NPTEL – Biotechnology – Gene Therapy

Module 3 Viral vectors

Lecture 15

Lentivirus

Lentiviruses are enveloped RNA viruses found in most of the vertebrates. The most common Lentiviruses used in gene therapy experiments are retroviruses. The virion contains two copies of RNA genome, which are covered by a cone shaped core. The viral genome contains three genes that code for different viral proteins. The gag gene encodes for capsid, matrix, and nucleocapsid protein. The pol gene is a viral polymerase which comprises of proteases, reverse transcriptase and integrase. The env gene encodes for glycosylated envelope protein which mediates the virus entry into the host cell receptor. The viral RNA genome is flanked by long terminal repeats (LTR) sequences which are responsible for packaging of viral RNA genome. Most of the retroviruses lead to persistent infection by integrating to the host cell genome. Lentiviruses also contain two additional proteins tatand rev which take part in transactivation of viral transcription and nuclear export of viral RNA, respectively.

Following the infection virion binds to the host cell receptor and viral genome enters the cell by fusion. Viral RNA is converted into double stranded DNA with the help of enzyme reverse transcriptase. The double stranded DNA then migrates to the nucleus and integrates to the host cell genome by the help of enzyme integrase. This stage of the virus is known as Provirus. The proviral DNA forms new viral genome by using cellular transcription factors. The immature viral proteins are processed by viral proteases, and assemble along with RNA genome to form an infectious virion, which then buds out from the host cell membrane.

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Most of the work based on the lentiviral vector focuses on modifying the human immune deficiency virus type 1 (HIV-1). HIV-1 encodes six accessory proteins namely tat, rev, vif, vpr, nef, and vpu in addition to an essential gag, pol, and env proteins (Figure 15.1).

Table 15.1 Different genes of lentivirus and their functions:

Gene Name Function

gag Group specific antigen Nucleocapsid core protein

pol Polymerase Encode reverse transcriptase, integrase, protease

vif Viral infectivity fector Virus infectivity

vpr Viral protein R Nuclear targeting

vpu Viral protein unique Help in virus budding

env Envelope Codes for surface coat protein

tat Transactivator of Virus gene expression transcription

rev Regulator of expression of Structural gene expression virion proteins

nef Negative regulatory factor Virus infectivity

Figure 15.1 Schematic diagram of lentivirus:

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Gene therapy vectors based on lentiviruses lack all the viral sequences except the LTRs, rev responsive element (RRE), and cis-acting elements. Usually viral rev proteins are added in transin order to facilitate the trafficking of viral RNA genome in the nucleus following efficient binding with RRE. The production of vector RNA is either directed by LTRs or by tissue specific promoter. Generally vectors are designed in such a way so as to utilize tissue specific promoter (CMV) and LTR hybrid promoter (CMV/LTR). The use of CMV/LTR hybrid promoter allows abundant vector RNA production independent of transactivation by tat protein. HIV accessory proteins, vif, vpr, nef, and vpucan be deleted during the lentiviral production. Sometimes polypurine tract present in the HIV genome can be included as a cis- active element during the viral production in order to enhance the nuclear trafficking. The HIV-1 infects only the cells that contain CD4 receptor and co-receptors such as CXCR4 and CCR5.The attachment of HIV to the receptor and co-receptors is largely mediated by viral glycoproteins. This specificity restricts the host range for HIV-1 infection. Scientists worldwide are trying to broaden the host range by various molecular biology techniques. One such technique is to generate a pseudo type HIV virus that contains vesicular stomatitis glycoprotein along with env protein, which is having broad tropism.

Figure 15.2 Generation of lentiviral vector:

Diagram depicts a HIV based vector system where viral essential genes have been replaced by the transgene. Cytomegalovirus promoter is used to transcribe the transgene as well as gag, RRE, and cPPT. RRE acts a cis-acting sequence essential for nuclear export of viral RNA while polypurine tract (cPPT) helps in the import of proviral DNA. The deletion in U3 region in the genome makes it inaccessible to use LTR for transcription. The packaging system consists of gag/pol, VSV-G, and rev expressing constructs. Presence of RRE along with gag/pol helps in nuclear export of the RNA by transcribing rev protein.Lentiviral vectors are produced by transfection of vector construct along with the packaging constructs in producer cells. Vector RNA genomes are packaged by precursor proteins atthe cellular membrane. The mature particles bud through the cellular membrane,containing the envelope derived from VSV-G glycoproteins.

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Lecture 16

Recombinant simian virus 40

Simian virus 40 (SV40)belongs to polyoma viruses group. The virus was first isolated as a contaminant from a monkey kidney culture used for the production of poliovirus vaccine. Its genome is very small and can be easily modified for gene therapy purpose.

