King Saud University College of Pharmacy Departments of

King Saud University College of Pharmacy Departments of Pharmaceutics/ Pharmacognosy PHG 424: Pharmaceutical Biotechnology Gene Therapy A new revolution towards best patient well-being Ibrahim A. Alsarra, Ph. D. Professor of Pharmaceutical Biotechnology

Outlines Terminology Introduction Historical Background Potential Target Diseases for Gene Therapy Vectors in Gene Therapy Problems and Ethics Concluding Remarks

Terminology

Allele (Pronounced al-eel) is any one of a number of viable DNA codings occupying a given locus (position) on a chromosome. Usually alleles are DNA sequences that code for a gene, but sometimes the term is used to refer to a non-gene sequence. An individual's genotype for that gene is the set of alleles it happens to possess.

Antisense therapy A form of treatment for genetic disorders or infections. When the genetic sequence of a particular gene is known to be causative of a particular disease, it is possible to synthesize a strand of nucleic acid (DNA, RNA or a chemical analogue) that will bind to the messenger RNA (m. RNA) produced by that gene and inactivate it, effectively turning that gene "off". This synthesized nucleic acid is termed an "antisense" oligonucleotide because its base sequence is complementary to the gene's messenger RNA (m. RNA), which is called the "sense" sequence (so that a sense segment of m. RNA " 5'-AAGGUC-3' " would be blocked by the anti-sense m. RNA segment: " 3'-UUCCAG-5' ").

Antisense therapy…cont. Antisense drugs are being researched to treat lung cancer, diabetes and diseases such as asthma and arthritis with an inflammatory component. Most potential therapies have not yet produced significant clinical results, though one antisense drug, fomivirsen (marketed as Vitravene), has been approved by the US Food and Drug Administration (FDA) as a treatment for cytomegalovirus retinitis.

Fomivirsen (brand name Vitravene) is an antiviral drug. It is used in the treatment of cytomegalovirus retinitis (CMV) in immunocompromised patients, including those with AIDS. It is synthetic 21 member oligonucleotide with phosphorothioate linkages (which are resistant to degradation by nucleases). It is an oligonucleotide that blocks translation of viral m. RNA by binding to a coding segment of a key CMV gene. It was the first antisense antiviral approved by the FDA. It is available as an intraocular injection in a concentration of 6. 6 mg/m. L.

Ex vivo (Latin: out of the living) means that which takes place outside an organism. In science, ex vivo refers to experimentation done in or on living tissue in an artificial environment outside the organism. The most common "ex vivo" procedures involve living cells or tissues taken from an organism and cultured in a laboratory apparatus, usually under sterile conditions for a few days or weeks. Ex vivo studies are usually performed in vitro, although the use of these two words is not synonymous.

Germline In biology and genetics, the germline of a mature or developing individual is the line (sequence) of germ cells that have genetic material that may be passed to a child. For example, sex cells, such as the sperm or the egg, are part of the germline. So are the cells that produce sex cells, called gametocytes, the cells that produce those, called gametogonia. Cells that are not in the germline are called somatic cells. Such are, for example, all cells of the liver. If there is a mutation or other genetic change in the germline, it will be/can be passed to offspring, but for a change in a somatic cell it will not be.

Germline…cont. Germline cells also have the distinction of being immortal, i. e. , they are supported by a special enzyme called telomerase. This enzyme is dedicated to lengthening the DNA primer of the chromosome, allowing for unending duplication. Somatic cells, by comparison, can only divide around 30 -50 times, as they do not contain telomerases.

Somatic cell Is generally taken to mean any cell forming the body of an organism: the word "somatic" is derived from the Greek word sōma, meaning "body". In mammals, germline cells are the sperm and ova (also known as "gametes") which fuse during fertilization to produce a cell called a zygote, from which the entire mammalian embryo develops. Every other cell type in the mammalian body is a somatic cell; internal organs skin, bones, blood and connective tissue are all made up of somatic cells.

