speak out now against possible future misuses of enhancements, without first having a description of their characteristics. However, it may be appropriate to speak out against any possible use of such procedures to diminish human capacities.

12.4 Germ line gene therapy

Germ line gene intervention has grabbed the world’s attention. All major statements about germ line intervention condemn its present use. That position is clearly correct. So far as we know ,there have been no efforts anywhere in the world to attempt germ line intervention on human beings.

The argument against germ line intervention in that it is somehow beyond human right to interfere with the fundamental process of life and that this prohibition cannot be overcome b the highly ambiguous notion of “diseased” genes.

The prohibition on germ line therapy is a matter of formal legislation in some nations ( e.g. Sweden ) and is accomplished through regularity controls in many others for example The United Kingdom and The United States. It’s time for Muslims scholars also to discuss the matter and formulate a clear Fatwa. Regarding germ line therapy.

The prospect of using germ line interventions to improve the basic traits of humanity is everywhere condemned. Human individuality and community is linked to the claim that you are who you are. It is wrong to burden individuals with a programmed destiny, and make them victims of genetics expectations.

12.5 Fair Distribution of Resources

If gene therapy has a chance to be an enabling technology, with wide spread uses, the oft-expressed concern that the economically developed world is spending it’s medical resources wastefully in developing them is misguided.

As with all technologies, costs drops the more widespread the use. Moreover, improvements in strategy and design may sharply reduce cost through improvements of product. It is not likely that gene therapies will be subject to comparable reductions in cost based upon scale of production, but there is no reason to suppose they will necessarily increase the total social costs spent on gabbling with the diseases they are used to treat.

These observations say nothing, however about whether and to what extent gene therapy applications most relevant in the developing world can be practiced there at reasonable expense, within a reasonable time.

12.6 Recommendations

In conclusion, our recommendation fit a familiar pattern of the following :-

1. Somatic cell gene therapy is permissible, regulated as an experimental therapy.

2. It’s use for enhancement purposes should be prohibited till we prove the safety of all somatic cell gene therapy.

3. Germ line interventions are indefensible and should be categorically prohibited in both therapy and enhancement.

4. Islamic world should work parallaly with the international committees in establishing the guidelines for gene therapy in accordance of the Islamic Shariah.

5. The Islamic community should formulate an organization for bioethics , to discuss all the new developed technologies.

6. Initiations of local, regional ethical committees to follow up the recent developments and formulate recommendations.

13. CONCLUSIONS

Gene therapy could be considered as a future approach of treating many genetic diseases and acquired diseases. Despite the great progress in the clinical trials, the practical reality is still out of reach since there is little evidence of the clinical efficiency of gene therapy.

One of the difficulties involves the discovery of new efficient vehicles, and to translate the gene therapy from the bench to the bedside. For examples success has been achieved in gene transfer for the treatment of Duchenn’s muscular dystrophy of cultured human myoblasts that incorporated into mouse myotubes after intramuscular injection (Mendell, et al 1995). Later studies showed little success in clinical trials mainly due to immune responses (Yang, et al 1994 and Dai et al 1995). More recent studies using newly developed better viral vehicles showed substantial progress (Karpate, et al 1997). Also, the success of gene therapy must involve, appropriate disease target with a gene-delivery system, that have efficient way of gene expression, that produce long-term therapeutic results and little or no toxicity.

However, many of successes of gene therapy involve those diseases, controlled by single recessive genes. This is mainly due to the fact that many of these genes have been cloned. Therefore, the long-term expression of a normal copy of such relevant gene would correct the abnormality. One of the aspects worth mentioning is the development of new systems of in vivo delivery. For example the intramuscular injection of DNA and DNA –based vaccines for infectious diseases. (Ulmer, et al 1993) and treating some coronary-artery resterosis (Ohno, et al 1994, Chang 1995). The future of gene therapy for diseases such cystic fibrosis, Dechenn’s muscular dystrophy and other diseases depend on developing and improving gene-delivery systems. New approaches including methods of modifying adenovirus vectors as well as immunosuppressive tools to prolong recombinant-gene expression in animals (Yang, 1994).

