Gene Therapy-Types, Strategies, Approaches, Methods, Vectors, Clinical Applications and Recent Advances

Gene Therapy
Types of Gene Therapy
Gene Therapy Strategies
Gene Therapy Approaches
Methods of Gene Therapy
Vectors for Gene Therapy
Clinical Applications
Recent Advances
chimeric antigen recipient t (car-t) cell therapy
crispr/cas9
induced pluripotent stem cells (ips)
micro-rna

1. GENE THERAPY Presented by: Aditya Sharma M.S. (Pharm) Pharmaceutical Analysis NIPER Guwahati 1

2. CONTENTS Introduction Types of Gene Therapy Gene Therapy Strategies Gene Therapy Approaches Methods of Gene Therapy Vectors for Gene Therapy Clinical Applications Recent Advances Conclusion References 2

3. INTRODUCTION The ability to make site-specific modifications to the human genome has been an objective in medicine since the recognition of the gene as the basic unit of heredity. Thus , gene therapy is understood as the ability of genetic improvement through the correction of altered (mutated) genes or site-specific modifications that target therapeutic treatment. It is a technique for correcting defective genes responsible for disease development. The first approved gene therapy experiment occurred on September 14, 1990 in US, when Ashanti DeSilva was treated for ADA-SCID. 3

4. TYPES OF GENE THERAPY There are several approaches for correcting faulty genes; the most common being the insertion of a normal gene into a specific location within the genome to replace a non functional gene. Gene therapy is classified into the following two types: Somatic gene therapy Germ line gene therapy 4

5. Somatic Gene Therapy :- In somatic gene therapy, the somatic cells of a patient are targeted for foreign gene transfer. In this case the effects caused by the foreign gene is restricted to the individual patient only, and not inherited by the patient's offspring or later generations. Germ Line Gene Therapy :- Here, the functional genes, which are to be integrated into the genomes, are inserted in the germ cells, i.e., sperm or eggs. Targeting of germ cells makes the therapy heritable. 5

6. GENE THERAPY STRATEGIES  Gene Augmentation Therapy (GAT ) Targeted Killing of Specific Cells  Targeted Inhibition of Gene Expression Targeted Gene Mutation Correction 6

7. Gene Augmentation Therapy (GAT) 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. 7

8. Targeted Killing of Specific Cells It involves utilizing genes encoding toxic compounds (suicide genes), or prodrugs (reagents which confer sensitivity to subsequent treatment with a drug) to kill the transfected/ transformed cells. This general approach is popular in cancer gene therapies. 8

9. Targeted Inhibition of Gene Expression This is to block the expression of any diseased gene or a new gene expressing a protein which is harmful for a cell. This is particularly suitable for treating infectious diseases and some cancers. 9

10. Targeted Gene Mutation Correction It is used to correct a defective gene to restore its function which can be done at genetic level by homologous recombination or at mRNA level by using therapeutic ribozymes or therapeutic RNA editing. 10

11. GENE THERAPY APPROACHES 1. Classical Gene Therapy :- It involves therapeutic gene delivery and their optimum expression once inside the target cell. The foreign genes carry out following functions. Produce a product (protein) that the patient lacks; Produces toxin so that diseased cell is killed. Activate cells of the immune system so as to help in killing of diseased cells . 2. Non-classical gene therapy :- It involves the inhibition of expression of genes associated with the pathogenesis, or to correct a genetic defect and restore the normal gene expression. 11

12. METHODS OF GENE THERAPY  There are mainly two approaches for the transfer of genes in gene therapy: 1 . Transfer of genes into patient cells outside the body (ex vivo gene therapy) 2 . Transfer of genes directly to cells inside the body (in vivo gene therapy). 12

13. Ex Vivo Gene Therapy In this mode of gene therapy genes are transferred to the cells grown in culture, transformed cells are selected, multiplied and then introduced into the patient. The use of autologous cells avoids immune system rejection of the introduced cells. The cells are sourced initially from the patient to be treated and grown in culture before being reintroduced into the same individual. This approach can be applied to the tissues like hematopoietic cells and skin cells which can be removed from the body, genetically corrected outside the body and reintroduced into the patient body where they become engrafted and survive for a long period of time. 13

