Treatment of Genetic Diseases and Human Gene Therapy

TREATMENT OF GENETIC DISEASES

Genetic diseases occur due to deficiency of an enzyme or protein. A major motivation for gene therapy has been the need to develop novel treatments for diseases for which there is no effective conventional treatment. The goal of treatment is to modify the phenotype. Methods of treatment include:

Replacement of Deficient Enzyme/Protein

Examples of replacement of deficient enzyme/protein include treatment of SCID with blood transfusions, treatment of mucopolysaccharidoses with bone marrow transplants, treatment of Hemophilia A with cryoprecipitate/Factor VIII, treatment of Gaucher’s disease with a-glucosidase, replacement of vitamin B₆ in homocystinuria, replacement of vitamin B-2 in methylmalonicacidemia, replacement of vitamin D in vitamin D resistant rickets, thyroxine in congenital hypothyroidism and cortisone in congenital adrenal hyperplasia.

Restriction of Toxic Substrates

Examples of restriction of toxic substrates include reduction of galactose in galactosaemia, reduction of cholesterol in familial hypercholesterolemia, restriction of phenylalanine in PKU, and reduction of protein in urea cycle disorders.

Therapy of Genetic Disorders with Drugs

Therapy of genetic disorders with drugs includes treatment of Wilson’s disease with penicillamine and treatment of malignant hyperthermia with dantrolene.

Surgical Approaches Such as Removal of Tissues or Organ Replacement

Removal of tissues includes colectomy in polyposis coli and organ replacement includes kidney transplants in adult polycystic kidney disease.

The term gene therapy describes any procedure intended to treat or alleviate disease by genetically modifying the genetic material of living cells to fight disease. One of the goals of gene therapy is to supply cells with copies of missing or altered genes, in an attempt to correct the disorder by altering the genetic makeup of some of the patient’s cells. Gene therapy can also be used to change how a cell functions, for example by stimulating the immune system cells to attack cancer cells, or by introducing resistance to human HIV

Cell Therapy

Cell therapy has emerged as a treatment for many diseases, and involves placement of characterized cells or embryonic stem cells in a target organ in sufficient numbers to restore the function of damaged tissue or organs. Differentiated cells may be replaced by regenerated cells or cycling stem cells, and these include hepatocytes, skeletal muscle and endothelial cells. The donor cell may be genetically engineered to synthesize and to secrete a missing entity. Examples of cell therapy also include the use of pancreatic cells and delivery of factor VIII cells engineered to secrete neurotropic factors.

The principle sources of hematopoietic stem cells for clinical transplantation include bone marrow. More recently, umbilical cord blood has been used as an alternative source of hematopoietic support. Bone marrow transplantation involves the replacement of enzymatically deficient cells with enzymatically normal cells. Allogenic bone marrow transplantation has been used to treat blood dyscrasias, hematological malignancies, and immunodeficiency states. Transplantation of haematopoietic stem cells alters course of some lysosomal and peroxisomal disorders. Hematopoietic stem-cell transplantation using bone marrow or umbilical cord blood has been the only effective long-term treatment for Hurler syndrome.

Somatic Gene Therapy

The range of disorders that might be considered amenable to this type of therapy has expanded from single-gene disorders to include cancer, AIDS, other infectious diseases, and atherosclerosis. In addition recombinant protein therapies with insulin, erythropoietin, or clotting factor could be converted for in vivo production via somatic gene therapy. During the past years than 300 clinical protocols and over 3000 patients have been subjected to somatic gene therapy. There are three approaches to somatic cell gene therapy: (1) ex vivo, where cells are removed from the body and incubated with a vector, and the gene-engineered cells are then returned to the body; (2) in situ, where the vector is placed directly into the affected tissues; and (3) in vivo, where a vector would be injected directly into the bloodstream.

Gene Therapy Strategies

For somatic gene therapy, at least three strategies for regulation of expression of the therapeutic DNA can be distinguished.