Gene therapy has shown great progress, in recent years and many patients worldwide are subjected to clinical trials in diseases such as cystic fibrosis, muscular dystrophy,

adenosine deaminase deficiency (During et al 1998). Also acquired diseases as cancer and AIDS were investigated. Other diseases, are recently considered as a target for gene therapy such as some central nervous system (During & Ashenden. 1998) and diabetes mellitus (Levine and Leibowitz, 1999 Nikol and Fling 1998). In spite of these progresses, the technology still needs to overcome two main problems: the mechanism of gene delivery and control expression of transferred gene.

Some of the recent trials started to investigate the gene therapy by integrating the genetic material into germ cells. This will introduce heritable changes into the offspring of patients. New development now underway to carry out gene therapy in utero i.e. prenatal gene therapy (Jones and Bui 1998, Senior, 1998, Senut, and Gage,. 1999). These issues have to be thoroughly discussed specially from the ethical point of view (Gordon, 1998).

It could be concluded that gene therapy is now grounded on good scientific principles and the negative results of early clinical trials was mainly due to the early nature of the field. However, recent progress in developing new systems of in vivo or in vitro gene delivery, showed good promise for treatment of a number of diseases. It is expected that new successes will show up in the near future. However gene therapy raises some important ethical issues specially the germline gene therapy.

14. SUMMARY

• Gene therapy is a new approach for treating many human diseases. It involves transferring normal gene that either replace or supplement the defected gene. Therefore the introduced gene will function normally forming the biologically active product to alter the course of the pathologic processes.

• The human traits are transmitted following the basic genetic rules. These are controlled by genes which are located on either the autosome or on the sex chromosomes. The genes are found in two different forms either dominant or recessive. The mode of inheritance of any characters depends on the nature of the gene and its location. Some traits are controlled by one gene i.e. monofactorial, while others are controlled by more than one gene i.e. multifactorial.

• Recent development of molecular biology has explained the nature of gene expression to show the phenotype. The gene is chemically composed of deoxyribonucleic molecule (DNA). It acts through its control to form protein. The process of gene expression depends on two major processes, namely the transcription and translation. The process of transcription involves the synthesis of three types of RNA. They are messengers RNA that carry the genetic information i.e. genetic code which is represented by the type, number and sequence of bases. The transfer RNA carries the different amino acids. The ribosomal RNA combines with the protein to form the ribosomes where protein synthesis takes place.

• The process of translation involves translating the genetic code to determine the type and the sequence of amino acids in the protein.

• Molecular biology has helped in manipulating the genes, and a new technology, appears that is the recombinant DNA technology. This involves cutting the DNA at a specific site using enzymes known as restriction enzymes. Ligate the DNA fragment i.e. the gene of interest in vehicle known as vector to form a chimerical DNA, which is the recombinant DNA. The vectors used could be either bacterial plasmid or virus. The recombinant DNA can then be introduced to a host such as bacteria. When bacteria divides, the recombinant DNA replicate. Therefore we increase the number of copies of the transferred gene and also its product. This is known as gene cloning. Gene cloning is very important in gene therapy.

• There are two main types of gene therapy. Somatic gene therapy where we introduce the gene into somatic cells. The other is the germline gene therapy, which involves introducing the gene into the germ line.

• Gene therapy can be carried either outside the human body that is known as ex-vivo gene therapy. Following transfer of the genetic material to the cells, the cells are implanted back into the host. The other type is the in-vivo gene therapy that is introducing the manipulated gene directly to the host.

• There are different types of target cells used in gene therapy such as bone marrow cells, skin cells, live cells, T cells and stem cell.

• The major steps in gene therapy is isolating the gene and transfer it using a number of delivery systems. The delivery systems are known as vectors. There are viral vectors and non-viral vectors. The viral vectors generally used are the retrovirus, Adenovirus, Adenoassociated virus and Herpes Simplex Virus.

• The retrovirus is an animal virus composed of protein coat and RNA as genetic material. It has a unique life cycle. When it enters the cell, a DNA copy is formed from it using the enzyme reverse transcriptase. It is then integrated with host genome randomly. To use the retrovirus as a vehicle for gene therapy requires the removal of all the genes that are responsible for viral replication. It only infects replicating cells.

• Adenovirus is not enveloped, and its genome is a double stranded DNA. When infection it does not integrate with the host genome. The adenoviral vector used in gene therapy constructed of two parts, the first is the viral DNA vector and the second is a packaging cell line. It is capable of infecting a broad range of cell types, dividing and non-dividing. Its advantage is ability of inserting large size DNA and is not integrative. However, its disadvantage that its expression is transient so the therapy would need to be replaced.