14. In Vivo Gene Therapy  In vivo method of gene transfer involves the transfer of cloned genes directly into the tissues of the patient. This is done in case of tissues whose individual cells cannot be cultured in vitro in sufficient numbers (like brain cells) and/or where re-implantation of the cultured cells in the patient is not efficient. Liposomes and certain viral vectors are employed for this purpose because of lack of any other mode of selection. In case of viral vectors such type of cultured cells were often used which have been infected with the recombinant retrovirus in vitro to produce modified viral vectors regularly. These cultured cells will be called as vector-producing cells (VPCs)). The VPCs transfer the gene to surrounding disease cells. The efficiency of gene transfer and expression determines the success of this approach, because of the lack of any way for selection and amplification of cells which take up and express the foreign gene. 14

15. VECTORS FOR GENE THERAPY  Vectors for gene therapy can be classified into two types: 1 . Viral vectors : Adenovirus , Retrovirus, Adeno- Associated Virus, Lentivirus, Vaccinia virus, Herpes simplex virus 2 . Non-viral : Physical methods – Electroporation, Gene Gun, Sonoporation , Magnetofection , Hydrodynamic delivery etc. Chemical methods – Oligonucleotides, Lipoplexes , Polymersomes , Polyplexes , Dendrimers, Inorganic Nanoparticles, Cell-penetrating peptides, etc. 15

16. CLINICAL APPLICATIONS The absence of certain cellular or biological factors, for example enzymes, in some diseases can be due to a gene being defective. Gene therapy can deliver to target cells genes that code for the missing biological factor. Cancer , infectious diseases, cardiac disease, neurological disorders and some inherited conditions are among the areas into which gene therapy research is being carried out. 16

17. Cancer Gene therapy strategies against cancer include the introduction of tumour suppressor genes, genes that induce apoptosis, genes that inhibit tumour angiogenesis and genes coding for an enzyme to convert prodrugs to active drugs, and immunotherapy. Cancers for which gene therapy products are being developed include glioblastoma, metastatic melanoma, head and neck cancer, non-small cell lung cancer, prostate cancer, renal cell cancer and colorectal cancer. TroVax , an experimental gene therapy, targets the tumour antigen 5T4 and is delivered by the “modified vaccinia Ankara” (MVA) vector. 17

18. Neurological disorders Neurological disorders that could be treated with gene therapy include Parkinson’s disease, Alzheimer’s disease and motor neurone disease. As an example, ProSavin is currently being trialed for Parkinson’s disease, which is caused by dopamine deficiency in the brain. ProSavin delivers into the brain, via a lentivirus vector, genes that encode for three enzymes required for dopamine synthesis. 18

19. Inherited diseases Haemophilia : T herapies could be designed to deliver genes that express the missing factors VIII and IX, meaning individuals would no longer need to inject exogenous clotting factors. Research is being undertaken with adeno-associated virus and lentivirus vectors. Cystic fibrosis : Cystic fibrosis is caused by mutations in the gene that encodes a cellular protein called “cystic fibrosis transmembrane conductance regulator” (CFTR). This mutation results in abnormal ion transportation within lung and other cells. The CFTR gene has been cloned and researchers are now developing gene therapies to enable expression of CFTR. Non-viral vectors are undergoing trials to deliver gene therapy locally to the lungs via nebuliser . Inherited retinal degeneration: Occular gene therapies could offer a treatment strategy for people with inherited retinal degeneration. Both viral and non-viral vectors are being researched. The genetic and biological backgrounds of ocular diseases are well defined and so gene therapy can be targeted appropriately. 19

20. Infectious diseases Gene therapy vaccines are being developed and trialed for tackling infectious diseases, including tuberculosis, malaria, HIV and influenza. Tuberculosis vaccines are being studied that use genetically modified vaccinia comprising a recombinant fowl pox virus and a recombinant MVA virus, each containing a tuberculosis antigen. Malaria vaccines undergoing trials utilise chimpanzee adenovirus containing a malaria antigen. 20

21. Cardiac disease Gene therapy has been investigated to target angiogenesis (the formation of new blood vessels) during cardiac surgery and to improve calcium-handling mechanisms in heart failure. 21

