Gene therapy with viral and non viral vectors.pptx

Gene therapy and genetic engg

1. GENE THERAPY 1

2. Genes Genes, which are carried on chromosomes, are the basic physical and functional units of heredity. Genes are specific sequences of bases that encode instructions on how to make proteins. it’s the proteins that perform most life functions and even make up the majority of cellular structures. 2

3. Why Genetic Disorders When genes are altered so that the encoded proteins are unable to carry out their normal functions, genetic disorders can result . 3

4. What is Gene Therapy Gene therapy is the insertion of genes into an individual's cells and tissues to treat a disease, such as a hereditary disease in which a deleterious mutant allele is replaced with a functional one. Although the technology is still in its infancy, it has been used with some success. 4

5. Genes are Medicine ? Gene therapy is ‘the use of genes as medicine’. It involves the transfer of a therapeutic or working gene copy into specific cells of an individual in order to repair a faulty gene copy. Thus it maybe used to replace a faulty gene, or to introduce a new gene whose function is to cure or to favourably modify the clinical course of a condition. 5

6. Gene therapy could be very different for different diseases Gene transplantation (to patient with gene deletion) Gene correction (To revert specific mutation in the gene of interest) Gene augmentation (to enhance expression of gene of interest) Targeted killing of specific cells by introducing killer gene Gene ablation – targeted inhibition of gene expression 6

7. Ex vivo gene therapy - Usually with blood cells (lymphocytes or blood stem cells) for diseases affecting the hematopoietic system In vivo gene therapy - Oncolytic adenoviruses for the treatment of cancer Adeno-associated vectors for the treatment of Duchenne muscular dystrophy or hemophilia Non-viral for cancer Different Routes of Gene Therapy 7

8. http://laxmi.nuc.ucla.edu:8237/M288/SChow_4_10/sld005.htm 8

9. Transgenes Integrated Not integrated - stable expression; may provide a cure - expression is transient; repeated treatments necessary - random insertions in heterochomatin can be inactivated; In euchromatin -- Can disrupt important host genes; Long-term consequences are unknown for episomes (plasmids) random mutagenesis not an issue 9

10. Episomes and integrated trasgenes behave differently in dividing cells Integral transgene Episome Loss of plasmid 10

11. Gene Therapy in Blood Cells 11

12. Therapeutic protection gene Gene Therapy in Blood Cells 12

13. Gene Therapy in Blood Cells 13

14. Gene Therapy in Blood Cells 14

15. Therapeutic protection gene Gene Therapy in Blood Cells 15

16. Influences on choice of vector high efficiency viral vectors for gene replacement short term gene expression To prime an immune response To sensitize cells to radiotherapy …Liposomal Delivery… therapy of monogenic diseases (cystic fibrosis; SCID; hemophilia…) 16

17. Desirable characteristics of gene delivery vector 1. High titer or concentrations (>10 8 particles/ml) 3. Precise and stable introduction of transgene 2. Easy and reproducible method of production 4. Vector should not elicit immune response in the host 6. Vector should be able to target specific cell types 5. Transgene should be responsible for its regulatory elements (on/off system) 17

18. Vectors Viruses eg retro viruses, adenoviruses (commonly used) Direct introduction (“golden bullets”) Liposomes Endocytosis of DNA bound to cell surface receptors (low efficiency) Artificial chromosome (under development)) 18

19. Methods of gene delivery (therapeutic constructs) -- Injection of naked DNA into tumor by simple needle and syringe -- DNA transfer by liposomes (delivered by the intravascular, intratracheal, intraperitoneal or intracolonic routes) -- DNA coated on the surface of gold pellets which are air-propelled into the epidermis (gene-gun), mainly non applicable to cancer -- Biological vehicles (vectors) such as viruses and bacteria. Viruses are genetically engineered They are currently the most efficient means of gene transfer . 19