• Adenoassociated viruses requires coinfection with either adenovirus or herpes simplex virus. Its genome is DNA and can be integrated to specific sites on chromosome number 19 in human. Its advantage, that they are not pathogenic, infecting non-dividing cells and shows longer lasting expression. But it cannot incorporate genes larger than 5kb.

• Herpes simplex virus (HSV) infects the nervous system. A number of HSV vectors have been developed. They are constructed by deleting al least one of the essential regulatory genes making the virus less cytotoxic.

• A number of non-viral vectors are developed for gene transfer. Among these are calcium phosphate, where plasmid DNA are precipitated with a buffer of phosphate and calcium chloride.

• The DNA can be introduced to the target cells by either microinjection or electroporation and Naked DNA injection.

• Non-viral techniques used for both in vitro and in vivo gene therapy applications are the liposomes, polycation conjugation. The liposomes are spheres of lipid, and could surround the DNA. The other type is polycation conjugation. This method involves the conjugation of DNA and receptor binding molecules that leads its way to the target cell. Then it enters inside the cell.

• One of the most important aspects of gene therapy is the ability to target a specific gene to a specific cell type and its regulation of its expression. This could be achieved either by making use of cell properties such as its rate of cell division. This is possible to use vectors that only attack the dividing cells. The other is to activate the expression of

the gene using a receptor. The third way is to target the cell by a liposome that are prepared to incorporate a specific proteins onto their surface, and combined with DNA. This liposome can recognize a specific cell to deliver the gene.

• Mice are developed to be used as a model for testing the gene therapy techniques. They have to be prepared in a way to generate genetically manipulated mice.

• There are large numbers of inherited human diseases are subjected for gene therapy. Most of them are caused by single gene defect and they are well defined. The candidate disease should have no effective conventional treatment e.g. sickle cell anemia, cystic fibrosis, Huntington’s disease, Duchenn’s muscular dystrophy and many others.

• Recently, gene therapy was used to treat other diseases such as lung diseases, e,g. cystic fibrosis, non heritable diseases such as HIV, and cancer diseases.

• Gene therapy could be considered as a future means of treating many genetic diseases and acquired diseases. Despite the great progress in the clinical trials but the practical reality is still out of reach. Some of the difficulties in gene therapy involves the discovery of new efficient vectors and expression of the transferred gene.

• Gene therapy raises some important ethical issues specially on the germline gene therapy.

15. REFERENCES

- Aldred, M.A., 1999. Gene therapy for eye cancer. Molecular Medicine Today 5 : 193.

- Anderson, W.F., Blease, R.M. and Culver K. 1990. The ADA human gene therapy clinical protocol; points to consider response with clinical protocols. Human Gene Therapy 1 (3 ) : 331-362.

- Anderson, W.F. 1992, Human gene therapy. Science 256 : 808-813.

- Aoki M. et al (1997). Survival of grafts of genetically modified cardiac myocytes transfected with FITC-labeled oligodeoxynucleotides and the b-galactosidase gene in the noninfarcted area, but not the myocardial infarcted area. Gene Ther., 4 : 120-127.

- Barinaga, P. (1994)“ Altering Human Genes: Social, Ethical, and Legal Implications” Perspective in Biology and Medicine 37 : 566-575.

- Brother, M. 1996 Block of tat – mediated transactivation of tumour necrosis factor beta gene expression by polymeric – TAR decoys. Virology 222 : 252-256.

- Caplen, N.J. 1998. TP 53 on trial for cancer gene therapy. Molecular Medicine Today 4 : 465.

- Caplen, N.J. 1998. Gene therapy: different strategies for different applications. Molecular Medicine Today 4 : 373-374.

- Coi,D.W., Mukhopadhyar, T. and Roth J.A. 1995. Suppression of lung cancer cell growth by ribozyme-mediated modifications of P53 pre-mRNA. Cancer Gene therapy 2 : 199-205.

- Chang, C.C. 1998. Cord blood stem cell transplantation. Clinician Reviews 8 (2): 67-70 proliferative disorders with a constitutively active form of the retinoblastoma gene product. Science, 267 : 518-522.

