Gene Expression System-viral gene delivery Mpharm(Pharamaceutics)

Gene therapy can be broadly defined as the transfer of genetic material to cure a disease or at least to improve the clinical status of a patient.
One of the basic concepts of gene therapy is to transform viruses into genetic shuttles, which will deliver the gene of interest into the target cells.
Safe methods have been devised to do this, using several viral and non-viral vectors.
In the future, this technique may allow doctors to treat a disorder by inserting a gene into a patient's cells instead of using drugs or surgery.
The biggest hurdle faced by medical research in gene therapy is the availability of effective gene-carrying vectors that meet all of the following criteria:
Protection of transgene or genetic cargo from degradative action of systemic and endonucleases,
Delivery of genetic material to the target site, i.e., either cell cytoplasm or nucleus,
Low potential of triggering unwanted immune responses or genotoxicity,
Economical and feasible availability for patients .
Viruses are naturally evolved vehicles that efficiently transfer their genes into host cells.
Choice of viral vector is dependent on gene transfer efficiency, capacity to carry foreign genes, toxicity, stability, immune responses towards viral antigens and potential viral recombination.
There are a wide variety of vectors used to deliver DNA or oligo nucleotides into mammalian cells, either in vitro or in vivo.
The most common vector system based on retroviruses, adenoviruses, herpes simplex viruses, adeno associated viruses.

1. Submitted To- Dr- Priyanka Kriplani Professor GENE EXPRESSION SYSTEM (VIRAL GENE THERAPY) Submitted To- Dr. Priyanka Kriplani Professor Submitted By- Muskan (M-506) M.Pharm (2 nd sem) GURU GOBIND SINGH COLEGE OF PHARMACY

2. Gene therapy Gene Transfer Gene Transfer Techniques Viral delivery systems Reference Contents

3. Gene therapy can be broadly defined as the transfer of genetic material to cure a disease or at least to improve the clinical status of a patient. One of the basic concepts of gene therapy is to transform viruses into genetic shuttles, which will deliver the gene of interest into the target cells. Safe methods have been devised to do this, using several viral and non-viral vectors. In the future, this technique may allow doctors to treat a disorder by inserting a gene into a patient's cells instead of using drugs or surgery. Gene Therapy

4. Gene Transfer It is defined as a technique of insertion of unrelated, therapeutic genetic information in the form of DNA into target cells.

5. Hurdles in Gene Therapy The biggest hurdle faced by medical research in gene therapy is the availability of effective gene-carrying vectors that meet all of the following criteria: Protection of transgene or genetic cargo from degradative action of systemic and endonucleases, Delivery of genetic material to the target site, i.e., either cell cytoplasm or nucleus, Low potential of triggering unwanted immune responses or genotoxicity, Economical and feasible availability for patients .

6. Gene Transfer Techniques- Based on Vectors

7. Viruses are naturally evolved vehicles that efficiently transfer their genes into host cells. Choice of viral vector is dependent on gene transfer efficiency, capacity to carry foreign genes, toxicity, stability, immune responses towards viral antigens and potential viral recombination. There are a wide variety of vectors used to deliver DNA or oligo nucleotides into mammalian cells, either in vitro or in vivo. The most common vector system based on retroviruses, adenoviruses, herpes simplex viruses, adeno associated viruses. Viral Gene Delivery System

8. Viral Gene Delivery System

9. Viral Gene Delivery System Provide greater gene transfer efficiency in both in vivo and in vitro environments Persist for longer periods of time in most cases Can target a large number of cells A large variety of viruses are available to choose from Innate ability of tropism toward infection Capable of evading endosomes by various mechanisms learned by evolution of viruses Can trigger severe immune responses and inflammatory reactions Their cloning capacity is very limited Produced by complex production methods Low capability of tropism to some specific target cells Can cause mutagenesis by inserting their exogenous DNA into the host genome Research is needed to further understand the mechanisms of molecular infection by viruses Advantages Disadvantages

10. Mechanism of Viral Gene Delivery

11. Viral Gene Delivery System Mechanism The mechanism of viral gene delivery starts with the incorporation of the transgene into the viral DNA, and then this modified DNA is injected into the viral vector. This vector, upon reaching the target site, attaches to the receptors found on the cell membrane of the target cells. After cellular internalization, the vector is packed into endosomes, followed by an acid breakdown of these endosomes that release the capsid containing the modified DNA. This capsid then travels to the nucleus and binds to nuclear pores to enter the nucleus, where the modified gene is integrated into the DNA of the target cell. After that, transcription and translation occur, which form the protein of interest and bring about gene expression 

12. Virus Size and Type genome Viral Proteins Physical Properties Disease in Animals Retrovirus 7–10kb of single stranded RNA Gaga, Polb, Envc 100nm diameter; enveloped Rapid or slow induction of tumors; acquired immunodeficiency syndrome (AIDS Adenovirus 36kb double stranded linear DNA Over 25 proteins 70–100nm in diameter; nonenveloped Cold; conjunctivitis; gastroenteritis Adenovirus associated virus 4-7 kb single stranded linear DNA Repd and Cape 18–26nm in diameter; nonenveloped No known disease Herpes simplex virus 1 (HSV1) 152kb of double stranded linear DNA Over 81proteins 110nm in diameter Mouth ulcers; genital warts; encephalitis Baculovirus 130kb of double stranded circular DNA Over 60 proteins 270 by 45 nm rectangles; enveloped None in mammals; insect pathogen