20. MOST COMMON VIRAL VECTORS Retroviruses Adenoviruses Adeno-associated viruses Herpes simplex viruses can create double-stranded DNA copies of their RNA genomes . Can integrate into genome. HIV, MoMuLV, v-src, Rous sarcoma virus dsDNA viruses that cause respiratory, intestinal, and eye infections in humans. Virus for common cold ssDNA viruses that can insert their genetic material at a specific site on chromosome 19 dsDNA viruses that infect a neurons. Cold sores virus 20

21. Gene Therapy- Vectors to deliver therapeutic genes LV- Lentivirus vectors RV- gammaretroviral vectors, AAV – adeno -associated vectors Adenovirus vectors Vectors are replication defective – so they cannot replicate and spread once they are inside the cells and after delivering the anti-HIV genes 21

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25. ADENOVIRUS (AV) AV was the first viral vector developed for gene therapy and was approved for clinical trials in 1990. It was isolated from human adenoid tissue-derived cell cultures for the first time in 1953, hence the term adenovirus, and included in a diverse family of non-enveloped double-stranded DNA ( dsDNA ) viruses called Adenoviridae . AV carries a linear dsDNA ranging from 26 to 45kb in a medium sized (∼100nm) non-enveloped icosahedral viral particle composed of penton and hexon subunits. 25

26. 1) The hexon subunits form a major part of the viral capsid coat and carry antigenic motifs, the penton subunits constitute fiber and knob domains required for infection. The fiber knob domain initiates AV infection by binding to a variety of proteins such as MHC-1 α2 subunit,CD46, sialic acid saccharides on glycoproteins , and AV receptor (CAR) expressed on cell surface 26

27. 2) The interaction between arginine - glycine -aspartic acid (RGD) sequence of the fiber penton subunit and αν integrins on the cell surface drives endocytosis of viral particle and completion of viral infection 27

28. A broad tissue tropism and a nodal for AV transduction efficiency , giving an opportunity to manipulate binding sites for CAR and other ligands to de-target AV infection, an essential feature of popular viral vectors used in gene therapy. 28

29. Based on the expression of AV genes during infection and multiplication, its genome is organized into early (E1, E2a, E2b, E3, and E4), intermediate (IVA2 and IX), and late genes (L1, L2, L3, L4, and L5). Also, its genome carries non-coding inverted terminal repeat (ITR) sequences, ψ packaging sequences, and many viral RNAs. The genome of AV has been manipulated many times to develop safe and efficient vectors for gene therapy applications. 29

30. The first -generation vectors with a partial deletion of E1 or E3 genes do not replicate or display oncogenicity but can deliver less than an 8kb gene and display leaky expression of viral proteins, strong immune response, and contamination with replication-competent virus 30

31. Second-generation vectors were created by deleting E2A, E2B, and E4 from the genome of the first generation AV vectors. However, their production has become complicated, and they do not prevent leaky expression of viral proteins and rapid loss of therapeutic gene expression, and thus have lost enthusiasm for their widespread use in gene therapy 31

32. The third-generation vectors , otherwise known as gutless or helper-dependent AV vectors, lack all viral genes except the ψ and ITR sequences. Thet have capacity to carry larger therapeutic genes (up to 37kb in size), their ability to display long-term transgene expression, and lesser contamination with replicating virus particles. They are also less immunogenic than first - and second-generation vectors. The third-generation vectors were successfully used to express transgenes for about 2years in animals with no adverse effects . 32

33. Adeno -associated virus: From defective virus to effective vector The small microbe was first isolated as a contaminant in the simian adenovirus preparation and then named adeno -associated virus (AAV) The 4.7-kb-long single-stranded DNA ( ssDNA ) packed inside a non-enveloped viral particle carries p5, p19, and p40 promoters as well as rep and cap genes flanked by two 145 nucleotide-long inverted terminal repeats (ITR) and no polymerase gene. a co-infecting helper virus (such as adenovirus or herpes simplex virus) or induction of cellular stress is usually required for a productive infection to occur 33