- Clayman G.L. et al 1998. Adenovirus – mediated p 53 gene transfer in patients with advanced recurrent head and neck squamous cell carcinoma. J. Clin, Onco. 16, 2221-2232.

- Cristiano RJ, Curiel DT: Strategies to accomplish gene delivery via the receptor-mediated endocytosis pathway. Cancer Gene Therapy 3 : 49-57, 1996.

- Culver, K. V., and Blaese, R.M. 1994, Gene therapy for cancer. Trends Genet, 10 : 174-178.

- Dachs, G.U. Dougherty, G.J. Stratford et al. 1997. Progress in gene therapy for cancer. Oncology Research g (6-7) 313-325.

- Dai Y, Schwarz, E.M., Gu, D., Zhang, W.W., Sarvetnick N., Verma, I.M. 1995, Cellular and humoral immune responses to adenoviral vectors containing factor IX gene : tolerization of factor IX and vector antigens allows for long – term expression Proc. Natl. Acad. Sci. USA 92 : 1401-1405.

- Davern, T.J. and Scharschmidt , B.F.1998. Gene therapy for liver disease. Digestive Diseases, 16 : 1 23-37.

- Drake, M.L., Mang Yu, 1997. Gene therapy for HIV / AIDS Readers : 7 (6) : 184-190.

- Drumm ML, Pope HA, Cliff WH, et al 1990: Correction of the cystic fibrosis defect in vitro by retrovirus – mediated gene transfer Cell 62 : 1227-1233.

- Dunaief, J.L. Kwan, R.C. Bhardway, N. Lopez, R., Gouras, P. and Goff, S.P. 1995. Retroviral gene transfer into retinal pigment epithelial cells followed by transplantation into retina. Human gene therapy 6 : 1225- 1229.

- During, J.M. and Ashenden L.A. 1998. Towards gene therapy for the central nervous system. Molecular Medicine Today 4 : 485-493.

- Edger H. and Tursz T..(1995) “ Report on Human Gene Therapy” Proceeding of International Bioethics committee of UNESCO, 1 : 29-48.

- Edger H. and Tursz T. (1996)“ Ethical considerations regarding access to experimental treatment and expermentation on human subject.”Proceeding of International Bioethics Committee of UNESCO 1 : 41-53.

- Ficazzola, M.A. and Taneja, S.S. 1998. Prospects for gene therapy in human prostate cancer, Molecular Medicine Today 4 : (11) : 494-504.

- Gloriosa, J.C., Deluca, N.A. and Fink, D.J., 1995. Development and application of herpes simplex virus vectors for human gene therapy. Annual Review of Microbiology; 49 : 675-710.

- Glorioso, J.C. Bender, M.A., Goins, W.F., Fink, D.J., and Deluca, N. 1995. HSV as a gene transfer vector for the nervous system. Molecular Biotechnology 4 : 87-99.

- Gilboa, E, Smith. C. 1994 Gene therapy for infectious diseases : The AIDS model. Trends Genet 10 : 139-144.

- Goldman MJ, Yang Y, Wilson JM: 1995 Gene therapy in a xenograft model of cystic fibrosis lung corrects chloride transport more effectively than the sodium defect. Nature Genetics 9 : 126-131.

- Goldman M, Su Q, Wilson JM: 1996 Gradient of RGD - dependent entry of adenoviral vector in nasal and intrapulmonary epithelia: implications for gene therapy of cystic fibrosis. Gene Therapy 3 : 811- 818.

- Gordon, J.W. 1998. Germline alteration by gene therapy: assessing and reducing the risks. Molecular medicine today, 4 : 468-470.

- Harris, J.D., and Lemoine, N.R. 1996. Strategies for targeted gene therapy. Trends Genet 12 : 400- 405.

- Hayashi H. et al 1999. An experimental application of gene therapy for human retinoblastema. Invest. Opthal mol.

- HoA, Lix, 1995. Stem cells as vehicles for gene therapy : Novel strategy for HIV infection. Stem cells 13 : 100-105.

- Ho, D., Neuman, A.U. Perelson, A.S. et al 1995. Rapid turnover of plasma virions and CD 4 lymphocytes in HIV infection. Nature 371 : 123-126.