Its genome has ds circular DNA wrapped up as nucleosome with the help of histone proteins (H1,H2 (A and B), H3, H4). It is roughly 40-45 nm in diameter. The chromosome is 5000bp long.The genome of SV 40 is like a minichromosome. The genome of SV40 consists of early proteins, late proteins and regulatory proteins. Early proteins are non structural while late proteins are structural proteins. Regulatory region consist of promoter, enhancer and origin of replication.The virions are made up of three capsid proteins namely VP1, VP2, and VP3. MHC class I molecules act as receptors for SV 40 virus. Following attachment to the cell surface by VP1 virus gets internalized by endocytosis.The viral genome replication takes place inside the nucleus (Figure 1). The host RNA polymerase II helps in the transcription of early gene products. The early proteins produced by the virus once it enters the cell consist of T antigen, t antigen and middle T antigen. The large T antigen migrates to the cytoplasm while majority of it gets back to the nucleus duringvirus formation before release. The capsid proteins are produced later which then forms viable virus and comes out of the cell.The early protein coding genes can be replaced by the gene of interest.The mutation in p53 promoter which leads to cancerous condition was discovered in the SV 40 virus.

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Figure 16.1 Replication of SV40 in the host cell:

16.1 SV40 as a vector

Gene therapy using recombinant SV40 is highly suitable means to transfer the gene in a number of conditions. The wild type SV40 virus encodes two early genes, a large T antigen and a small t antigen. The large T antigens are important for virus replication and synthesis of viral late proteins. The genome of SV40 can be modified in order to carry foreign gene by deleting large T antigen (approximately 2.5 kb). The most alarming situation in a wild type SV40 is regarding the ability of large T antigen to bind with tumor suppressor genes such as p53 and retinoblastoma (pRb) protein. The binding of large T antigen may lead to cancer because of inactive p53 and pRb proteins. To avoid this complication all SV40 recombinant viruses lack large T antigen.

The late proteins of SV40 are synthesized from the opposite strand. Out of four structural proteins VP1 is the most abundant one. The minimum viral specific sequence required to assemble the virion consists of origin of replication and encapsidation sequences.The origin of replication and encapsidation sequences overlaps with the early promoter region in the genome. The transcription of the genome and the transgene is governed by pol III promoter. The transcription of the transgene is terminated by the incorporation of a polyadenylation signal downstream to the gene of interest. Most of the vectors available for the expression of transgene requires SV40 early and late polyadenylation signal.

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Recombinant SV40 viral vectors are made in some plasmid background such as pGEMT plasmid vector. The permissible cells are transfected with the plasmid along with the support plasmid needed to package the viral genome. The recombinant virus containing the transgene is then recovered by the lysis of transfected cell. The recombinant virus is then further amplified in the next round of infection in the cell lines. This approach is used to package the transgene of about 5.5 kb without affecting the productivity.

16.2 Features of a SV40 based vector

Some of the specific features of an SV40 vector are as follows

 Capacity of the transgene is limited to 5 Kb.  Both resting as well as dividing cells are equally transduced by SV40 based vectors.  High level of transduction efficiency.  Long lasting transgene expression. It has been reported that the transgene expression once established will remain lifelong.  Neutralizing antibody against recombinant SV40 has not been reported yet. Therefore it is easy to administer multiple injection of recombinant SV40 vector without neutralizing its effect.  SV40 readily integrates with the host cell genome both in dividing and non dividing cells.  The recombinant SV40 is quite stable and can be stored in lyophilized form.  Protein production from a transgene is optimum (not high like adenovirus).  Production titer of recombinant SV40 is very high so a large quantity of cells can be transduced by the vector.

Many cell types have been tested successfully for the transgene expression by the recombinant SV40, such as hepatocyte, lymphocytes, kidney cells, brain cells, skin, and lungs etc. However, the cell types such as cardiac and smooth muscles of blood vessels are known to transduce less efficiently by SV40 vectors.

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16.3 Safety issue with recombinant SV40 based vectors

The major issue of using recombinant SV40 vectors is contamination of wild type SV40 in the recombinant population and insertional mutagenesis. The first issue is largely related with the cell line used for the production of recombinant virus. Mostly COS7 cells are used to produce the recombinant SV40 because of the high yield of the virus. However, COS7 cells are reported to produce wild type population during the subsequent passage. The appearance of wild type population in the samples can be avoided by using packaging cell lines expressing the viral structural proteins designed in such a way as not to overlap with the wild type sequences.