13. Adenoviral Vectors They contain a DNA genome that is double-stranded and has a size of 35 kb. They are non enveloped viruses. There are three generations of adenoviral vectors that are based on the level of attenuation achieved by the deletion of genes. In the first-generation adenoviral vectors, the E1A and E1B genes are deleted. In the second-generation adenoviral vectors, a large number of the early genes are deleted. In third-generation adenoviral vectors, the complete genome or genetic information of the virus is deleted, which is why they are also called gutless vectors . The adenoviral genome is quite large in size, and the prospect of complete genome deletion greatly renders these viruses a high coding capacity. First-generation vectors can allow ~3.2 kb of genome insertion, while third-generation vectors allow up to 30 kb. An advantage of adenoviral vectors is that there is a very negligible possibility of integration of their genome into the genome of the host cell, which makes them rather safe and nontoxic for use. However, long or sustainable gene expression is difficult to achieve with adenoviruses because their life cycle is not adapted to it.

14. Applications of Adenoviral Vectors In vivo gene therapy differentiated cells. transduce non dividing and terminally Transfect cells in vivo in the intact organ. Gene therapy for cystic therapy. Gene therapy of muscle in liver and therapy of disease of CNS.

15. Retrovirus Vector Commonly employed vectors derived from Murine Leukemia Virus(MuLV). Virus genome has two single copy RNA molecules, complexed with viral core proteins, surrounded by lipid envelope. The main benefit that retroviral vectors offer is their capacity to change their ssRNA genome into a dsDNA particle that steadily incorporates into the genome of its host cells. T Applications Ex-vivo gene therapy. In-vivo gene transfer using retro viral vectors for suicide genes used in brain tumour. Treatment of T-lymphocyte deficiency (ADA), Tumour Infiltrating Lymphocytes (TIL), Bone marrow cells(ADA deficiency, Gauchers disease), hepatocytes (LDL receptor deficiency) and melanoma

16. Adeno Associated Virus Vector Members of Parvo virus family. Heat stable and resistant to various chemicals. Depend on virus - cannot replicate its own, another virus is necessary for replicate,. Major disadvantages of these vectors are complicated process of vector production and the limited transgene capacity of the particles. Applications Used in haematopoietic stem cells for treatment of ß- thalassemia and sickle cell anaemia. ẞ-thalassemia erythrocyte contains insufficient ẞ- globin chain whereas, mutant ẞ- globin chains are produced in sickle cell.

17. Belonging to the Poxviridae family of viruses, they are the most complicated and largest of all viruses that infect humans and cause diseases. Their genome is double-stranded and has a size of approximately 180–220 kb. The smallpox virus is the most popular virus belonging to this family and represents its group very well. The vaccinia virus belonging to the poxvirus family is the virus which was used for the development of smallpox vaccine. There are two features unique to this virus: its capability to carry out its life cycle in the cell cytoplasm due to which it does not insert its genome into the host cell genome, and its occurrence in two different infectious variants i.e., an intracellular mature virus (IMV) and an extracellular enveloped virus (EEV) Pox viruse vector

18. A class of double-stranded DNA viruses that infect a particular cell type, neurons. Herpes simplex virus type 1 is a common human pathogen that causes cold sores. It is a human neurotropic virus, which is mostly used for gene transfer in nervous system. It has a large genome compared to other viruses, which enable scientist to insert more than one therapeutic gene into a single virus, paving the way for treatment of disorders caused by more than one gene defect. HSV makes an ideal vector as it can infect a wide range of tissues including muscle, liver, pancreas, nerve and lung cells. Herpes simplex viruses (HSV)

19. Baculoviruses that belong to the Baculoviridae family are also being explored for their potential to be used as vectors in gene delivery. These viruses possess a reasonable cloning capacity of about 38 kb, and they allow insertion of about 100 kb of genomic material in their capsid. These viruses also do not replicate in mammalian cells, which reduces their risk of causing toxicity. Several studies are exploring the use of these viruses for gene delivery in treatment of certain lymphomas  Other Viruses

20. References Butt MH, Zaman M, Ahmad A, Khan R, Mallhi TH, Hasan MM, Khan YH, Hafeez S, Massoud EES, Rahman MH, et al. Appraisal for the Potential of Viral and Nonviral Vectors in Gene Therapy: A Review.  Genes . 2022; 13(8):1370. Seow Y, Wood MJ. Biological gene delivery vehicles: beyond viral vectors. Molecular therapy. 2009 May 1;17(5):767-77. Pan X, Veroniaina H, Su N, Sha K, Jiang F, Wu Z, Qi X. Applications and developments of gene therapy drug delivery systems for genetic diseases. Asian journal of pharmaceutical sciences. 2021 Nov 1;16(6):687-703.

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