GENE THERAPY Dr. S. Moses Arunsingh BIRO 25.03.2011
Gene – a short history• The concept of gene came into vogue when Gregor Johann Mendel from Austria conducted his famous Mendelian Experiments in the 1860s.• But he didn’t call it Gene at that time, instead he used the term Elements or Traits.• In 1909, Danish biologist Wilhelm Johannsen coined the term “Gene" to refer to discrete determiners of inherited characteristics.
Gregor Johann Mendel 1822 - 1884
Classical• In order to provide a structure for understanding the concept of gene, its history is divided into classical, neoclassical, and modern periods.• The classical view prevailed into the 1930s, and conceived the gene as an indivisible unit of genetic transmission, recombination, mutation, and function.
Neo Classical• Establishment of DNA as the physical basis of inheritance led to the neoclassical concept of the gene, which prevailed until the 1970s.• In this view the gene (or cistron, as it was called then) was believed to be responsible for the synthesis of a single mRNA and hence for one polypeptide. This hypothesis prevailed from 1955 to the 1970s.
Modern• Starting from the early 1970s, DNA technologies have led to the modern period of gene conceptualization.• Modern discoveries include those of repeated genes, split genes and alternative splicing, assembled genes, overlapping genes, transposable genes, complex promoters, multiple polyadenylation sites, polyprotein genes, editing of the primary transcript, and nested genes.• We are currently left with a rather abstract, open, and generalized concept of the gene, even though our comprehension of the structure and organization of the genetic material has greatly increased.
Modern• The gene is no longer a fixed point on the chromosome producing a single messenger RNA. Rather, most eurkaryotic genes consist of split DNA sequences, often producing more than one mRNA by means of complex promoters and/or alternative splicing.• Moreover, in certain cases the primary transcript is edited before translation, using information from different genetic units and thereby demolishing the one-to-one correspondence between gene and messenger RNA.
• Philosopher of science Philip Kitcher concludes: A gene is anything a competent biologist has chosen to call a gene.
DNA1844 - 1895
DNA• Frederick Miescher discovered DNA in 1869 whilst studying pus ridden bandages that were being sent to the laboratory by the local clinic.• The discovery was published in 1871.• The man who discovered DNA did not know that it was the hereditary material and he also did not call it DNA. He referred to it as nuclein because it had come from the nucleus.
DNA as the Messenger
DNA• The demonstration that DNA contained the genetic information was first made in 1944 by a series of experiments by Avery, Macleod and McCarty.• They did this by transferring the purified DNA from one Pneumococcus to another and found that the Capsule type of the former was transferred to the latter.
Structure Of DNA- April 25, 1953Watson & Crick Maurice Wilkins Rosalind Franklin NOBEL PRIZE - 1962
• The DNA is a right handed helix.• The backbone of the strand is formed by sugar moiety 2-deoxy D-ribose.• Purines – Adenine and Guanine• Pyrimidines – Cytosine and Thymine• Purines and pyrimidines bind with the sugar by a glycosidic bond forming nucleoside.
• Addition of a phosphoryl group to this nucleoside forms the nucleotide.• The nucleotides on a strand are held together by phosphodiester bonds.• The two strands are linked by hydrogen bonds• Adenosine with thymine by double bonds.• Guanine with cytosine by triple bonds• CHARGAFF’s RULE: A=T G=C
Human Genome Project 1989 – 2003 – to date• GENOME - The full DNA sequence of an organism• The Human Genome Project (HGP) is an international scientific research project with a primary goal of determining the sequence of chemical base pairs which make up DNA and to identify and map the approximately 20,000–25,000 genes of the human genome from both a physical and functional standpoint.• The best estimates of total genome size indicate that about 92.3% of the genome has been completed.
Human Genome Project• The human genome contains 3164.7 million chemical nucleotide bases (A, C, T, and G).• The total number of genes is estimated at 30,000.• Less than 2% of the genome codes for proteins.• The functions are unknown for over 50% of discovered genes.• Almost all (99.9%) nucleotide bases are exactly the same in all people.
Gene Therapy• Gene therapy is an experimental technique that uses genes to treat or prevent disease. – Replacing a mutated gene that causes disease with a healthy copy of the gene. – Inactivating, or “knocking out,” a mutated gene that is functioning improperly. – Introducing a new gene into the body to help fight a disease.
Inception• On September 14, 1990 researchers at the U.S. National Institutes of Health performed the first (approved) gene therapy procedure on four-year old Ashanti DeSilva, born with a rare genetic disease called severe combined immune deficiency (SCID).