- Ivanov, V. 1993 “Ethics and Human Genetics”, in council of Europe, “Second Symposium on Bioethics”, Stasbourg, 30/11-2/12.

- Jaffee, EM., Pardoll DM 1997. Considerations for the clinical Development of Cytokine. Gene – Transduced Tumor Cell Vaccines. Methods 12 (2) : 143-53.

- Johnson LG, Olsen JC, Sarkadi B, et al 1992: Efficiency of gene transfer for restoration of normal airway epithelial function in cystic fibrosis. Nature Genetics 2 : 21-25.

- Johnson LG, Boyles SE, Wilson J, et al 1995: Normalization of raised sodium absorption and raised calcium – mediated chloride secretion by adenovirus-mediated expression of cystic fibrosis transmembrane conductance regulator in primary expression of cystic fibrosis transmembrane conductance regulator in primary human cystic fibrosis airway epithelial cell. J Clin Invest 95 : 1377-1382.

- Jones , D.R. and Bui, T.H.1998. Fetal therapy : prospects for transplantation early in pregnancy. Molecular Medicine Today 4 : 10-11.

- Kaplitt, M.G., Darachiev, B. and During, M.J. 1998, Prospects for gene therapy in pediatric neurosurgery Pediatric Neruosurgery, 28 (1) : 3-14.

- Karpati, G., Gilbert, R., Petrof, B.J., Nalbantoglu, 1997. Gene therapy research for Duchenne and Becker muscular dystrophies. Current opinion in Neurology, 10 : 430-435.

- Kasahara N, Dozy AM and Kahn YW 1994 tissue specific targeting of retroviral vectors through ligand-receptor interactions. Science 255 : 1373-1376.

- Kaplitt, M.G., Darakchiev, B. and During M.J, 1998. Prospects for gene therapy in pediatric neurosurgery. Pediatric Neurosurgery, 28 : (1) : 3-14.

- Kay, M.A., and Woo, S.L.C. 1994. Gene therapy for metabolic disorders. Trends Genet. 10 : 253-257.

- Kerr W.G. 1998. Genetic modification of the hematolymphoid compartment for therapeutic prposes.

- Klug, W. and Cummings, M.R. 1997. Concepts of genetics 5th ed. Pub. Prentice Hall.

- Kotin, R.M. 1994. Prospects for the use of adeno – associated virus as a vector for human gene therapy. Human gene therapy ; 5 : 793-801.

- La Face, D.M. and Mang Yu 1997. Gene therapy for HIV/AIDS : Where are we going . The AIDS Reader 7 (6) : 184-190.

- Lamarch, P., Ari, J.H. and Danel, C. et al 1992. Adenovirus mediated transfer of a recombinant human 1-antitrypsin cDNA to human endothelial cells, Proc Notl. Acad. Sci. USA 89 : 6482-6486.

- Lasic, D.D. 1998. Novel applications of liposomes. Trends in Biotechnol. 16 : 307-321.

- Ledley, F.D. 1995. Non-viral gene therapy: The promise of genes as pharmaceutical products. Human Gene Therapy 6 : 1129-1144.

- Levine, F. and Leibowitz, G. 1999. Towards gene therapy of diabetes mellitus. Molecular Medicine Today 5 : 165-171.

- Licht, T, Herrmann F, Gottesman, M.M. et al 1997. In vivo drug-selectable genes. A new concept in gene therapy. Stem cells 15 (2) : 104-111.

- Liu, J. Woffendin, C. Yang, Z. et al : 1994. Regulated expression of a dominant negative form of Rev improves resistance to HIV replication in T cells. Human Gene Therapy 1 : 32-37.

- Marcel T., Grausz JD: The TMC, 1997 worldwide gene therapy enrollment report, end 1996. Human Gene Therapy 8 : 775-800.

- Mendell, J.R. Kissel, J.T., Amato, A.A. et al 1995. Myoblast transfer in the treatment of Duchenn’s muscular dystrophy N. Engl. J. Med, 333 : 832-838.

- Mettal S.K., McDermott M.R., Johnson D.C. et al 1993. Monitoring foreign gene expression by human adenovirus based vector using the fire luciferase gene as a reporter. Virus Res. 28 : 67-90.

- Morgan, R.A. and Anderson, W.F. 1993. Human gene therapy. Annu Rev. Biochem-62 : 191-217.