(1) A cDNA under the control of a foreign promoter can be utilized so that the product is synthesized at high levels, but without normal regulation. (2) Alternatively, genomic DNA including the sequences necessary for proper regulation of the level and tissue specificity of expression of the therapeutic gene can be used. (3) Lastly, artificial minigenes that link genomic regulatory regions with cDNA encoding the entire open reading frame, provide constructs that are of manageable size and are properly regulated. All of these strategies would typically involve random insertion of DNA sequences into the genome of the recipient. An alternative strategy would be to use site-specific recombination so that the region of a gene containing the mutation would be replaced by the normal DNA sequence. The choice of strategy will vary depending on the expression requirements of the disorder to be treated. In the case of enzyme or other protein deficiencies, where a modest increment in function may result in much improved homeostasis, any of the three nonhomologous approaches might suffice. In the case of haemoglobin disorders, the relative accessibility of bone marrow stem cells and the advantages of maintaining all the normal regulatory mechanisms make homologous recombination an extremely attractive goal. Several single­gene disorders are now candidates for gene therapy and a number of phase I clinical trials are in process. Life threatening, recessive diseases involving marrow-derived cells (e.g., adenosine deaminase deficiency, chronic granulomatous disease, and leukocyte adhesion deficiency) are considered the preferred targets for somatic gene therapy, as are disorders in which extracellular products such a hormones, clotting factors, or other serum proteins might be produced by transfected cells.

The strategies for gene therapy

1. Gene augmentation therapy: Addition of functional alleles used to treat inherited disorders caused by genetic deficiency of a gene product
2. Targeted killing of specific cells: Using genes encoding toxic compounds (suicide genes) or prodrugs (reagents which confer sensitivity to subsequent treatment with a drug)
3. Targeted inhibition of gene expression: In treatment of infectious disease
4. Targeted mutation correction: Using homologous recombination, antisense oligonucleotides and TFOs (triplex forming oligonucleotides)

As seen above, the genetic material may be transfused directly into cells within a patient (in vivo gene therapy) or cells may be removed from the patient and the genetic material inserted into the cells in vitro, and the modified cells transplanted back into the patient (ex vivo gene therapy). Ex vivo gene transfer involves the transfer of cloned genes into cells grown in culture. The cells that have been transformed are selected, expanded in cell culture in vitro and then introduced into the patient. In in vivo gene transfer, cloned genes are transferred into the tissues of the patient, and liposomes and viral vectors are used for this purpose.

Principles of gene transfer

Classical gene therapy required efficient transfer of cloned genes into disease cells so that introduced genes are expressed at suitably high levels. The sizes of the DNA fragment to be transferred are limited, and therefore an artificial minigene may be used, which a cDNA sequence is containing the complete coding DNA sequence, flanked by appropriate regulatory sequences to ensure a high level of expression.

Following gene transfer, the inserted genes may integrate into the chromosomes of the cell or remain as extra chromosomal genetic elements (episomes). Gene integration into chromosomes allows perpetuation by chromosomal replication following cell division. As the progeny cells contain the introduced genes, long-term stable expression may be obtained. Stem cells are an immortal population of undifferentiated precursor cells, which give rise to mature differentiated cells and are very efficient cells to target. Because normally insertion occurs randomly, the disadvantages of chromosomal integration may be that the location of the inserted genes may result in death of the host cell due to insertion into and inactivation of a gene. Aletrnatively, inserted genes may not be expressed due to integration into a highly condensed heterochromatic region, or an inserted gene may cause activation of an oncogene or inactivate a tumor suppressor gene and cause cancer.

Methods of gene delivery

In order to modify a specific cell type or tissue, the therapeutic gene must be efficiently delivered to the cell, in such a way that the gene can be expressed at the appropriate level and for a sufficient duration. The gene replacement used in gene therapy is delivered to the cell using a carrier vector. Two broad approaches have been used to deliver DNA to cells, namely viral vectors and non-viral vectors, which have different advantages as regards efficiency, ease of production and safety. The repertoire of delivery systems, which began with retroviral vectors, has expanded to include vectors based on adenovirus, adeno-associated virus, herpes virus, vaccinia, and other agents, and nonviral systems such as liposomes, DNA-protein conjugates, and DNA-protein-defective virus conjugates.