34. ITRs having palindromic sequences base pair to allow synthesis of cDNA rep and cap genes undergo alternate splicing to express replication proteins (Rep78, Rep68, Rep52, and Rep40), capsid or virion proteins (VP1, VP2, and VP3), and an assembly activating protein (AAP), respectively Unlike other viruses, AAV requires a few other helper proteins, agents or viruses such as AV, herpes simplex virus type I/II, pseudorabies virus, cytomegalovirus, genotoxic agents, UV radiation, or hydroxyurea to infect cells and complete replication 34

35. AAV2 attachment is primary mediated by heparan sulphate proteoglycans , while internalization is aided by the co-receptors, such as avb5 and FGFR1 etc. The use of ubiquitous heparan sulphate proteoglycans as docking sites explains in part the well-known broad tropism of this virus. However, the events and processes that regulate the trafficking of AAV particles into the nucleus are still not fully understood. 35

36. After entry into the host cell nucleus, AAV can follow either one of two distinct and interchangeable pathways of its life cycle: the lytic and the lysogenic . The former develops in cells infected with a helper virus such as adenovirus or herpes simplex virus (HSV), whereas the latter is established in host cells in the absence of a helper virus. 36

37. When AAV infects a human cell alone, its gene expression program is auto-repressed and latency is ensued by preferential integration of the virus genome into a region of roughly 2-kb on the long arm (19q13.3-qter) of human chromosome 19, designated AAVS1. This site-specific integration involves the AAV ITRs and Rep proteins (Rep78, Rep68). 37

38. AAV can also be generated by providing the missing genes E1a, E1b, E2a, E4orf6, and VA that are needed for viral infection. These genes are often cloned in pXX6 helper plasmid and used to co-transfect HEK293 cells along with AAV expression plasmid (rep-cap plasmid) to produce AAV. Therapeutic genes are cloned in the AAV expression plasmid carrying ITR sequences, and their size can be increased by co transfecting another plasmid carrying rep cap genes or by generating virus in rep-cap stable cells. AAV inserts a therapeutic gene in the genome of target cells to provide long-term transgene expression. For instance, the gene expressing F IX blood coagulation factor in one individual of a cohort persisted for more than 10 years during a clinical trial AAV in gene therapy 38

39. AAV inserts a therapeutic gene in the host genome at a specific location on the q arm of chromosome 19. Despite having no large homology regions, more than 70% of the transgene integration events occur within this site; However,the underlying mechanism remains unknown. But AAV lacking its rep-cap genes can deliver a therapeutic gene in the episomal form without inserting into the genome of the target cells. The therapeutic gene in the episomal form can develop into a chromatin-like structure and remain quiescent in cells for months to years without damaging the patient’s body. 39

40. AAV1 displays high transduction efficiency of muscles, neurons, heart, and retinal pigment epithelium. AAV2 has been shown to infect many types of cancer cells, neurons, kidney, retinal pigment epithelium, and photoreceptorcells . The majority of these genomes then form concatemers that persist extrachromosomally as linear episomes within nondividing cells. In the presence of helper virus, wild-type AAV initiates a productive viral infection, while in the absence of helper, AAV can establish latency in the human genome through Rep-mediated integration. 40

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42. Herpes simplex virus 1, mild disease in human, no risk Linear ds DNA, 152 kb, about half of the total 81 genes are non-essential for virus replication 40-50 kb of foreign DNA can be accommodated Neurotropic virus, target to nervous system Replication defective amplicon particles Herpes simplex virus 1 42

43. Herpes simplex virus and pox/ vaccinia vectors also show promise for their effectiveness as oncolytic vaccines. Since its emergence onto the gene therapy scene nearly 25 years ago, the replication-defective Herpes Simplex Virus Type-1 (HSV-1) amplicon has gained significance as a versatile gene transfer platform due to its extensive transgene capacity, widespread cellular tropism, minimal immunogenicity, and its amenability to genetic manipulation. 43