Stem cell Primal cells common to all multicellular organisms that retain the ability to renew themselves through cell division and can differentiate into a wide range of specialized cell types. The two categories of human stem cells are embryonic stem cells, derived from blastocysts and adult stem cells, derived from umbilical cord blood or bone marrow. In a blastocyst of a developing embryo, stem cells differentiate into all of the specialized embryonic tissues. In adult organisms, stem cells and progenitor cells act as a repair system for the body, replenishing specialized cells. As stem cells can be readily grown and transformed into specialized tissues such as muscles or nerves through cell culture.

Introduction Gene therapy is the insertion of genes into an individual's cells and tissues to treat a disease, and hereditary diseases in particular. Gene therapy typically aims to supplement a defective mutant allele with a functional one. Although the technology is still in its infancy, it has been used with some success. Antisense therapy is not strictly a form of gene therapy, but is often lumped together with them.

Introduction…cont. Gene therapy may be divided into germ-line gene therpay and somatic gene therapy. Germ-line gene therapy aims at the introduction of genes into germ cells or embryonal cells. For ethical reasons, human germ-line gene therpy is presently not accepted worlwide.

Introduction…cont. Somatic gene therapy: is the introduction of a gene into somatic cells. The ethical considerations of somatic gene therapy have been widely discussed and a consensus emerged which allows to genetically manipulate a patient’s somatic cells for the purpose of correcting severe disorders.

Background In the 1980 s, advances in molecular biology had already enabled human genes to be sequenced and cloned. Scientists looking for a method of easily producing proteins such as insulin, the protein deficient in diabetes mellitus type 1 investigated introducing human genes to bacterial DNA. The modified bacteria then produce the corresponding protein, which can be harvested and injected in people who cannot produce it naturally. On September 14, 1990 researchers at the U. S. National Institutes of Health performed the first approved gene therapy procedure on four-year old. She was born with a rare genetic disease called severe combined immunodeficiency (SCID).

Background… cont. She lacked a healthy immune system, and was vulnerable to every passing germ. Children with this illness usually develop overwhelming infections and rarely survive to adulthood; a common childhood illness like chickenpox is life-threatening. In gene therapy procedure, doctors removed white blood cells from the child's body, let the cells grow in the lab, inserted the missing gene into the cells, and then infused the genetically modified blood cells back into the patient's bloodstream.

Background… cont. Laboratory tests have shown that therapy strengthened the child’s immune system; she no longer has recurrent colds, she has been allowed to attend school, and she was immunized against whooping cough. This procedure was not a cure; the white blood cells treated genetically only work for a few months, and the process must be repeated every few months.

Basic process In most gene therapy studies, a "normal" gene is inserted into the genome to replace an "abnormal, " disease-causing gene. A carrier molecule called a vector must be used to deliver therapeutic gene to the patient's target cells. Currently, the most common vector is a virus that has been genetically altered to carry normal human DNA. Viruses have evolved a way of encapsulating and delivering their genes to human cells in a pathogenic manner.

Basic process…cont. Target cells such as the patient's liver or lung cells are infected with the vector. The vector then unloads its genetic material containing therapeutic human gene into the target cell. The generation of a functional protein product from therapeutic gene restores the target cell to a normal state.

Ex vivo Vs. In vivo Gene Therapy Cells from a number of organs or tissues (e. g. skin, liver) or from tumors can be removed from the patient and cultured ex vivo in the laboratory. Ex vivo gene therapy is currently the most widely used in clinical trials. In the majority of cases, retroviral vectors are used to insert therapeutic gene into the recipient cells.

Ex vivo Vs. In vivo Gene Therapy Other organs (e. g. lung, brain) are less suited for ex vivo gene therapy as culture of the target cells or re-transplantation are not feasible. Somatic gene therapy can only be performed by in vivo gene transfer. It can be performed by administering the gene of interest either locally or systematically.