Viral Vectors Used for Gene Therapy

Viruses are obligate intracellular parasites, designed through the course of evolution to infect cells, often with great specificity to a particular cell type. They tend to be very efficient at transfecting their own DNA into the host cell, which is expressed to produce new viral particles. By replacing genes that are needed for the replication phase of their life cycle (the non-essential genes) with foreign genes of interest, the recombinant viral vectors can transduce the cell type it would normally infect. To produce such recombinant viral vectors the non-essential genes are provided in trans, either integrated into the genome of the packaging cell line or on a plasmid. As viruses have evolved as parasites, they all elicit a host immune system response to some extent. Mammalian virus vectors have been the preferred vehicles for gene transfer because of their high efficiency of transduction into human cells. Introduced viruses can recombine with endogenous retroviruses resulting in recombinant progeny that can undergo productive infection. Adenoviruses need repeated infections as they are non-integrating and repeated infections may provoke severe inflammatory responses (this was seen in gens therapy trials for cystic fibrosis). The most common type of vectors used in gene therapy are viruses. These viruses are genetically disabled and unable to reproduce. Mammalian virus vectors have been the preferred vehicles for gene transfer because of their high efficiency of transduction into human cells. A number of viruses have been developed, and these include retroviruses, adenoviruses, adenoassociated viruses and herpes simplex virus.

The Retroviral Systems

Retroviruses contain RNA as their genetic material instead of DNA. Retroviruses produce reverse transcriptase, which transforms their DNA into RNA. The first step in development of replication defective retroviruses involves replacement of gag gene (for group specific antigen), pol gene (encodes reverse transcriptase), and env gene (encodes the envelop protein) with the gene of interest. This replication-defective recombinant viral vector is transfected into a packaging cell line. Following injection, retroviruses deliver a nucleoprotein (preintegration) complex into the cytoplasm of infected cells. This complex reverse transcribes the viral genome and then integrates the resulting DNA copy into a single site in the host cell chromosomes. In absence of viral genes, the recombinant DNA or therapeutic gene is transcribed by using viral LTRs or in some cases, it is under control of the internal promoter and protein of interest is synthesized. Retroviral vectors efficiently integrate at random sites in the genome of dividing cells, permanently altering the recipient. Typically one or a few integrated copies of the recombinant vectors are found in each transduced cell. Retrovirus has a broad range of infectivity to different types of cells preferably mitotic cells. The major advantage of using retrovirus vectors is that one can determine the number of copies of gene per host cell. Retroviral vectors have several disadvantages: first, they require dividing cells as a target; second, they are difficult to produce at titers high enough for most in vivo approaches; and, third, depending on its location, retroviral integration may adversely alter the expression of a gene in the area (e.g., a proto-oncogene) and produce a transformed cellular phenotype. Currently, about 60% of approved clinical protocols for somatic gene therapy use retroviral vectors.

The Adenoviral Systems

Adenoviruses have been extensively studied, especially the Ad2 and Ad5 serotypes. The protein encoded by E1 gens is very important for viral replication. DNA up to 3.2 kb in size can replace the E1 gene to produce a replication defective recombinant vector. DNA up to 7.5 kb in size can be inserted into the genome of the adenovirus by deleting other non­essential genes. This virus has a natural tropism for respiratory epithelium. Therefore, this became a model vector for developing gene therapy in respiratory disorders. In contrast to retroviruses, adenoviruses can infect mitotic as well as post­mitotic cells. Adenovirus vectors, in contrast to retroviral vectors, offer a high titre and a better ability to infect large numbers of cells in vivo, but there is a concern about toxic effects on infected cells. In addition, the therapeutic effect is transient, with expression for only days or weeks.

The Adeno-associated Virus (AAV) Systems

This is a non-pathogenic human parvovirus and requires a helper virus for viral infection. In the absence of helper virus (adenovirus, cytomegalovirus, herpes virus), the virus integrates into human genome at specific site, 19q13.3-qter. The therapeutic gene is cloned between the two inverted terminal repeats. This recombinant plasmid is transfected along with another plasmid, which expressed viral structural proteins into the cells infected with a helper virus. virus have the potential to provide high titre, safety, and long-term expression. It is believed that the recombinant virus persists as an episome in these cells, reducing the risk of malignant transformation.

The Vaccinia Systems

The vaccinia virus is a double stranded DNA poxvirus. It infects vertebrates as well as some non-vertebrate cells. It replicates in the cytoplasm of cells. Because the vaccinia virus genome is very large, transfer vectors are constructed which contain the vaccinia virus DNA flanking the gene of interest and a selectable marker such as thymidine kinase (TK). Recombinant vaccinia virus is produced by recombination between transfer vector DNA and vaccinia DNA introduced into the cell by infection. Resulting viral rDNA can be selected based on their TK^- phenotypes. The major limitation of this viral system is that vaccinia vectors provide only transient expression, since they do not provide for DNA integration. Transient expression may be applicable in eliciting an altered immune response to malignant cells or treatment of an acute disease process.