Wild type SV40 virus is known to integrate with the cellular genome for a long time. Although the site of the integration of SV40 is not fixed and varies each time it infects the host cell. It is quite obvious that the activity of the genes changes because of the integration of any foreign DNA fragments. The insertion of foreign gene may activate or inactivate the gene. The activity of gene becomes down- regulated if the insertion occurs in between the open reading frame of the gene. The insertion of the foreign DNA may lead to death of the cells if the function of the affected gene is critical for the cell cycle. The mostbothering part about the integration is regarding the activation of a cellular gene because of the activation of promoter. The activation is more alarming when the activation is of cellular oncogene. The effect is more pronounced in retroviral vector based vectors.

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Lecture 17

Non viral vectors

17.1 Introduction

Therapeutic gene expression has emerged as a potent tool in modern medicine. The main goal of gene therapy is to treat the disease caused by loss of function/ mutation, by introducing specific gene and its regulatory elements. For stable expression at physiological levels the therapeutic gene must be maintained within the nucleus, replicated and passed on to subsequent generations. Viruses particularly retro viruses are preferred systems for gene delivery owing to their invivotransfection efficiency. But immunogenicity and cytoxicity of viral vectors have limited their clinical use. Moreover, the phenomenon of insertional mutagenesis associated with use of viruses is another cause of concern. Non viral vectors on the contrary are much safe, in terms of reduced pathogenicity and capacity of insertional mutagenesis as well as their low cost and ease of production. Current inability of such vectors to achieve proper gene delivery and sustained gene expression, without disturbing host gene expression and signaling pathways needs to beovercome before they are used in human gene therapy.

17.2 Traditional non viral methods of transgenesis

With the advent of using exogenous genetic element for treating diseases various methods for deliveringtransgenehave been developed. These conventional methods can be broadly classified into physical and cationic polymer methods.

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17.3 Physical methods of transgenesis

a. Hydrodynamic pressure techniques: In this technique intravascular injection ofplasmid DNA is given to drive DNA molecules out of the blood circulation and into the tissue. Thismethod oftransgenesisislimited to locations which can tolerate temporary increase in pressure. b. Electroporation: In this method controlled electric shocks are given to cells resulting in formation of large cytoplasmic pores, through which polynucleotide can move into the cell. Such methods are mostly restricted to in vitro applications but development of new electrodes designed for in vivo applications has allowed its use in certain areas of the body, such as tumors, muscles and liver tissue in animal models. c. Ballistic delivery: In ballistic delivery or particle bombardment, DNA-coated metal microparticles are allowed to penetrate cell membranes at high velocity.In vivo this technique is limited to cutaneous applications. d. Microinjection: In this technique DNA is injected into cellsresulting in efficient transgene expression but is laborious and limited to ex vivo applications, such as for the delivery of artificialchromosomes.

17.4 Cationic polymer method of transgenesis a. Lipofection: This method involves the use of cationic lipids mixed with DNA in aqueous solution formingliposomes that encapsulate DNA.Such DNA preparations are taken in either by pinocytosis or phagocytosis, depending on the cell type.Use of liposomes in vivo is limited by its rapid plasma clearance and high toxicity. b. Cationic peptides: Theseare chain of basic amino acids, which compact DNA into spherical complexes, or chromatin components such as histones or protamine, which compact DNA in a structured manner allowing it to enter cell by their interaction with sulfated membrane-bound proteoglycans.

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c. Polyethylenimine: Polyethylenimine is a linear or branched polymer with many protonable amino nitrogen atoms that allows efficient DNA condensation and cell entry. The polymer behaves as an effective ‘proton sponge’ that causes the rupture of endosome by osmotic swelling and releases the polyethylenimine–DNA complexes into the cytoplasm. d. Receptor-mediated delivery: In this methodpolycations conjugated DNA bound to acell-specific ligand is targeted to cells via receptor-mediated internalization.

All these non viral vectors have proven well in in vitro transfection but lacks in in vivo transfection efficiency and only allow transient transgene expression. This inefficiency of non viral vectors can be attributed to the following: • Interaction of the non viral–DNA complex with blood plasma proteins, undesirable cells and extracellular matrix. • Inability to escape from liposome or endosome enclosed moiety. • Vulnerability to cytoplasmic degradative enzymes. • Inability to pass through the double membrane nuclear envelope and subsequent degradation during breakdown of nuclear membrane at mitosis.