Vectors• Vectors are the vehicles used to carry the desired gene to the target cells.• A perfect vector would target specific cell types, insert their genetic information into a safe site in the genome, and be regulated by normal physiological signals.• They may be – Viral vectors – Non viral vectors. A completely reliable, safe and efficient vector is not yet available
Non Viral Vectors• Lipoplexes - 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.• Polyplexes - Complexes of polymers with DNA are called polyplexes.
Non Viral Vectors• Oligonucleotides - Antisense specific to the target gene to disrupt the transcription of the faulty gene or siRNA to signal the cell to cleave specific unique sequences in the mRNA transcript of the faulty gene.• Naked DNA – Plasmid(1-20 kbp) and Cosmids (37 to 52 kbp) , BAC (150-350 kbp) and YAC(100 kb to 3000 kb)
Non Viral Vectors• Research are also experimenting with introducing 47th (artificial human) chromosome into target cells. This chromosome could exist autonomously alongside the standard 46, not affecting their working or causing any mutations.
Viral Vectors• A virus is used to deliver a gene of interest to the cells. In this case the virus should not be replication competent.• Otherwise a virus is designed to cytotoxic. In this case the virus should be specific to the cancer cells.
VIRAL VECTORSRETROVIRUS CONVENIENT TO WORK WITH BUT INFECTS ONLY DIVIDING CELLS CAN HOLD 8 KBPADENOVIRUS INFECT ALL CELLS BUT HAVE AN IMMUNE RESPONSE SO REPEATED USE IS DIFFICULT CAN HOLD 7.5 KBP OF DNAADENO A unique animal virus that requires coinfection with an unrelatedASSOCIATED helper virus, such as an Adenovirus, for infection into a cell.VIRUS Without a helper virus they insert into a host genome and remain as a provirus. Therefore this virus is less likely to produce a immune response in the host.HERPES VIRUS BIG VIRUS SO MORE GENE CAN BE PACKAGED BUT MORE PATHOGENIC 30 KBP
Viral Vectors• Parvovirus and simian virus 40 are being considered for gene therapy in CNS and smooth muscle cells respectively.• Studies undertaken on adeno-associated virus (AAV) have revealed the following merits 1. They form a stable preparation 2. They lack pathogenicity 3. They have high efficiency of integration
Methods of Gene Transfer• Transduction: A virus takes up a piece of DNA from its bacterial host and incorporates it into its own viral genome.
Methods of Gene Transfer• Transformation: Here pieces of genetic instructions are released by a bacterium into its environment. Another bacterium, not necessarily of the same strain, picks up the DNA and incorporates it into its own genome.• Transfection is the process of deliberately introducing nucleic acids into cells. The term is used notably for non-viral methods in eukaryotic cells.
• Transfection of animal cells typically involves opening transient pores or "holes" in the cell membrane, to allow the uptake of material.• Transfection can be carried out using calcium phosphate, by electroporation, or by mixing a cationic lipid with the material to produce liposomes, which fuse with the cell membrane and deposit their cargo inside.
Different Approaches• Suicide- Gene therapy• Cytotoxic virus targeted to p-53 deficient cells• Molecular immunology ( Cancer Vaccines)• Tumor-Suppressor Gene Therapy• Radiation Inducible gene linked to a cytotoxic agent• Targeting signal transduction pathways
Suicide- Gene therapy• Transducing into the cells a gene that converts a prodrug into a toxic agent.• Both the gene and prodrug are inert by itself.• The toxic therapeutic molecule is produced within the tumor cell thereby avoiding the systemic toxicity.
HSV-tk and Ganciclovir• HSV thymidine kinase gene packaged into adenovirus.• The virus is injected into the tumor.• Ganciclovir is administered systemically.• The thymidine kinase converts ganciclovir into a nucleoside analogue resulting in inhibtion of DNA synthesis leading to cell death.
• More cells are killed than are transduced initially.• There is a substantial bystander effect.• The virus cannot be administered systemically because of hepato-tropism.• Tried for pancreatic tumor, brain tumor, mesotheliomas, liver metastasis.
• Tumor growth reductions are impressive but cure rates are low.• Can be used as adjuvant to radiation or chemotherapy.
b-CD and 5-FC• The virus carries the bacterial cytosine deaminase and is injected intratumorally.• 5-FC is administered systemically.• The b-CD converts 5-FC into 5-FU in the tumor cells resulting in DNA damage and cell death.•
Drawbacks• The delivery is too low.• Tumor specificity is too poor.