Potential target diseases Inherited Disorders Cancer

Potential target diseases Inherited Disorders For somatic gene therapy of inherited disorders an intact version of the affected gene is introduced into these cells in which the inadequate expression of the gene is determining the major symptoms of the diseases. A genetic disorder, or genetic disease, is a condition caused by abnormal expression of one or more genes resulting in a clinical phenotype. Some disorders may confer an advantage, at least in certain environments. There are genetic disorders caused by the abnormal chromosome number, as in Down syndrome (extra chromosome 21).

Potential target diseases Inherited Disorders Defective genes are often inherited from the parents. In this case, the genetic disorder is known as a hereditary disease. This can often happen unexpectedly when two healthy carriers of a defective recessive gene reproduce, but can also happen when the defective gene is dominant. Currently around 4, 000 genetic disorders are known, with more being discovered. Most disorders are quite rare and affect one person in every several thousands or millions. Cystic fibrosis is one of the most common genetic disorders; around 5% of the population of the United States carry at least one copy of the defective gene. Additionally, some people in Japan developed genetic disorders in part due to the effects of radiation from the atomic bombs dropped on Hiroshima and Nagasaki.

Cystic fibrosis (CF) A common hereditary disease that affects the entire body, causing progressive disability and early death. Difficulty breathing is the most common symptom and results from frequent lung infections, which are treated, though not always cured, by antibiotics and other medications. A multitude of other symptoms, including sinus infections, poor growth, diarrhea and male infertility, commonly result from the effects of CF on other parts of the body. A wide variety of other symptoms can occur in some individuals with CF including Cystic Fibrosis Related Diabetes, a unique form of arthritis, asthma, and female infertility.

Cystic fibrosis (CF)…cont. CF is one of the most common fatal inherited diseases. It is most prevalent among Europeans; one in twenty-two people of European descent carries one gene for CF, making it the most common genetic disease among them. Individuals with cystic fibrosis can be diagnosed prior to birth by genetic testing or in early childhood by a sweat test. There is no cure for CF, and most individuals with cystic fibrosis die young - many in their 20 s and 30 s from lung failure. Ultimately, lung transplantation is often necessary as CF worsens. CF is caused by a mutation in a gene called the cystic fibrosis transmembrane conductance regulator (CFTR).

Potential target diseases Cancer is a class of diseases or disorders characterized by uncontrolled division of cells and the ability of these cells to invade other tissues, either by direct growth into adjacent tissue through invasion or by implantation into distant sites by metastasis. Metastasis is defined as the stage in which cancer cells are transported through the bloodstream or lymphatic system. Cancer may affect people at all ages, but risk tends to increase with age, due to the fact that DNA damage becomes more apparent in aging DNA. It is one of the principal causes of death in developed countries.

• When normal cells are damaged beyond repair, they are eliminated by apoptosis. • Cancer cells avoid apoptosis and continue to multiply in an unregulated manner.

Potential target diseases Cancer There are many types of cancer. Severity of symptoms depends on the site and character of the malignancy and whethere is metastasis. A definitive diagnosis usually requires the histologic examination of tissue by a pathologist. This tissue is obtained by biopsy or surgery. Most cancers can be treated and some cured, depending on the specific type, location, and stage. Once diagnosed, cancer is usually treated with a combination of surgery, chemotherapy and radiotherapy.

Potential target diseases Cancer The unregulated growth that characterizes cancer is caused by damage to DNA, resulting in mutations to genes that encode for proteins controlling cell division. Many mutation events may be required to transform a normal cell into a malignant cell. These mutations can be caused by chemicals or physical agents called carcinogens, by close exposure to radioactive materials, or by certain viruses that can insert their DNA into the human genome. Mutations occur spontaneously, and may be passed down from one generation to the next as a result of mutations within germ lines. However, it should be noted that some carcinogens also appear to work through non-mutagenic pathways that affect the level of transcription of certain genes but do not necessarily mutate the bases themselves.

Gene Transfer Methods Viral Non Viral

Broad Methods There a variety of different methods to replace or repair the genes targeted in gene therapy: A normal gene may be inserted into a nonspecific location within the genome to replace a nonfunctional gene. This approach is most common. An abnormal gene could be swapped for a normal gene through homologous recombination. The abnormal gene could be repaired through selective reverse mutation, which returns the gene to its normal function. The regulation (the degree to which a gene is turned on or off) of a particular gene could be altered.