NONVIRAL SYSTEMS FOR GENE THERAPY

Liposomes

Liposomes are spherical vesicles composed of lipid bilayers, which mimic the synthetic structure of biological membranes. The DNA lipid complexes are easy to prepare and there is no limit to the size of DNA that is transfected. However, the efficiency of gene transfer is low and the introduced DNA does not integrate into chromosomal DNA resulting in transient expression of the inserted genes.

Direct Injection

An example of this is intramuscular injection of a dystrophin minigene into a mouse model of DMD, mdx. There is a poor efficiency of gene transfer and a low level of stable integration of injected DNA.

Particle Bombardment (Gene Gun) Techniques

A micro projectile gene gun used to shoot DNA coated micro projectiles (tungsten or golden particles which are inert) into cells. The gun propels DNA-coated particles through the cell wall due to velocity and gets accommodated into the nucleus of the cells.

Receptor Mediated Endocytosis

The DNA is coupled to a targeting molecule that can bind to a specific cell surface receptor inducing endocytosis and transfer of DNA into cells. Coupling is achieved by covalently linking polylysine to the receptor molecule and then arranging for reversible binding of the negatively charged DNA to the positively charged polylysine component. A more generalised approach utilizes the transferring receptor, which is expressed in many cell types but is relatively enriched in proliferating cells and hematopoietic cells. This method has high gens transfer efficiency but does not allow integration of transformed genes. The protein-DNA complexes are not stable in serum and the DNA conjugates may be entrapped in endosomes and degraded in the lysosomes.

Targeted inhibition of gene expression in vivo

Selective inhibition of expression of a gene in vivo, without interfering with normal cell function is the approach for treating cancer, infectious disease, and some immunological disorders. Methods of blocking gene expression without mutating it can be accomplished at three levels.

1. At the DNA level by blocking transcription: Targeted inhibition of expression at the DNA level can be achieved using triple helix methods. A gene specific oligonucleotide is designed that will base pair with a defined double stranded DNA sequence of a target gene to inhibit transcription.
2. At the RNA level by blocking mRNA ribosome attachment or mRNA attachment: This includes antisense methods, which involve binding of gene specific oligonucleotides or polynucleotides to RNA.
3. At the protein level by blocking post-translational processing includes use of intracellular antibodies and oligonucleotides designed to bind and inactivate a selected protein.

Gene modification

Gene modification involves correction of the defective gene without introducing new gene into the cells so that it will function normally. There are various ways to modify defective gene expression. These include Gene correction, in which only the defective portion of mutant gene is altered so that it will start functioning normally; Gene replacement, in which the mutant sequence of a gene is removed from the host genome and replaced with a normal functional gene, and Gene augmentation, in which introduction of a normal genetic sequence into host genome modifies the expression of mutant gene, and the defective host gene remains unaltered.

Gene therapy for mitochondrial disorders

Several methods are under investigation to develop effective carrier system for mitochondrial diseases. Some of these include bombardment of DNA-coated tungsten particles to the whole cells to insert the therapeutic gene into mitochondrial DNA, electroporation of exogenous plasmid DNA up to 7.2kb into the matrix compartment of mitochondria, and delivery of DNA by peptide targeting, which involves tagging a therapeutic gene with nuclear coded proteins, which are naturally imported into mitochondria. This technique of hijacking protein pathway has proved successful in vitro. Effective dissociation of the tagged DNA from proteins with the help of mitochondrial processing peptidase after entry into mitochondria is very essential without disturbing conformation of DNA. Other methods include introduction of engineered mitochondria into the cells using endocytosis, or receptor mediated therapy.