- Mulligan, R.C. 1993. The basic science of gene therapy. Science 260 : 926-932.

- Morgan, R.A. and Anderson, W.F. 1993. Human gene therapy. Annu. Rev. Biochem, 62 : 191-217.

- Mulligan, R.C. 1993. The basic science of gene therapy. Science 260 : 96-932.

- Muzycka N: (1992). Use of adeno-associated virus as a general transduction vector for mammalian cells. Curr Microbiol and Immunol 158 : 97-128, 1992.

- Nikol,S. and Fling, B.H. 1998, Gene therapy for restenosis: Progress or frustration. J. Invas Cardiol 10 (8) : 506-514.

- Ohno, T. Gordon, D. San, H. et al 1994. Gene therapy for vascular smooth muscle cell proliferation after arterial injury. Science, 265 : 781-784.

- Overturf K. et al. 1998. Ex vivo hepatic gene therapy of a mouse model of hereditary tyrosinemia type I. Hum. Gene Ther., 9 : 295-304.

- Perales, J.C., Ferkol, T. Molas, M. and Hanson R.W. 1994. An evaluation of receptor-mediated gene transfer using synthetic DNA-ligand complexes. European Journal of Biochemistry 226 : 255-266.

- Poeschla, E. 1995-1996. Gene therapy and HIV disease. AIDS clinical reviews : 1-45.

- Paulus, W. Baur, I. Boyce, F.M. Breakefield, and Reeves, S.A. 1996. Self-contained, tetracycline- regulated retroviral vector system for gene delivery to mammalian cells. Journal of Virology 70 : 62-67.

- Prazeres, D.M.F., Ferreira, G.N.M., Monteiro G.A. et al 1999. Large scale production of pharmaceutical- grade plasmid DNA for gene therapy: problems and bottlenecks. Trends Biotechnol. 17 ( 4 ) : 169-174.

- Reynolds, P.N., Feng, M. and T. David. 1994. Chimeric viral vectors the best of both worlds. Molecular Medicine Today 5 : 25-31.

- Rich DP, Anderson MP, Gregory RJ, et al 1990: Expression of cystic fibrosis transmembrane conductance regulator corrects defective chloride channel regulation in cystic fibrosis airway epithelial cells. Nature 347 : 358-363.

- Riordan JR, Rommens JM, Kerem B et al1989: Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245 (4922) : 1066-73.

- Robbins, P.D., Tahara, H. and Ghivizzani S. 1998. Viral vectors for gene therapy. Trends in Biotechnol. 16: 35 -40.

- Rock, K. L., Gramm, C., Rothstein, L. et al 1994. Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class 1 molecules. Cell 78 : 761-771.

- Rommens JM, Lannuzzi MC, Kerem B, et al 1989: Identification of cystic fibrosis gene: chromosome walking and jumping. Science 245 (4922) : 1059-1065.

- Rosenfeld, M.A., Siegfried, Yoshimura K et al 1990. Adenovirus mediated transfer of a recombinant a-antitrypsin gene to the lung epithelium in vivo. Science, 252 : 431-434.

- Roth, J.A., Cristiano, R.J. 1997. Gene therapy for cancer: What have we done and where are going? Journal of National canc. Inst. 89 (1) : 21- 39.

- Roth, J.A. et al 1998. Gene therapy for non-small cell lung cancer: a preliminary report of a phase 1 trial of adenoviral p. 53 gene replacement. Semin. Onco. 25, 33-37.

- Salmons, B. and Gunzburg WH. 1993. Targeting of retroviral vectors for gene therapy. Hum. Gene Ther. 4 : 129-141.

- Schiedner G. et al 1998. Genomic DNA transfer with a high-capacity adenovirus vector results in improved in vivo gene expression and decreased toxicity. Nat. Genet., 18 : 180-183.

- Senior, K. 1998. In utero gene therapy: one step close to reality. Molecular Medicine Today 4 : 505.

- Senut, M.C. and Gage, F.H. 1999. Prenatal gene therapy: can the technical hurdles be overcome? Molecular Medicine Today 5 : 152-156.

- Simons, K. H. Garoff, and A. Helenius, 1982. How an animal virus gets into and out of its host cell. Sci. Am. Feb : 58.

