Gene therapy

This presentation focuses on the science of Gene Therapy, the techniques of germ-line and somatic gene therapy and the mechanism of curing diseases and disorders using gene therapy. The presentation starts by discussing some common basic terms from genetics and moves on to the historical development of gene therapy techniques in chronological order. The different types of gene therapy techniques and their mechanisms have been discussed in detail subsequently. In concluding slides, some commercially available gene therapy products are mentioned and challenges of gene-therapy techniques have been highlighted.

1. PRESENTATION BY TATHAGAT SAH BTech. Biotechnology URN - 1901181

2. Gene Therapy Table of Contents Some Basic Terms from Genetics What is Gene Therapy Historical Perspective Gene Mutation Types of Gene Therapy Germ-Line Gene Therapy Somatic Gene Therapy Ex-Vivo Gene Therapy In-Vivo Gene Therapy Methods for Gene Therapy Curing Diseases and Disorders Challenges of Gene Therapy

3. Some Basic Terms Gene – Sequence of nucleotides. Genome – All genetic material of an organism. The genome includes both the genes, the coding regions and the noncoding DNA, as well as mitochondrial and chloroplast DNA. Genetic Engineering – Genetic engineering is the artificial modification of an organism’s genetic composition. Gene Editing – Type of genetic engineering in which DNA is inserted, deleted, modified or replaced in the genome of a living organism. Gene Cloning –  process of isolating a DNA sequence of interest for the purpose of making multiple copies of it. Genome Sequencing – Determination of entirety of the DNA sequence of an organism's genome at a single time.

4. What is Gene Therapy Gene therapy is a therapeutic technique that uses genes to treat or prevent disease. Gene therapy involves altering the genes inside our body's cells in an effort to treat or stop disease although, not all medical procedures that introduce alterations to a patient's genetic makeup can be considered gene therapy. Various approaches to gene therapy include: Replacing a mutated gene that causes disease with a healthy copy of the gene. Inactivating, or “knocking out” or fixing a mutated gene that is functioning improperly. Introducing a new gene into the body to help fight a disease.

5. Historical Development of Gene Therapy Important milestones that have brought us to where we are with gene therapy today: 1990 – The first gene therapy clinical trial was conducted using new viral vector technology. Two patients with SCID received treatment using novel gamma retrovirus vector technology. Michael Blaese and French Anderson National Institutes of Health (NIH) 1999 – Jesse Gelsinger , an 18-year-old boy with ornithine transcarbamylase (OTC) deficiency, died while participating in an adenoviral gene therapy trial due to a severe immune reaction to the vector.  University of Pennsylvania

6. 2003 – CFDA approved the world’s first commercially available gene therapy to treat squamous cell carcinoma. 2009 – In a clinical trial, a genetic eye disease was treated using an adeno -associated virus (AAV) vector. 2010 – A self-inactivating lentiviral vector was first used in clinical trials of gene addition therapy in hemoglobinopathies . 2012 – The EMA approved the first AAV-based gene addition therapy for the treatment of lipoprotein lipase deficiency. 2016 – The EMA approved the first gamma retrovirus-based gene addition therapy to treat ADA-SCID. This therapy contains CD34+ cells transduced with retroviral vector that encodes for the human ADA cDNA sequence.

7. 2017 – The FDA approved the first in vivo gene addition therapy to treat patients with an inherited blindness called bi-allelic RPE65 mutation-associated retinal dystrophy. 2018 – The first clinical trial using CRISPR/Cas9 was initiated. This study is investigating the use of CRISPR/Cas9 for gene disruption in beta hemoglobinopothies .

8. Gene Mutation The perpetuation of the genetic material from generation to generation depends on maintaining a low mutation rate. A high mutation rate in the germ-line would destroy the species, and high mutation rates in the soma would destroy the individual. Sources of gene mutation include: Inaccuracy in DNA replication Chemical damage to the genetic material Damage by environmental factors, such as radiation and mutagens Spontaneous hydrolysis of DNA by water Transposition – Insertion of transposons

9. Types of Gene Therapy Gene therapy is most applicable to the correction of single gene disorders, especially recessive diseases where a functional copy of the defective gene will restore the activity of the non-functional protein. The insertion of the transgene can be targeted to either germ (egg and sperm) or somatic (body) cells. Germ-line gene therapy Germ-line gene therapy is when DNA is transferred into the cells that produce reproductive cells, eggs or sperm, in the body. This type of therapy allows for the correction of disease-causing gene variants that are certain to be passed down from generation to generation.

10. Pros Enables the generation being treated to lead a healthy life If germ-line gene therapy has the potential to remove a disease completely from the population it will remove the long term healthcare costs of treating the disease Cons Effects of gene therapy are too unpredictable Germ-line gene therapy involves more steps and introduces additional risks to the embryo compared with IVF and screening for healthy embryos before implantation Not easily affordable

11. Somatic Gene Therapy The genome of the recipient is altered, but this change is not passed to the next generation. Somatic gene therapy can be classed as being performed either in vivo or ex vivo: In vivo therapy involves the addition of a gene directly to a patient. Ex vivo therapy involves the removal of cells from the patient and their culturing and genetic manipulation in vitro before the return of the modified cells to the patient. The type of therapy used depends on the sorts of cell that need to be modified. If the cells in which the gene defect is apparent can be easily cultured, then the ex vivo route is advantageous.