44. Core of the virion contains a 152-kb double-stranded linear DNA genome Encodes ~80-85 viral genes that are arranged as unique long (UL) and unique short (US) segments These regions in-turn are flanked by inverted repeat sequences (designated ab , b’a ’, ac, c’a ’) “a” sites contain sequences required for cleavage/packaging of the HSV-1 genome. Additionally, the HSV-1 genome harbors three lytic origins of replication, with two located within the unique short ( oriS ) segment and one in the unique long segment ( oriL ). Approximately half of the viral genes have been shown to be dispensable for replication of the virus in cultured cells, and thus can be replaced by exogenous genetic material, which has been the premise for the development of HSV-1-based vectors for gene therapy. 44

45. The mature wild-type HSV-1 virion is composed of 4 sub-compartments: envelope, tegument, capsid , and the 150-kb linear double-stranded DNA genome. The envelope contains glycoprotein molecules involved in the cellular binding and viral entry processes of HSV-1 infection. The HSV-1 genome is enclosed within an icosahedral capsid , which consists of 162 capsomers made up of four capsid proteins: VP5, VP26, VP23, and VP19C 45

46. The HSV-1 derived amplicon plasmid contains a single oriS or oriL and an “a” site and is devoid of all viral genes. A bacterial origin of replication (ColE1) and an antibiotic resistant gene ( Ampr ) is included for bacterial propagation of the plasmid. A transgene unit-of-interest can be cloned into the HSV-1 amplicon using standard molecular cloning techniques and packaged into HSV-1 amplicon viral particles using helper virus-based or helper virus-free packaging methodologies. 46

47. The HSV-1 genome contains a significant portion of viral genes that are considered “non-essential” and can be deleted without affecting viral replication in cultured cells. These findings have paved the way for the generation of a number of HSV-1-derived vectors: conditionally replicating vectors, replication-defective vectors, and amplicon -based vectors 47

48. Conditionally replicating HSV-1 vectors are capable of replicating only in certain cell types and tissue types in vivo due to the deletion of non-essential viral genes (e.g. thymidine kinase and ICP34.5). Such vectors have been typically used in the development of therapies for malignant brain tumors (e.g. glioblastoma multiforme , GBM), and are referred to as oncolytic HSV-1 vectors. Since replication of these vectors is restricted to rapidly dividing cancer cells, it has been possible to employ suicide gene therapy for the targeted destruction of malignant cells. Replication-defective recombinant HSV-1 vectors have been deleted in the viral genes essential for lytic replication and reactivation (i.e. the immediate early genes ICP0, ICP4, ICP27, and ICP47), but retain the ability to establish latency. 48

49. The first hybrid amplicon vector to be generated was the HSV-1/ adeno -associated virus (AAV) hybrid amplicon , which incorporated the inverted terminal repeat (ITR) elements and the Rep gene from AAV into the HSV-1 amplicon backbone. These elements are directly involved in the integration of the AAV genome into the AAVS1 site located on human chromosome 19q13.3-qter during the latent phase of wild-type AAV infection. 49

50. Pox virus based vectors Vaccinia virus (VACV or VV) is a large, complex, enveloped virus belonging to the poxvirus family. It has a linear, double-stranded DNA genome of approximately 190 kb in length, which encodes for around 250 genes. The genome is surrounded by a lipoprotein core membrane. The poxviruses are the largest known DNA viruses and are distinguished from other viruses by their ability to replicate entirely in the cytoplasm of the host cell, outside of the nucleus. 50

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52. Vaccinia virus is well-known for its role as a vaccine that eradicated the smallpox disease, making it the first human disease to be successfully eradicated by mankind. This endeavour was carried out by the World Health Organization (WHO) under the Smallpox Eradication Program. Post eradication of smallpox, scientists study Vaccinia virus to use as a tool for delivering genes into biological tissues (gene therapy and genetic engineering). 52