• But still this can be combined with radiation as the target in both are same , there will be atleast an additive effect.• Combining the two suicide therapies, one will result in dna breaks and the other will prevent dna repair.• Proved to be most effective in invitro tumors.
Cytotoxic virus targeted to p53 deficient cells• Adenovirus has two genes E1a and E1b.• The E1a gene targets the Rb gene and the E1b targets and inactivates p53 gene when infecting a cell.• In this therapy, the E1b gene is eliminated by some means.• So that it can infect only the p53 deficient cells and cause cell lysis by replication.
• A genetically engineered type 5 adenovirus (Ad-p53) which is replication-defective, with a human wild-type p53 cDNA placed in the deleted E1 region of the vector.• This virus induced apoptosis in squamous cell carcinoma of the head and neck cell lines without significant effects on normal cell lines.• Ad-p53 also prevented the establishment of tumors after subcutaneous injection of squamous cell carcinoma of the head and neck cell lines in a nude mouse model.
Cancer Vaccines• To provoke a cellular immune response to the tumor which will be effective against metastasis.• Vaccination with tumor cells expressing cytokines necessary for immune response has shown tumor arrest in animal models.• It can be combined with suicide gene therapy to cause rapid necrosis and generate large amount of tumor antigens combined with an immune gene strategy.
Tumor Suppressor Gene Therapy• Gene Replacement Therapy.• To replace a mutant gene with the correct one which is responsible for the malignant phenotype.• This can result in modification of the cell growth, invasiveness, metastasis etc.
• The p53 gene is the most common target in this method.• Trials have shown growth arrest and growth reduction.• Phase I /II trials in nonmetastatic NSCLC are ENCOURAGING.
• As with other modalities needs to used with other methods such as radiation.• This is because of the technical difficulty of transducing a large quantity.• Mitigated to an extent by the bystander effect.
Signal Transduction Pathways• Many tumors have EGFR and Raf 1 in high expression. They are growth regulators.• NFkB is a cellular transcription factor that plays an important role in stress response.• These genes are targeted in this method.• They are also the target of may MAB
Radiation inducible gene linked to a cytotoxic agent• A chimeric gene containing the human DNA sequence of TNFa and parts of the Early Growth Response Gene (Egr1) enhancer is created.• Packed into a nonreplicating adenovirus.• It is injected into the tumor.• Then the tumor is irradiated.
Radiogenetic therapy• The radiation induces the Erg1 gene and it enhances the TNF a gene resulting in production of TNF a in the irradiated field alone.• It causes vascular destruction as well as apoptosis of tumor cells.
The Future Goals of Gene Therapy• The ideal gene transfer systems of the future will combine the best features of the different vectors being experimented with today.• Each system will be tailored to the specific tissue or cell type requiring treatment.• A way to alter the level of gene expression at will, and a way to shut off, or remove induced genes will further the wide spread use of gene therapy
Unsuccessful Trials• September 2000, University of Pennsylvania: An 18-year-old boy with inherited liver disease was treated with an adeno-virus form of gene therapy. He died four days later from acute respiratory distress and multi organ failure.• March 2003: FDA temporarily halted 27 gene therapy trial is US, after two children who had been treated with gene therapy in France developed a life-threatening illness
Do Future benefits outweigh the Current Costs?• With any new medical treatments there is bound to be failures and major setbacks. When heart transplants were first introduced the survival rate was very low, but today thousands have been saved.• Unlike a heart transplant, gene therapy, when perfected, will provide a relatively inexpensive and non-invasive treatment to numerous diseases, and make treatment available to many.
Conclusion• Some progress has been made in the treatment of cancer in humans using gene therapy but it is not impressive enough to date.• The main problem to be countered is transducing to sufficiently larger cell proportion in a tumor.
• At the end of August 2010, there have been (worldwide) * More than 3,500 patients that received gene therapy treatment; * 1644 gene therapy protocols, of which• 1060 addressed cancer
• In October 2003, the State Food and Drug Administration (SFDA) of China approved Type 5 Ad bearing the human wild-type p53 gene (Ad-p53) for the treatment of head and neck cancer.• The US FDA has not approved any gene therapy to date.• Gene-based treatments for cancer, cystic fibrosis, heart disease, hemophilia, wounds, infectious diseases such as AIDS, and graft- versus-host disease.
On the horizon• OncoVEX GM-CSF (HSV) developed by BioVex is currently in phase III for advanced melanoma, and likely to become the first approved oncolytic agent in the western world. OncoVEX GM-CSF is also currently being tested in a pivotal phase 3 trial in head and neck cancer.