Thus to increase the in vivo stability of non viral vectors novel modular vectors have been developed. These vectors have reduced affinity for intracellular proteins and cell surfaces and posses ligands for receptor-mediated endocytosis, peptide sequences that assist DNA compaction, endosomal disruption sequences and nuclear-import signals. Modular vectors can be easily modulated and they also mimic the ability of viruses to overcome the cellular barriers to DNA delivery. GD5 is one such modular vectors, which forms complex with DNA using DNA binding domain (DBD) of GAL4 transcription factor, attaches to tumor cells via a single chain antibody against ERBB2 antigen leading to receptor mediated endocytosis and facilitates endosomal escape using translocation domain of diphtheria toxin in acidic pH.

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Modular vectors can provide efficient delivery of therapeutic gene complexed to non viral vectors but stable nuclear maintenance and replication of such vectors can be achieved by either site specific integration into safe genomic regions or maintenance of extra chromosomal segments.

17.5 Safe integration of corrective genes to human genome The problem of insertional mutagenesis in gene therapy can be avoided by directing transgene into safe genomic locations that are not associated with cell proliferation or tumor suppression. Site specific recombination used by prokaryotic viruses may be helpful in this regard. Prokaryotic viruses express site specific recombinases (SSRs) that recognize unique genomic sequences called recombination sites within pathogen and host genome and mediate recombination at those sites. SSRs recognize defined but degenerate sequences and thus can be manipulated for integration into eukaryotic genome. Thus combination of SSR and non viral vectors can be useful in making safe integrations resulting in permanent expression of the therapeutic gene.For proper integration SSR must recognize specific recombination sites, for example loxP in case of bacteriophage Crerecombinase. Thus for use in human gene therapy these SSRs must be able to recognize endogenous sites in human genome called pseudosites that resemble phage recombination sites. Mammalian genomes contain several pseudo sites, e.g, bacteriophage Crerecombinase is capable of integrating between phage loxP site and pseudo loxP site in human genome albeit with 4 fold lower efficiency.

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Figure 17.1Site specific recombination by Crerecombinase:

SSRs from phages (Cre) and from yeast (Flp) catalyses both excision and integration in equilibrium and because the excision reaction isfavored this results in inefficient integration and subsequent expression. Other SSR like phageγintegrase create hybrid recombination sites and require separate excisionase for its function. Some phage SSR like those from λ, HXO22 require cofactors like IHF for recombination whereas other SSR like those from phage R4, TP901 and ΦC31 donot require cofactors and are capable of stable integration.

ΦC31 SSR is the most efficient non mammalian SSR for human gene therapy. This recombinase recognizespseudositepsA in human genome and mspA and mspL1 in mice and is capable of treating recessive genetic disorders. ΦC31 is capable of integrating large gene like alpha typeVII collagen (COL7A1) and dystropin gene to be used in the treatment of RDEB and Duchenne muscular dystrophy.

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Figure 17.2 Mechanism of integration of therapeutic gene in human genome using site specific recombinases:

Prokaryotic origin of SSRs limits their application in eukaryotic hosts. Efficient transgene expression might be improved by using exogenous nuclear localization signal. Moreover, directed evolution is another approach to improve transgenesis by prokaryotic SSRs. In this technique the SSRs are subjected to multiple rounds of random mutagenesis and screening. Flpe, a derivative of Flprecombinase from yeast was developed using directed evolution. Various high throughput methods have now been developed to improve SSR activity that includes FACS and PCR based methods. An attractive alternative of prokaryotic SSRs is Adeno Associated Virus (AAV). AAVs are non pathogenicmammalian virus and used viral encoded Rep protein to target transgene into specific sites called AAVS1, present on chromosome 19 in humans. The rep and cap genes in wild type AAV are bounded by 145 bp inverted terminal repeats (ITR). Rep is a replication protein with auxiliary recombinase function. Of the five alternatively spliced rep products only Rep68 and Rep78 catalyze site specific recombination by forming complexes with AAV ITRs

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and AAVS1 locus, followed by DNA strand cleavage and non homologous recombination.

Figure 17.3 Rep protein mediated site specific recombination in Adeno- associated virus:

The use of AAV is limited by certain level of immunogenicity it induces in humans and restriction in packaging 5 kb gene fragment. Recombinant AAV can be efficiently used in human gene therapy where the rep and cap genes are replaced by therapeutic genes. Plasmid containing Rep68 and Rep78, delivered by non-viral vectors along with ITR flanked transgene can efficiently integrate the corrective gene in AASV1 site in human genome. Constitutive expression of Rep is cytotoxic and cytostatic and sometimes results in abortive rearrangements of AASV1. To overcome this problem Rep can be provided in trans, from a cotransfected plasmid that lacks the Rep binding element. Alternatively the expression of Rep in host cells can be modulated by using ligands capable of inducing Rep or the protein can be externally delivered along with the plasmid to catalyze single integration event.