ANIMAL DISEASE MODELS FOR GENE THERAPY

An animal disease model is used for many human diseases to check the efficacy and safety of gene therapy. Several natural disease models have been found in nature, or have been generated by random mutagenesis, which does not take place at a predetermined locus. Some of these animal models include the Watanbe heritable hyperlipidaemic (WHHL) rabbit, which has a deletion of four codons of the LDL-receptor gene and as a result is hyperlipidemic and a model for human familial hypercholesterolemia, the mdx mouse which has mutations in the dystrophin gene and is a model for Duchenne muscular dystrophy, the Gunn rat, which shows deficiency of gene for enzyme UDP-glucuronyl transferase, the enzyme being absent in hereditary Crigler Najjar Syndrome resulting in increased bilirubin levels, the NOD mouse, which is diabetic and a model for human insulin dependant diabetes mellitus, and the hemophiliac dog, which has a missense mutation in the factor IX gene, and is a model for human hemophilia B. Animal models of disease can also be generated by genetic manipulation by insertion of foreign DNA. These models can be used to study gene function, and to create animal models for human diseases. In order to create such genetically modified animals, the DNA of germline cells is modified. This DNA is heritable and therefore certain cells that have the capacity to differentiate into different cell types seen in the adult are considered optimal targets for introduction of foreign DNA. Such cells include the fertilized oocyte or embryonic stem cells, which are capable of giving rise to both somatic, and germline cells. When a foreign DNA molecule is artificially introduced into the cells of an animal, the animal is called a transgenic animal and the inserted DNA the transgene. In gene targeting, the mutation is introduced into a preselected endogenous gene within an intact cell. The mutation may result in inactivation of gene expression, termed a knockout mutation, or altered gene expression, and is useful for studying gene function. Transgenic animals have been used to analyse human genes by investigating gene expression and its regulation, by investigating gene function by targeted gene inactivation, by investigating gene function, and by investigating dosage effects and ectopic expression. Examples of transgenic or gene-targeted mouse models of human disease include models for cystic fibrosis, P-thalassaemia, hypercholesterolemia, Gaucher’s disease, and fragile X-syndrome all produced by insertional inactivation by gene targeting. The ability to produce transgenic mice and gene targeting has permitted the design of many new animal models of disease. Another approach is genetic manipulation of animals using somatic cell nuclear transfer into an enucleated oocyte. In 1997, this approach allowed cloning of an adult mammal, a sheep called “Dolly”. The successful cloning of an adult animal has major implications for research, medicine and society.

Gene therapy for inherited disorders

Recessively inherited disorders, where disease results from a simple deficiency of a specific gene product can be treated by high-level expression of introduced normal alleles.

Examples of gene therapy for inherited disorders include:

1. Alteration of T-cells and hematopoietic stem cells in ADA deficiency. The gene therapy involves an ex vivo strategy using recombinant retroviruses containing the ADA gene.
2. Alteration of liver cells in familial hypercholesterolemia using an ex vivo strategy using retrovirus to deliver the LDL receptor gene.
3. In cystic fibrosis, the cells altered are the respiratory epithelial cells using an in vivo strategy using recombinant adenovirus or liposomes to deliver the CFTR gene.
4. Alteration of hematopoietic stem cells using an ex vivo strategy with retroviruses delivering the GBA gene in Gaucher’s disease due to glucocerebrosidase deficiency.

An example of an inherited disorder for which gene therapy has proved successful

Severe Combined Immunodeficiency (SCID)

This disease is mainly caused by deficiency of housekeeping gene adenosine deaminase enzyme (ADA) that is mainly produced by T-lymphocytes. If ADA is not present in the body, enzyme kinase converts one of the metabolic by-products into a toxin, which destroys the T-lymphocytes. T-lymphocytes are important to the body’s immune systems. They not only directly participate in immune responses, but controls activity of B-lymphocytes, cells that produce antibodies. Thus, deficiency of ADA will affect body’s immune system. In 1990, the National Institute of Health (NIH) received the first approval for gene therapy testing for SCID disease. Here researchers isolated lymphocytes from the patient and exposed them to recombinant retroviruses carrying genes for ADA production. These engineered lymphocytes were then replaced into the patient where they started secreting ADA enzyme. The first patients of SCID reported to have been benefited from successful gene therapy in 1990 and 1991 were age 4 and age 9. The children are progressing well and essentially leading a healthy life.