53. Vaccinia virus can accept as much as 25 kb of foreign DNA, making it useful for expressing large genes. Foreign genes are integrated stably into the viral genome, resulting in efficient gene expression. Vaccinia viruses re-engineered to express foreign genes are robust vectors for production of recombinant proteins. Vaccinia viruses have been engineered to express immunizing antigens of herpesvirus , hepatitis B, rabies, influenza, human immunodeficiency virus (HIV), and other viruses. 53

54. Retroviral Vectors A retrovirus is any virus belonging to the viral family  Retroviridae . All 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 part of the genetic material of the host cell. 54

55. Retrovirus genomes commonly contain these three open reading frames that encode for proteins that can be found in the mature virus. Group-specific antigen ( gag ) codes for core and structural proteins of the virus, polymerase ( pol ) codes for reverse transcriptase, protease and integrase , and envelope ( env ) codes for the retroviral coat proteins 55

56. The process of producing a DNA copy from an RNA molecule is termed reverse transcription. 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 . 56

57. Now that the genetic material of the virus is incorporated and has become part of the genetic material of the host cell, we can say that the host cell is now modified to contain a new gene. If this host cell divides later, its descendants will all contain the new genes. Sometimes the genes of the retrovirus do not express their information immediately. 57

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59. Retroviral vectors are created by removal of the retroviral gag, pol , and env genes. These are replaced by the therapeutic gene. Packaging cell lines provide all the viral proteins required for capsid production and the virion maturation of the vector. These packaging cell lines have been made so that they contain the gag, pol and env genes 59

60. 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). If the gene happens to be one regulating cell division, uncontrolled cell division (i.e., cancer) can occur. 60

61. Gene therapy trials to treat severe combined immunodeficiency (SCID) were halted or restricted in the USA when leukemia was reported in three of eleven patients treated in the French X-linked SCID (X-SCID) gene therapy trial. Ten X-SCID patients treated in England have not presented leukemia to date and have had similar success in immune reconstitution. Gene therapy trials to treat SCID due to deficiency of the Adenosine Deaminase (ADA) enzyme continue with relative success in the USA, Italy and Japan. 61

62. While pioneer work was performed on avian-infectious alpha retroviral Rous sarcoma virus, Moloney murine leukaemia virus ( MoMLV ) belonging to gamma retroviruses was initially used for the preparation of therapeutic vector and until now, MoMLV -derived constructs along with human immunodeficiency virus (HIV)-derived vectors are most frequently used. The construction of mutant Moloney murine leukaemia virus defective in the packaging of genomic RNA into virions represents an important step towards the development of retroviral vectors. The first gene delivery systems based on HIV-1 were prepared in the early 1990s. 62

63. Since MoMLV lacks elements necessary for active transport of genetic information through nuclear membrane, integration of viral DNA is possible only during the mitosis. MoMLV derived vectors transduce only dividing cells. Integration of viral genome is mediated by the pre-integration complex (PIC) consisting of integrase , capsid , p12, proviral DNA and host cell proteins. 63

64. The cloning capacity of retroviral vectors is up to 10 kbps , but the size of transgene significantly influences its expression and viral titre . Envelope protein coded by gene env is responsible for the tropism of MoMLV . 64

65. Lentiviral vectors In comparison to MoMLV -derived vectors, lentiviral vectors are more complex. The main difference between lentiviral vectors and vectors derived from other retroviruses is their ability to infect/ transduce quiescent non-dividing cells. They are able to pass through nucleopores into the intact nucleus. The mechanism of this phenomenon has not been completely clarified; yet, it is known that both viral and cellular proteins participate in this process. In addition to HIV vectors , vectors based on feline immunodeficiency virus (FIV), simian immunodeficiency virus (SIV) or equine infectious anaemia virus (EIAV) have also been prepared. 65