Retroviruses are another major source of integrase for human gene therapy. Retroviral integrase can be fused with sequence specific DNA binding proteins, such as phage γ repressor, E.coliLexA repressor, the Ty3 tRNA binding domain or designed Zn finger protein E2C for use in gene therapy.

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17.6 Extra chromosomal replicating units in transgenesis DNA molecules that can be replicated and segregated to daughter cells in an autonomous, extrachromosomal form avoiding insertional mutagenesis can be developed as another method of transgenesis in gene therapy.DNA viruses such as SV40 and Epstein–Barr virus (EBV) normally replicate episomally in mammalian cells and transfer viral episomesto daughter cells during cell division. Episomal plasmid vectors with viral ori sequence and requisite viral proteins (like Tag and EPVNA1) have been generatedthat can sustain the expression of a therapeutic transgene. For example, EBV-basedepisomal plasmid with 115 kbhypoxanthine phosphoribosyltransferase (HPRT)locus that was stably expressed in HPRT- deficienthuman lung fibroblast cells.Unfortunately, use of such episomes in human gene therapy is restricted as viral proteins such as Tag, alter the function of other key host cell proteins interfering with the retinoblastoma and p53 tumour- suppressorpathways. To overcome this problem ‘pEPI’ plasmid containing an SV40-ori sequence and scaffold/matrix attachment region (S/MAR) from the human β-interferon gene cluster has been developed that can propagate episomally, for several hundred divisions in CHO cells and HeLa human cervical cancer cells. Mitotic stability in pEPI results from the interaction of the S/MAR element with components of the nuclear matrix which is crucial for the organization of ‘boundary elements’ to protect their coding regions and to allow transcription factors to access enhancer and/or promoter sequences. Interaction of pEPI is mediated by binding of S/MAR to scaffold attachment factor A proteins, allows co-segregation of pPEPI with mitotic chromosomes and brings it closer to host replication machinery facilitating its own replication along with the host cell cycle. Transcription and replication are linked in pPEPI. Transcription of the AT rich S/MAR sequence is important in opening of chromatin structure to allow replication of pEPI and thus replication of this plasmid is not solely dependent on the SV ori. This property of chromosomal components like S/MAR can thus be exploited to develop novel episomally replicating plasmids for sustained expression of therapeutic genes in gene therapy.

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Figure 17.3 Plasmid map of pEPI:

17.7 Human artificial chromosome

Artificial chromosomes are replica of normal chromosomes that are capable of replication and retention at low, defined copy number and within host cells. They have three main parts: a centromere, telomeres at both termini, and ori of replication. These chromosomes have exceptionally high cloning capacity and thus provide an excellent approach to be used as vectors in gene therapy. The first prototype HAC was generated in1997.

Human Artificial Chromosomes (HAC) can be synthesized de novo by assembly of the major components or by truncation of mammalian chromosome down to mitotically stable minichromosomes of 1-10 Mb, consisting of alpha satellite DNA arrays. Also neocentromeres based chromosomes provide another alternative of synthesizing HAC. Neocentromeres are functional variant of human centromeres and naturally present in non-centromeric region that are devoid of alpha satellite DNA. Neocentromere based human minichromosomes are 0.7-2 Mb in size that bind to centromere protein and are mitotically stable.HAC have been used in several gene therapy studies, for example in long term expression of 40 kb HPRT gene in Lesch- Nyhan syndrome, 250 kb CFTR gene and regulatory sequence in treating cystic fibriosis.

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Because of its large size, microcell mediated chromosome transfer and microinjection are used to transfect HAC into different cell types in ex vivo approaches. Conventional non viral approaches make use of liposomes along with polyethylenimine or polylysine to deliver artificial DNA but are of lesser efficiency in vivo. The use of lipids with ultrasound has recently been demonstrated to induce better membrane pore formation allowing entry of DNA. Addition of NLS sequence either to the delivery agent or HAC might further improve nuclear entry or help stable transfection.

17.8 Maintenance of transgene expression in host system

With evolution eukaryotic systems have developed mechanisms to maintain genome integrity and prevent foreign gene expression. Transient expression of transgenes introduced by viral or non viral vectors and episomal plasmids is in part due to de novo cytosine methylation by DNA methyltransferases that transcriptionally silent chromatin structures. Transgene expression is more favored by viral promoters as compared to human housekeeping promoters, but cytosines in viral promoters are main targets for cellular methyltransferases. Thus promoter development has been focused by mix-matching transcription factor binding sites for strong and long term expression. Also the addition of S/MAR sequence can provide position independent transcription and inhibit transgene methylation. In nonviral gene therapy the bacterial plasmid backbone used also contributes to transgene silencing. To overcome this problem phage recombination system have been used to form minicircle DNA that can be maintained as episomesin vivo and facilitate 560 fold higher transgene expression. mRNA splicing is another barrier to stable transgene expression that can be reduced by altered codon used to remove splice junctions. Moreover, immune response against accumulated foreign proteins in host circulation might restrict transgene stability which can be reduced by employing specific T cells, but further study is required in this aspect.