- Snowden, M.M. and Grove, R.I. 1998. Gene therapy for cardiovascular diseases. Expert Opinion on Therapeutic Patents, 8 : 509-520.

- Stratford – Perricaudet L.D., Makeh I, Perri caudet M. et al 1992. Widespread long term gene transfer to mouse skeletal muscles and heart J. Clin. Invest. 90 : 626-630.

- Tanswell, A.K. and O Brodovich, 1997. The present and future role of gene therapy in new born.

- Tsukui T. et al 1996. Transgenesis by adenovirus – mediated gene transfer into mouse zone – free eggs. Nat. Biotechnol., 14 : 982-985.

- Varmus, H.E. 1982. Form and function of retroviral proviruses. Science 216 : 812.

- Varmus, H.E. 1988. Retrovirus Science 240 : 1427.

- Vile, R.G. 1996. Gene therapy for treating cancer: hope or hype? Chemistry and industry, 8 : 285-289.

- Ulmer, J.B., Dommelly, J.J., Parker, S.E. et al 1993. Heterologous protection against influenza by injection of DNA encoding a viral protein. Science, 259 : 781-784.

- United State President’s Commission for The Study of Ethical Problems in Medicine and Biomedical Behavioral Research “ Splicing Life” (1982).

- Wagner E., Zatloukal K, Cotton M, Kirlappos H, Mechtler K, Curied D T and Birnstiel ML. 1992. Coupling of adenovirus to transferrin-polylysine /DNA complexes greatly enhances receptor – mediated gene delivery and expression of transfected genes. Proc. Natl. Acad. Sci. USA 89 : 6099-6103.

- Wagner, J.A. and Gardner, P. 1997. Toward cystic fibrosis gene therapy. Annu. Rev. Med. 48 : 203-216.

- Walters L. (1991) “Human Gene Therapy : Ethics and Public Policy” Human Gene Therapy 2 : 115-122

- Welch, P. Hampel A. , Barber J. et al 1996. Inhibition of HIV replication by the hairpin ribozyme. In Eckstein, F., Lilley (eds) : Nucleic acids and molecular biology vol 10 Berlin Heidlberg, Springer – Verlag, pp 315-327.

- Wei, X, Ghosh, S.K., Taylor, M.E. et al, 1995. Viral dynamics in human immunodeficiency virus type –1 infection. Nature 371 : 117- 122.

- Welsh , M. J. and Ostedgoard, L.S. 1998. Cystic fibrosis problem probed by proteolysis. Nature Structural Biology 5 : 167-169.

- Wich MS. 1998. The prospects for development of gene therapy for treatment and prevention of breast cancer 21st Annual San Antonio Breast Cancer Symposium, San Antonio, TX.

- Wilson, J. M. 1996. Adenoviruses as gene – delivery vehicles. N Engl. J. Med. 334 : 1185-1187.

- Wilson JM 1995: Gene therapy for cystic fobrosis: challenges and future directions. J Clin Invest 96 : 2547-2554.

- Woffendin, C., Yang, Z. Y, Udaydumar, Xul et al : 1994. Nonviral and viral delivery of human immunodeficiency virus protective gene in primary human Tcells. Proc. Natl Acad. Sci. USA 91 : 11581-11585.

- Wolf, J. H. 1994. Recent progress in gene therapy for inherited diseases. Current opinions in Pediatrics; 6 : 213-218.

- Wu, J. Wu, G.Y. and Zern, M.A. 1998. The prospects of hepatic drug delivery and gene therapy. Expert Opinion on Investigational Drugs, 7: 1795-1817.

- Yang Y, Ertl, H.C., Wilson J.M. 1994, MHC class 1-restricted cytotoxic T lymphocytes to viral antigens destroy hypatocytes in mice infected with E1 – deleted recombinant adenoviruses. Immunity; 1 : 433 -442.

- Yang, Y., Nunes, F.A., Berencsi, K., Gonczol, E., Engelhardt, J. F., Wilson, J.M. 1994. Inactivation of E2a in recombinant adenoviruses improves the prospect of gene therapy in cystic fibrosis. Nat Genet, 7 : 362-369.

- Yu, M, Ojwang, J. Yamada, O. et al 1993. A hairpin ribozyme inhibits expression of diverse strains of human immunodeficiency virus type 1. Proc. Natl. Acad. Sci. USA 90 : 6340-6344.