66. Replication-competent retroviral vectors Retroviral vectors can be constructed as replication-defective to transduce target cells and enable long-term expression of transgene (immunology disorders, genetic diseases), or they carry transgenes inducing cell death (cancer gene therapy). On the other hand, the replication competent vectors (RCV) are prepared in order to replicate in the target ( tumour ) cells. Their progeny infects surrounding malignant cells. Since the targeting is an inevitable characteristic of RCV, they can be engineered to express ligands to tumour cell-specific markers. The advantage of MoMLV -derived vectors is their natural preference to tumour cells. MoMLV is unable to infect quiescent cells, making them suitable vehicles for the treatment of brain tumours . 66

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68. Example of 3 manufacturing platforms for the generation of modified cells for ex vivo gene therapy via viral vectors produced by transfection . 68

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71. There are two systems for the delivery of transgene into the cell – viral and non-viral. The nonviral approaches are represented by polymer nanoparticles , lipids, calcium phosphate, electroporation / nucleofection or biolistic delivery of DNA-coated microparticles . The safety is mentioned as the major advantage of non-viral approaches. In general, non-viral delivery of transgene is less effective in comparison to viral systems. 71

72. The non-viral vectors are Naked DNA, particle based and chemical based. They are administered by direct administration (plasmid DNA/Naked DNA)/ chemical /physical. Most of cardiovascular clinical trials use non-viral vectors as a mode of gene transfer. 72

73. Non-viral vectors are generally used to transfer following types of nucleic acids . Small DNA ( Oligodeoxynucleotides ) or related molecules synthesized chemically. Large DNA molecules (Plasmid DNA:p DNA) RNA( Ribozymes , Si RNA, m RNA) 73

74. Various delivery Systems: I. Physical Methods:  Gene therapy researchers are more attracted towards physical means of transferring gene material as it is simpler. These methods employ physical force to counteract the membrane barrier of the cells thus facilitating intracellular delivery of the genetic material. 1. Needle:  The genetic material of interest is administered through a needle carrying syringe into tissue or systemic injection from a vessel. Without any carrier it is the simplest and safest method of gene transfer. Attractive candidate tissues are muscle, skin, liver, cardiac muscle and solid tumours . However, the efficiency is low due to rapid degradation by nucleases in serum and cleared by mononuclear phagocyte system  74

75. Ballistic DNA:  Particle bombardment, micro projectile gene transfer or gene gun are the other terms used for ballistic DNA. This method was first used as gene transfer technique to plants. The method is based on the principle of delivering DNA coated heavy metal particles by crossing target tissue at a certain speed. The sufficient speed is achieved by high voltage electronic discharge, spark discharge or helium pressure discharge. Gas pressure, particle size, dose frequency are the critical parameters in determining the efficiency of gene transfer. 75

76. Electroporation :  The other terms used for electroporation are gene electro injection, gene electro transfer, electrically mediated gene therapy, electro gene transfer. Applying an electric field that is greater than the membrane capacitance will cause charges of opposite polarity to line up on either side of cell membrane thus forming a potential difference at a specific point on the cell surface. As a result membrane breakdown form a pore and allows the molecule to pass.  Electroporation has emerged as a reliable physical method for delivering plasmid DNA. The therapy can be delivered by intradermally , intramuscularly or as intratumoural 76

77. Sonoporation :   Sonoporation is a noninvasive site specific technique which utilizes ultrasound wave to temporarily permeablize the cell membrane to allow cellular uptake of DNA. Genetic material of interest is incorporated within micro bubble and administered into systemic circulation. This is followed by external application of ultrasound. The ultrasound wave’s cavitate the micro bubble within the microcirculation of target tissue, produces bio effects that result in deposition of targeted transfection of therapeutic gene.  77

78. Photoporation :   This physical method utilizes single laser pulse to generate transient pores on a cell membrane to allow DNA to enter into the cell. Focal point and pulse frequency of the laser controls the efficiency. It is claimed that the level of transgene expression is identical to that of electroporation . This technique lacks documented evidence 78