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17.9 Future prospects Non viral procedures of gene therapy mimic the basic viral mechanisms to achieve long term expression of the corrective gene without disturbing host gene expression and signaling pathways. The efficiency of targeted delivery is the key to clinical success of non viral gene therapy. The effectiveness and difficulties of such delivery can now be well determined in terms of quantification, magnitude and duration of reporter gene expression, using small animal imaging technologies in vivo. Safe and long term transgene expression is the ultimate requirement of human gene therapy. Today gene therapy is not only restricted in proving for a missing gene but being explored in various aspects like anti-cancer gene therapy etc. and tailoring non- viral gene delivery strategies might help in realizationof the potentials of human gene therapy.

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Lecture 18

DNA Vaccines

The earliest form of protection against the diseases has been in the form of vaccines. Discovery of vaccines by Edward Jenner in the year 1796 witnessed a revolution in human medicine. A vaccine is a biological formulation that confers immunity to a specific disease. The role of the vaccine is to stimulate the immune system, recognize the attacking agent, eradicate it and keep it in memory so that if there is any repeated exposure of the same disease then the destruction of such agent would be easier. There are several types of vaccines like killed, attenuated, live, subunit, conjugated and Nucleic acid vaccines.

Nucleic acid vaccine means the vaccination done using RNA or DNA vaccines. Here we are going to deal with DNA vaccines in full detail and RNA part will be summarized briefly. In a layman’s words DNA vaccine can be defined as the process of injecting manipulated DNA material in an organism to generate an immune response. In general DNA vaccines may be defined as those vaccines that provide immunity by transfecting host cells with DNA that encodes an antigen. Following transfection the specific immune response generated is along similar lines as responses that occur against conventional vaccines and this immune response arises by the protein produced by the host cell. The DNA vaccine seems to be a promising candidate as vaccines because they are very cost effective but still these are not listed for human use. There are some DNA vaccines for veterinary use though.

The reasons for announcing DNA vaccine as potential candidate are many but also when compared to other forms of vaccine it proves to be less cumbersome and offers many advantages too enlisted below-

1) It is easy to form and construct the DNA vaccines as compared to attenuated viruses and subunit protein vaccines. 2) It is cheap and cost effective. 3) It is quite stable at room temperature as compared to attenuated viral vaccines, whose repository and international transfer becomes difficult by the urgency to keep the vaccines cold.

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4) The protection offered by DNA vaccines favors bias towards cellular immunity, which is trusted to be vital for successful vaccination against intracellular pathogens.

The classic methods for synthesizing vaccines are given in Table 1 which highlights their similarities or differences with DNA vaccines.

Table 18.1 Comparison of different vaccine technologies: Live attenuated viruses Very effective Potential risk for certain ones, Manufacturing challenge Viral vectors Potential risk Resistance/pre-existing antibody, Inflammation Recombinant proteins Effective antibody response, Effective, Non-native forms sometimes do not induce cytolytic T lymphocytes DNA vaccines Inevitable need for increased potency Designer immune responses Specificity: avoid deleterious or diversional antigens Relative stability Safety Generic manufacturing Cost advantage

Despite the fact that viral vectors and DNA vaccines have parallel attributes as given in Table 1, they are only in their initial stages of clinical progress.Currently researchers worldwide are working on designing the vaccines in which the induction of CD8+ cytolytic T lymphocytes (CTL) responses and antibodies is emphasized due to the prominent role of CTL in such vaccines. Similarly designing of vaccines that can induce specific types of T helper responses, Th1 or Th2 are encouraged for the same reasons.

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Figure 18.1 Mechanism of cytotoxic T cells, helper cells and antibodies:

The diagram depicts the intracellular and intercellular interactions needed for the antigen to produce both cytotoxic and T helper cell responses as well as the production of antibodies. The general fate of a recombinant protein or an inactivated vaccine is that it is recognized by antigen presenting cells, then degraded into peptides and further allies with the major histocompatibility complex (MHC) class II molecules and this is the reason why such vaccines do not produce a required CTL response. In future these peptide or MHC complexes trigger T helper cells. For the production of CTL response, the viral infected cell must enter the cellular processing pathway from the cytoplasm so that the peptide gets associated with MHC class I molecules. These peptides subsequently are identified by specific cytolytic T cells and then are stimulated to kill the infected cell. Thus after successful delivery of a gene encoding an antigen into the cell, cytoplasm would be the site where the synthesized protein would be located and if such protein enters the intracellular processing pathway it can result in desired CTL response after getting associated with MHC class I molecules. Live vaccine virus is the best example to achieve this gene transfer with desired CTL response. Somehow this delivery method is not applicable to many virulent diseases as the live attenuated virus vaccines have the tendency to revert back to the wild type or they may successfully down-regulate the immune response. HIV live virus vaccine for example can revert back to the virulent type and thus can be more dreaded and fatal.