GENE THERAPY FOR INFECTIOUS DISEASE

Gene therapy for infectious disorders involves strategies like provoking a specific immune response or specific killing of infected cells by insertion of a gene encoding a toxin or prodrug. Current gene therapy trials for infectious disease are aimed at treating patients with AIDS. The gene therapy strategies interfere with the HIV virus life cycle at three levels. By blocking HIV-1 infection, by inhibition at the RNA level using antisense, ribozymes/approaches or inhibition at the protein level involves designing intracellular antibodies against HIV proteins such as the envelope proteins. The main target for gene therapy is the expression of genes that can interfere with virus replication in CD4+ T cells or stem cells. Potential therapeutic genes which will be either antisense version of the HIV tar gene or a mutated HIV rev gene that blocks the transport of HIV RNA from the nucleus and marker genes are introduced into CD4+ T cells. These recombinant cells will be cultured and can be given to HIV positive persons. Other strategies include provoking an immune response against the HIV virus by transferring a gene that encodes an HIV-1 antigen such as the envelope protein gp120 and expressing it in a patient, or boosting the patients’ immune system by transfer and expression of a gene encoding a cytokine such as an interferon using retroviral mediated methods.

GENE THERAPY FOR CANCER

Cancer therapy includes targeted killing of disease cells by inducing genes that encode toxins or by provoking enhanced immune responses. Some approaches focus on targeting single genes such as Tp53 gene augmentation therapy or antisense K-ras genes in lung cancer, where the translation of the mutant gene mRNA to the final oncoprotein product which is responsible for involuntary cell growth is prevented. In case of lung cancer, this strategy has been used against K-ras genes. In B-cell lymphomas, bcl-2 is over expressed as a result of translocation of bcl-2 gene to the immunoglobulin heavy-chain locus. This can be also hybridised with an antisense oligonucleotide, which is complementary to the bcl-2 gene.

Potential applications of gene therapy for the treatment of cancer include approaches like artificial killing of cancer cells using genes encoding toxins or conferring drug sensitivity for example by insertion of the multiple drug resistance gene (MDR1). Another approach is insertion of genes encoding foreign antigens or cytokines to enhance immunogenicity of the tumor or increase anti-tumor activity of immune cells, induce normal tissues to produce anti-tumor substances like interleukin-2 or interferon and production of recombinant vaccines for prevention and treatment of malignancy. This approach involves the use of tumor infiltrating lymphocytes (TIL) as a vehicle to carry recombinant viruses containing human cytokine genes by infecting lymphocytes with a recombinant virus. These tumor-infiltrating lymphocytes attack cancerous cells and produce cytokines causing lysis of tumor cells. It is possible to enhance TIL response by transfecting recombinant viruses carrying genes such as interleukins (IL) 2,3,4,6,7 and tumor necrosis factor (TNF) whose product induces strong immunogenic response against tumor cells. For tumors arising from oncogene activation, inhibition of expression of oncogenes can be carried out using antisense oligonucleotides or triple helix oligonucleotides or intracellular antibodies can be used to bind to the oncoprotein. For tumors arising from inactivation of tumor suppressors, gene augmentation therapy can be used. Another strategy used in cancer therapy is suicide vector gene therapy. Here suicide viral vector has been used to infect the tumor cells. This recombinant viral vector contains suicide gene, which encodes an enzyme that converts non-toxic prodrug into cytotoxic product, which regresses tumor mass. Cells that get transfected with these suicide genes commit metabolic suicide. For example, herpes simplex virus thymidine kinase (HSVtk), cytosine deaminase and varicella zoster virus thymidine kinase are a few examples of suicide genes.

Examples of cancer gene therapy trials

1- Alteration of tumor cells ex vivo or in vivo to deliver the HSV-tk gene in brain tumors

2- Use of fibroblasts ex vivo and retroviruses to deliver the MDR1 (multiple drug resistance gene) gene in breast cancer. Retroviruses to deliver the MDR1 gene have also been used to alter tumor cells in vivo in colorectal cancer.

3- Use of retroviruses deliver IL2 genes to fibroblasts ex vivo or tumor cells in vivo in malignant melanoma

4- Use of retroviruses to deliver the IL4 gene to tumor cells or T cells ex vivo in myelogenous leukemia, or fibroblasts ex vivo in small cell lung cancer

5- Use of retroviruses to deliver the TNFA gene to tumor cells for the treatment of neuroblastoma

References

Purandarey , H. (2009) . Essentials of Human Genetics. Second Edition. Jaypee Brothers Medical Publishers (P) Ltd.