12. Technique for Ex-Vivo and In-Vivo Somatic Gene Therapy

13. In vivo versus ex vivo gene therapies for the treatment of genetic diseases and cancer In vivo is useful i n the treatment of eye diseases, neurological disorders, and haemophilia Various inherited metabolic and immunological disorders and different types of cancers have been successfully treated with ex vivo gene therapy. AADC, ADA-SCID, ALL, CLL, LCA II, LHON, MLD, SCID-X1, WAS.

14. Gene Therapy Techniques Gene augmentation therapy This is used to treat diseases caused by a mutation that stops a gene from producing a functioning product, such as a protein. This therapy adds DNA containing a functional version of the lost gene back into the cell. The new gene produces a functioning product at sufficient levels to replace the protein that was originally missing. This is only successful if the effects of the disease are reversible or have not resulted in lasting damage to the body. For example, this can be used to treat loss of function disorders such as cystic fibrosis by introducing a functional copy of the gene to correct the disease.

15. Gene inhibition therapy Suitable for the treatment of infectious diseases, cancer and inherited disease caused by inappropriate gene activity. The aim is to introduce a gene whose product either inhibits the expression of another gene interferes with the activity of the product of another gene. The basis of this therapy is to eliminate the activity of a gene that encourages the growth of disease-relate cells. For example, cancer is sometimes the result of the over-activation of an oncogene. So, by eliminating the activity of that oncogene through gene inhibition therapy, it is possible to prevent further cell growth and stop the cancer early. 

16. Killing of specific cells Suitable for diseases such as cancer that can be treated by destroying certain groups of cells. The aim is to insert DNA into a diseased cell that causes that cell to die. This can be achieved in one of two ways: The inserted DNA contains a “suicide” gene that produces a highly toxic product which kills the diseased cell. The inserted DNA causes expression of a protein that marks the cells so that the diseased cells are attacked by the body’s natural immune system. It is essential with this method that the inserted DNA is targeted appropriately to avoid the death of cells that are functioning normally.

17. Mechanism of DNA Transfer A section of DNA/gene containing instructions for making a useful protein is packaged within a vector, usually a virus, bacterium or plasmid. The vector acts as a vehicle to carry the new DNA into the cells of a patient with a genetic disease. Once inside the cells of the patient, the DNA/gene is expressed by the cell’s normal machinery leading to production of the therapeutic protein and treatment of the patient’s disease.

18. Curing Diseases and Disorders Following are some of the FDA approved gene therapies: Zolgensma Onasemnogene Abeparvovec-Xioi ‎ Disease:  Spinal Muscular Atrophy Method:  Utilizes NAV AAV9 to deliver functional copies of the SMN1 gene to neurons. Yescarta Axicabtagene Ciloleucel Disease: Large B-cell Lymphoma Method:  The patient sits for leukapheresis , and the product is sent to a lab. There, the T cells are genetically enhanced to attack cancerous cells and expanded.

19. Luxturna Voretigene neparvovec-rzyl Disease:  Retinal Dystrophy Methods:  A functional copy of RPE65 is introduced to patients via a subretinal injection of Adeno -associated viral vector solution. Zynteglo Disease:  Beta-Thalassemia  Methods:  Hematopoietic Stem Cells (HSCs) are collected from the patient, then treated with a lenti -viral vector to introduce functional genes for beta-globulin. HSCs are then re-introduced to the patient.

20. MB-107 Disease:  X-linked Severe Combined Immunodeficiency (XSCID) Methods:  Inherited non-functional interleukin genes on the X chromosome are replaced by functional copies via lenti -viral delivery. Strimvelis Disease:  Adenosine Deaminase Deficiency (ADA-SCID) Methods:  A sample of the patient’s bone marrow is taken, and the CD34+ cells are transduced with a retroviral vector in order to restore gene function before being reintroduced to the patient.  

21. Disorder Symptoms Cystic fibrosis α 1- antitrypsin deficiency Phenylketonuria Tay-Sachs disease Sickle cell anaemia Thalassemia Autosomal Recessive: Recurrent lung infection, increased mucus production Liver failure, emphysema Mental retardation Neurological degeneration, blindness, paralysis Anaemia Anaemia Neurofibromatosis type 1 Huntington’s disease Mytonic dystrophy Familial retinoblastoma Autosomal Dominant: Tumours of peripheral nerves Involuntary dance-like movements, dementia Heart defects and cataracts Tumours of the eye Haemophilia Duschenne muscular dystrophy Fragile-X syndrome X-Linked: Deficient blood clotting Progressive muscle wasting Mental retardation Examples of some single-gene human genetic disorders

22. Other gene therapy trails are currently on-going for both genetic and non-heritable diseases: • Haemophilia B . sufferers lack the gene for factor IX, a critical agent in the blood clotting process. Parvoviruses have been used to insert the missing gene into skeletal muscle cells (High, 2001). The cells then generate the missing factor, thereby removing the need for daily injections of the protein itself. • Cancer . Modified viral vectors can be used to prime the immune system to attack cancer cells, while other approaches employ viruses to carry suicide genes into the cancer cells. • HIV . Specifically engineered HIV may eventually be recruited to help control HIV-1 infection (Statham and Morgan, 1999).

23. Challenges of Gene Therapy Delivering the gene to the right place and switching it on. Avoiding the immune response: Sometimes new genes introduced by gene therapy are considered potentially harmful intruders which can spark an immune response in the patient, that could be harmful to them. Making sure the new gene doesn’t disrupt the function of other genes. The high cost of gene therapy.

24. END OF PRESENTATION!