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18.1 Features of DNA vaccines

It is the efficient machinery of the virus that allows it to introduce its genetic material into the host cell. This is the reason why it was unbelievable for many researchers that a simple plasmid could enter the mammalian cell and synthesize the desired protein. Use of DNA plasmids as gene delivery vehicles is very convenient as compared to other methods as it does need any modification except a promoter active in mammalian cells. Still the use of DNA vaccines is a topic of debate because of its safety concerns.

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Figure 18.2 Schematic representation of a DNA vaccine:

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Figure 18.3 Antigen presentation after DNA vaccination:

Following DNA vaccination communication between surfaces of the T-cell and the antigen presenting cells (Co-stimulatory molecules) as well as recognition of MHC class I complex by the T-cell receptor is must for a successful antigen presentation to a naïve T cell to stimulate the cell. Co-stimulatory molecules are absent in muscle cells and hence they do not behave like efficient antigen presenting cells. Usually if a naïve T-cell confronts a cell carrying a right antigen- MHC Class I complex in the absence of the co-stimulatory molecules or antigen presenting cells then the T-cells turn insensitive to this antigen if there is any repeated exposure to the same antigen.

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18.2 Trials with DNA vaccines:

After several failures the first fairly successful DNA vaccination result in humans was attained with a malaria- specific DNA vaccine. This trial validated the rise of vaccine specific T cells in the peripheral blood of 11 out of 20 malaria-naive volunteers after three intramuscular pDNA (plasmid DNA) inoculations. However the investigation did not evaluate clinical gain. Several trials have offered evidence of principle for the ability of DNA vaccines to induce humoral and cellular immune responses in humans. Although, the immune responses analyzed were not as good as expected from the preclinical studies in any of these trials. For example, HIV-infected patients with huge viral counts generated a normal T-cell response against HIV Nef following DNA vaccination with a DNA vaccine encoding several HIV antigens, with no effect on viral counts.In an attempt to blend the qualities of DNA vaccines with those of adenoviral- or MVA-based vaccines, so-called ‘heterologous prime-boost’ systematic plans have been developed. In these plans the potent but broad immunity induced by MVA- or adenoviral- based vaccines gets focused on the appropriate antigens by DNA vaccination which is highly specific. Recently investigations based on DNA vaccines have shifted largely to tumor antigens and the reason behind it might be the excelling funding opportunities in this field.

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Figure 18.4 Techniques of delivering DNA vaccine:

Due to discouraging results in the clinical trials, the DNA vaccination field is putting a lot of attention on upgrading the delivery methods, carrier molecules and genetic optimization of the construct used. Generally administration of DNA vaccines is done either by intramuscular (IM) or by intradermal (ID) route. Following IM route the antigen mainly is generated in muscle cells and this route promises high antigen expression but does not prove to be very immunogenic because muscle cells generally lack antigen presenting cells. On the contrary ID route has scanty antigen expression but it is much more immunogenic as compared to IM because skin serves as the essential gate for the entry of pathogens and is always abundant with antigen presenting cells.Various other devices have been designed for efficient DNA delivery like ‘Gene Gun’. Along with the gene gun techniques like electroporation and “Particle mediated epidermal delivery” (PMED) are also in use.These are also known as Second generation DNA vaccines. Multiple projects working on DNA vaccine rely

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on different methods. Various other techniques require the use of naked pDNA wherein it is worked out in a nanoparticle composed of lipids or polymers so that pDNA stability increases and thus resulting in higher cellular uptake.

18.3 Attempts to improve DNA vaccines

Efficacy of DNA vaccines can be improved by enhancing the immunogenicity of the encoded antigen. This can either be done by adding a genetic adjuvant or by altering the gene encoding the antigen itself. Usually a genetic adjuvant is added to increase the immunogenicity of the antigen and enhance the effects of vaccine. HGMB162 is an example of a genetic adjuvant. Immunogenicity of the antigen can also be increased by codon optimization, addition of signal sequences and genetic fusion to an entire protein referred to as carrier protein.Codon optimization is defined as the technique in which the gene encoding the antigen is edited for optimal transcription and translation in the species that the vaccine is meant for. With the repetition of the genetic code the optimal tRNA for any amino acid differs from species to species. Specifically when innate prokaryotic or viral genes are used in DNA vaccines, codon optimization can considerably enhance its transcription in the eukaryotic cells of the vaccinated host. Addition of signal sequences aims the antigen to various subcellular compartments, hence increasing the immunogenicity of DNA vaccines. Fusion of the antigen to a carrier protein is a device that is often used and required in the construction of DNA vaccines. (Table 2).