Vectors in gene therapy Viral methods: 1 - Retroviruses The family of viruses that includes HIV, the virus that causes AIDS). This incorporates the genes of that virus among the genes of the host cell for the life span of that cell. The genetic material in retroviruses is in the form of RNA molecules, while the genetic material of their hosts is in the form of DNA. When a retrovirus infects a host cell, it will introduce its RNA together with some enzymes into the cell. This RNA molecule from the retrovirus must produce a DNA copy from its RNA molecule before it can be considered for part of the genetic material of the host cell. The process of producing a DNA copy from an RNA molecule is termed reverse transcription.

Vectors in gene therapy 1 - Retroviruses …cont. It is carried out by one of the enzymes carried in the virus, called reverse transcriptase. After this DNA copy is produced and is free in the nucleus of the host cell, it must be incorporated into the genome of the host cell. That is, it must be inserted into the large DNA molecules in the cell (the chromosomes). This process is done by another enzyme carried in the virus called integrase. One of the problems of gene therapy using retroviruses is that the integrase enzyme can insert the genetic material of the virus in any arbitrary position in the genome of the host. If genetic material happens to be inserted in the middle of one of the original genes of the host cell, this gene will be disrupted (insertional mutagenesis).

Vectors in gene therapy 1 - Retroviruses …cont. If the gene happens to be one regulating cell division, uncontrolled cell division (i. e. , cancer) can occur. This problem has recently begun to be addressed by utilizing: 1 - Zinc finger nucleases or 2 - by including certain sequences such as the beta-globin locus control region to direct the site of integration to specific chromosomal sites. Examples of diseases targeted using these viruses include: severe combined immunodeficiency (SCID) and leukemia.

Vectors in gene therapy Viral methods: 2 - Adenoviruses Viruses that carry their genetic material in the form of doublestranded DNA. They cause respiratory (especially the common cold), intestinal, and eye infections in humans. When these viruses infect a host cell, they introduce their DNA molecule into the host. The genetic material of the adenoviruses is not incorporated into the host cell's genetic material. The DNA molecule is left free in the nucleus of the host cell, and the instructions in this extra DNA molecule are transcribed just like any other gene.

Vectors in gene therapy The only difference is that these extra genes are not replicated when the cell is about to undergo cell division so the descendants of that cell will not have the extra gene. As a result, treatment with the adenovirus will require readministration in a growing cell population although the absence of integration into the host cell's genome should prevent the type of cancer seen in the SCID trials. This vector system has shown real promise in treating cancer and indeed the first gene therapy product to be licensed is an adenovirus to treat cancer.

Vectors in gene therapy Viral methods: 3 - Adeno-associated viruses Small viruses with a genome of single stranded DNA. These viruses can insert genetic material at a specific site on chromosome 19. There a few disadvantages to using AAV, including the small amount of DNA it can carry (low capacity) and the difficulty in producing it. Several trials with AAV are on-going or in preparation, mainly trying to treat muscle and eye diseases. Recent clinical trials have also been initiated where AAV vectors are used to deliver genes to the brain. This is possible because AAV viruses can infect non-dividing cells, such as neurons in which their genomes be expressed for a long time.

Vectors in gene therapy Non-viral methods: 1 - Naked DNA This is the simplest method of non-viral transfection. Clinical trials have been carried out of intramuscular injection of a naked DNA plasmid have occurred with some success, however the expression has been very low in comparison to other methods of transfection. In addition to trials with plasmids, there have been trials with naked PCR product, which have had similar or greater success, however this success does not compare to that of the other methods, leading to research into more efficient methods for delivery of the naked DNA such as electroporation and the use of a "gene gun", which shoots DNA coated gold particles into the cell using high pressure gas.