79.   Magnetofection :  It is based on the hypothesis of magnetically targeted drug delivery. The technique is based on coupling therapeutic gene to magnetic nanoparticle . This complex is introduced in the cell culture. 79

80. Hydroporation :   It is also called as hydrodynamic gene transfer. The technique uses hydrodynamic pressure to penetrate the cell membrane. Hydrodynamic pressure is created by injecting large volume of DNA solution in a fraction of time. This creates increased permeability of capillary endothelium and forms pores in plasma membrane encircling parenchyma cells. The therapeutic gene of interest can reach the cell through these pores and these membrane pores are closed later thus keeping the genetic material inside the cell.  80

81. Chemical Carriers:  Chemical vectors are broadly classified into inorganic particles, lipid based, polymer based and peptide based. They are generally categorized as  Those forming condensed complex with therapeutic gene to protect them from nucleases and other blood components. Those designed to target specific cells. Those designed to increase the delivery of genetic material to cytosol or nucleus. Those designed to disintegrate from DNA/RNA in the cytosol . Those designed for sustained or controlled release of therapeutic gene in tissue. Chemical non-viral nucleic acid delivery systems are generally DNA/Cationic lipid ( Lipoplexes ), DNA/cationic polymer ( Polyplexes ) and DNA/cationic Polymer/cationic Lipid ( Lipopolyplexes ) 81

82. Inorganic particles :  They are generally nanoparticles that can be engineered by varying in size, shape and porosity in order to escape from  reticulo endothelial system or to protect an entrapped molecule from degradation. Calcium sulphate , silica, gold, magnetic compounds, quantum dots. Carbon nanotubes , fullerenes, supra molecular system are most studied in this category. 82

83. Gold:  The properties like ease of preparation, unlimited surface characterization and inert nature attracted researchers towards gold nanoparticles . Gold nanoparticles have strong absorption of light near infra-red region. The near infra-red light can penetrate deeply into tissues. Modifying the surface of gold with DNA can be used to transfect the cell by using photo thermal effect. Thermal denaturation induced by photo thermal effect helps to control the release of gene. Studies had proved that transfection efficiency with gold is comparable to lipoplexes comparatively with lower toxicity in vitro. However, major concern is its high chemical stability, so it is not easily dissolved in cell resulting in accumulation in cell which may harm the cell growth. 83

84. B. Synthetic/natural biodegradable: Cationic lipids:  Hundreds of lipids have been developed for gene transfer. All of them share the common structures of positively charged hydrophilic head and hydrophobic tail with linker structure that connects both. The positively charged head group binds with negatively charged phosphate group in nucleic acids and form uniquely compacted structure called lipoplexes . Transfection efficiency depends on overall geometric shape, number of charged group per molecules, nature of lipid anchor and linker bondage. Lipoplexes due to their positive charge electrostatically interact with negatively charged glycoproteins and proteoglycans of cell membrane which may facilitate cellular uptake of nucleic acids.  84

85.  Peptide based:   Peptide based vectors are considered advantageous over other non-viral vectors in tight compact and protecting DNA, target specific cell receptor, disrupting endosomal membrane and delivering genetic cargo into nucleus. Cationic peptides are rich in basic residues like lysine and/or arginine . Attaching peptide ligands to polyplex or lipoplexes enables vector to achieve specific target. Short peptide sequence taken from viral protein enables the vector to provide nuclear localization signal that assist transport of genetic material into nucleus. Due to these advantages peptides are frequently used to functionalize cationic lipoplexes or polyplexes 85

86. Polymer based vectors Cationic polymers mix with DNA to form nanosized complex called polyplexes . Polyplexes are more stable than lipoplexes . Polymers are categorized into natural and synthetic polymers. Natural - proteins, peptides, polysaccharides. Synthetic- Polyethylene mine (PEI), Dendrimers , and Polyphosphoesters . 86