22. RECENT ADVANCES CRISPR/Cas9 Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is a family of short palindromic DNA sequences that are essential for prokaryote antiviral Defense. The versatility of the CRISPR system allows for gene inactivation (knock-out), integration of exogenous DNA (knock-in), allele substitution and more. Several studies have supported the effectiveness and safety of in vivo use of CRISPR/Cas9 genome editing. One such study demonstrated the efficacy of CRISPR/Cas9 in reducing the mutant Huntingtin (mHTT) gene in a mouse model via striatal injection of an AAV vector with Cas9 and mHTT-gRNA. CRISPR technology is also posed to have relevant future applications in cancer therapeutics and mutation detection via improved precision and programmability. Animal models have shown promising in vivo excision of HIV-1 provirus via AAV delivery of Cas9 and single guide RNAs that is a major step towards human clinical trials for a gene therapy-based cure for neuroHIV. 22

23. Chimeric antigen recipient T (CAR-T) cell therapy Chimeric antigen recipient T (CAR-T) cell therapy involves reprog - ramming a patient’s own immune cells, namely T-lymphocytes, to recognise and attack tumor T-cells. CAR-T immunotherapy utilizing autologous T-cells expressing CD-19 specific CARs has yielded impressive clinical trial results in treatment of B-cell malignancies, such as acute lymphoblastic leukemia, chronic lymphocytic leukemia and non-Hodgkin’s lymphoma. Recent studies have demonstrated successful CAR-T therapy against multiple myeloma and improved treatment of non-Hodgkin’s lymphomas and other hematological malignancies. 23

24. Induced pluripotent stem cells ( iPS ) Gene therapy targeted at stem cells has the potential for life-long integration and self-replication of gene products. For instance, patients with chronic liver disease further afflicted by the hepatitis virus that only receive transplantation of induced stem cells will be able to generate healthy hepatocytes, but these cells are still vulnerable to reinfection. Thus, tandem administration of a gene therapy vector encoding for a short hairpin RNA (shRNA) against the virus confers immunity to reinfection, effectively restoring normal hepatic function Combination therapy utilizing stem cells has shown substantial promise in treating retinal diseases including age-related macular degeneration; cardiovascular diseases through a focus on regeneration of the myocardium to treat heart failure; HIV, by re-engineering host immunity to target the HIV-1 virus and more. 24

25. Micro-RNA Micro-RNA (miRNA) are small, noncoding RNA sequences that can directly modulate gene expression by altering the post-transcriptional function of a specific, corresponding messenger RNA (mRNA) sequence Delivery of therapeutic miRNA has the potential for adverse off-target effects; however, miRNA binding sites can be utilized to regulate and inhibit overexpression of a therapeutic gene Several studies have shown the viable application of miRNA in controlling tropisms of oncolytic viruses (including adenovirus, HSV and measles virus, (among others) to achieve superior antitumor activity by preventing immunogenicity and inflammation. 25

26. CONCLUSION The power and versatility of gene transfer strategies are such that there are few serious disease entities for which gene transfer therapies are not under development. The development of new classes of therapeutics typically takes two to three decades; monoclonal antibodies and recombinant proteins are recent examples. Gene therapeutics, which entered clinical testing in the early 1990s, traversed the same time course. Examples of clinical success are now ample, and gene therapy approaches are likely to become increasingly important. A central question is the long-term safety of gene transfer, and regulatory agencies have mandated a 15-year follow-up for subjects enrolled in many gene therapy trials . 26

27. REFERENCES Friedmann T. A brief history of gene therapy .  NatGenet . 1992;2(2):93–98. Review . Gardlík R, Pálffy R, Hodosy J, Lukács J, Turna J, Celec P. Vectors and delivery systems in gene therapy. Med Sci Monit . 2005;11(4):RA110–RA121. Review . Hanania , E. G., Kavanagh, J., Hortobagyi , G., Giles, R. E., Champlin , R., & Deisseroth , A. B. (1995). Recent advances in the application of gene therapy to human disease. The American Journal of Medicine, 99(5), 537–552. doi:10.1016/s0002-9343(99)80232-0    27