Vectors in gene therapy Non-viral methods Gene gun

Electroporation, or electropermobilization, is a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by an externally applied electrical field. It is usually used in molecular biology as a way of introducing some substance into a cell, such as loading it with a molecular probe, a drug that can change the cell's function, or a piece of coding DNA.

Vectors in gene therapy Non-viral methods: 1 - Naked DNA…cont. Advantages of such a gene transfer: Easy, safe and suited also for the transfer of large gene constructs. It works with muscle and skin.

Vectors in gene therapy Non-viral methods: 2 - Oligodeoxynucleotides The use of synthetic oligodeoxynucleotides in gene therapy is to inactivate the genes involved in the disease process. There are several methods by which this is achieved. One strategy uses antisense specific to the target gene to disrupt the transcription of the faulty gene. Another uses small catalytic molecules of RNA called si. RNA to cleave specific unique sequences in the m. RNA transcript of the faulty gene, disrupting translation of the faulty m. RNA, and therefore expression of the gene.

Vectors in gene therapy Non-viral methods: 3 - Lipoplexes and polyplexes To improve the delivery of the new DNA into the cell, the DNA must be protected from damage and its entry into the cell must be facilitated. To this end new molecules, lipoplexes and polyplexes, have been created that have the ability to protect the DNA from undesirable degradation during the transfection process. Plasmid DNA can be covered with lipids in an organized structure like a micelle or a liposome. When the organized structure is complexed with DNA it is called a lipoplex.

Vectors in gene therapy Non-viral methods: 3 - Lipoplexes and polyplexes There are three types of lipoplexes, anionic (negatively charged), neutral or cationic (positively charged). Initially, anionic and neutral lipids were used for the construction of lipoplexes for synthetic vectors. However, although there is little toxicity associated with them, they are compatible with body fluids and there was a possibility of adapting them to be tissue specific, they are complicated and time consuming to produce so attention was turned to the cationic versions.

Vectors in gene therapy Non-viral methods: 4 - Hybrid methods Due to every method of gene transfer having shortcomings, there has been some hybrid methods developed that combine two or more techniques. Virosomes are one example; they combine liposomes with an inactivated HIV or influenza virus. This has been shown to have more efficient gene transfer in respiratory epithelial cells than either viral or liposomal methods alone. Other methods involve mixing other viral vectors with cationic lipids or hybridizing viruses.

Problems and ethics Short-lived nature of gene therapy - Before gene therapy can become a permanent cure for any condition, therapeutic DNA introduced into target cells must remain functional and the cells containing therapeutic DNA must be long-lived and stable. Problems with integrating therapeutic DNA into the genome and the rapidly dividing nature of many cells prevent gene therapy from achieving any long-term benefits. Patients will have to undergo multiple rounds of gene therapy. Immune response - Anytime a foreign object is introduced into human tissues, the immune system is designed to attack the invader. The risk of stimulating the immune system in a way that reduces gene therapy effectiveness is always a potential risk. Furthermore, the immune system's enhanced response to invaders it has seen before makes it difficult for gene therapy to be repeated in patients.

Problems and ethics Problems with viral vectors - viruses, while the carrier of choice in most gene therapy studies, present a variety of potential problems to the patient --toxicity, immune and inflammatory responses, and gene control and targeting issues. In addition, there is always the fear that the viral vector, once inside the patient, may recover its ability to cause disease. Chance of inducing a tumor - If the DNA is integrated in the wrong place in the genome, for example in a tumor suppressor gene, it could induce a tumor.

Problems and ethics Multigene disorders - Conditions or disorders that arise from mutations in a single gene are the best candidates for gene therapy. Unfortunately, some of the most commonly occurring disorders, such as heart disease, high blood pressure, Alzheimer's disease, arthritis, and diabetes, are caused by the combined effects of variations in many genes. Multigene or multifactorial disorders such as these would be especially difficult to treat effectively using gene therapy.

Concluding Remarks

Remark: Although in the last decade gene therapy has evolved from “possible in theory” to “clinical testing in Humans”, we are still at the beginning of gene therapy era.