87. Polyethylenimine (PEI):  PEI is considered as a gold standard for in vivo and invitro gene transfer. Cationic polymers have high density amine groups which exert protein sponge effect that ultimately stops the acidification of endosomal pH. This leads to the influx of chloride within the compartment and increases the osmotic pressure, leading to the swelling and rupture of endosomal membrane. b.Chitosan :  It is a natural polymer based on cationic polysaccharide. One of the most studied non-viral vectors. It is nontoxic even at high concentrations. It is a linear cationic polysaccharide composed of glucosamine. The positive charge of chitosan electrostatically bind with negative charged DNA. On account of its mucoadhesive properties chitosan /DNA polyplexes are widely used in oral and nasal gene therapy. To effectively negotiate intracellular barriers, chitosan is conjugated to folic acid. 87

88.  Poly (DL- Lactide ) (PLA) and Poly ( DL- Lactide - co- glycoside) (PLGA):  They are biodegradable polyesters undergo bulk hydrolysis thus providing sustained delivery. The degradation products are removed by citric acid cycle. PLGA is approved by FDA as vehicle for protein delivery. Less than 10 μm in size, they are easily phagocytosed by antigen presenting cell and inducing immune reaction d. Dendrimers :   Dendrimer molecules are symmetrical in size and shape with terminal group functionality. It binds to genetic material when positively charged peripheral groups interact with nucleic acids in physiological pH. due to nanometric size it can interact effectively with cell membranes, organelles, and proteins. The terminal amino group and positive charge density determine the toxicity profile. e. Polymethacrylate   Polymethacrylate are vinyl based polymer able to condense polynucleotides into nanometer size particle. But the transfection is limited due to their low ability to interact with membranes. 88

89. Delivery efficiency is the major hurdle for almost all of the non-viral vectors.  89

90. Liposomes possessed excellent biocompatibility, low immunogenicity, ability to deliver large piece of nucleic acid and ease of handling. Due to positive charge liposomes may undergo nonspecific interaction with negatively charged serum protein, enzymes and result in decrease cell adhesion, hemolysis and low transfection . To overcome this positive charge heterocyclic ring like imidazolium , pyridinum , and polyamine groups were added. But the progress is not great enough and full of difficulties and challenges 90

91. PEGylation is one of the popular mechanism considered to reduce the opsonization and aggregation of liposomes in reticuloendothelial system. However the drawback of PEGylated surface is reduced biological activity because of decreased uptake by target cells. Neutral helper lipid is proposed to improve the target uptake of PEGylated liposomes . But all this exhaustive and extensive efforts yield very limited improvements clinically 91

92. Over the past decade several strategies have been developed to improve the poor outcome of non-viral vectors by focusing; extracellular stability (polynucleotide degradation in extracellular space), internalization (internalization of carrier), intracellular trafficking ( endosomal rupture and polynucleotide release), nuclear entry (dissociation of polynucleotide from the carrier and entry of polynucleotide into the nucleus). 92

93. The potential of mRNA for therapeutic protein expression  in vivo  has been investigated as an alternative to DNA-based gene therapy owing to its unique advantages. Recent advances in chemical modifications of mRNA reduce stimulation of the immune system and improve stability when it is delivered  in vivo . Small interfering RNA ( siRNA ) has great therapeutic potential, as it can silence nearly any targeted gene after introduction into cells. Lipid- and polymer-based siRNA nanoparticles and conjugate systems enable successful delivery of chemically modified siRNAs in humans. 93

94. Delivery of genome editing systems — including zinc-finger proteins, transcription activator-like effectors and CRISPR– Cas (clustered regularly interspaced short palindromic repeat–CRISPR-associated) systems — facilitates gene editing at desired sites in the genome. Recent proof-of-concept studies in model organisms have shown that this approach may be used to cure genetic diseases, which is in contrast to the temporary expression or random insertion of a DNA fragment in conventional gene therapy. 94