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Table 18.2 Popular carrier proteins used for fusions with HPV-16 E6 and E7:

Carrier protein Antigen Proposed mode of action

Mycobacterium E7 Provision of CD4+ T-cell tuberculosis, HSP-70 help, Increased antigen uptake by DC Heat shock protein 60 E6, E7 Increased antigen uptake by DC Calreticulin E6, E7 Targeting of antigen into the antigen presentation pathway Extracellular domain of E7 Altered subcellular localisation Flt3 ligand HSV VP22 E7 Antigen spreading , Improved antigen stability TTFC E6, E7 Increased antigen stability IP-10 E7 Enhanced antigen presentation, chemoattraction Invariant chain with E6 Provision of CD4+ T cell PADRE epitope insertion help Pseudomonas aeruginosa E7 Enhanced cross exotoxin A (domain II) presentation E. coli β-glucoronidase E7 Enhanced stability

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18.4 How safe are DNA vaccines

Usually DNA vaccines are granted safe for both patient and environment. So far limited local reactivity at the injection site is the only drawback in the studies done. However due to the use of genetic vaccines there is a potential threat that the genome of the plasmid may get integrated into host genome of somatic cells and may lead to the production of transformed cells or oncogenes. To evaluate the potency of the associated risk, the parameter generally set is to compare the integration rate of pDNA with the spontaneous mutation frequency of autologous genes.This mutation rate may vary from person to person but overall, 2 x10-6 spontaneous gene- inactivating, mutations per gene is usually approved as the standard value.There has been no evidence of elaborated studies carried out for genomic integration of DNA vaccines in humans which might be partly due to hurdles to obtain a biopsy from the administration site. However such studies have been carried out in many lab animals and all these studies have shown that the integration rates are always several folds less than the spontaneous integration rate.

18.5 Secondary role of DNA vaccines

Although DNA vaccines are still not exposed to human population openly but it has many other roles to carry out. It may be because the plasmid DNA can be used in vitro or in vivo. This property of DNA vaccines enables them to design monoclonal and polyclonal antibodies. Other experiments that require the use of DNA vaccines are the making of Knock-out mice models and also for developing DNA vaccine libraries to use them to know which genes encode protective antigens without any info about its corresponding protein.

18.6 RNA vaccines

RNA vaccines have been known in the field of vaccine development is quite well known. Over the past 25 years, many clinical trials of DNA in the form of plasmid and viral vector based vaccines have showed us a safe and efficacious way to deliver many foreign antigens. Yet, plenty and satisfactory potency for general efficacy in humans has remained elusive for DNA and RNA vaccines and the practicality of repeated use of viral vectors has been compromised by anti-vector immunity. RNA vaccines, including those based on mRNA and self-amplifying RNA

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replicons, have the tendency to surpass the restrictions of plasmid DNA and viral vectors. Possible difficulties related to the cost and feasibility of synthesizing RNA vaccines are being addressed, increasing the likelihood that RNA-based vaccines will be commercially available. Proof of concept for RNA vaccines has been shown in humans and the prospects for further progress into commercial products are very motivating.

18.7 Conclusion

The brief study done here indicates that the science of DNA vaccines is spreading rapidly with second generation formulations, delivery vehicles and a promising approach towards the development of new vaccines and immunotherapeutics. Their role has witnessed an encouraging participation as research tools and diagnostics. Further it is expected that with the ongoing human trials and biological engineering of genomes many emerging and fatal diseases will disappear totally.

Genetic Adjuvant

A genetic adjuvant is a protein with adjuvant properties that is encoded by the pDNA together with the antigen and hence co-expressed with the antigen, enhancing the immune response towards this antigen.

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Lecture 19

Lipoplexes and polyplexes (part I)

19.1 Introduction

It is very challenging to deliver nucleic acids to target cells and tissues. Non viral and viral methods are being used for this purpose, former being a potential alternative for the latter because of certain disadvantages of the viral vectors like immunogenicity, high cost, limited amount of DNA which can be carried for recombination. However, non viral vectors too suffer in certain areas like low efficiency for gene delivery as in for in-vivo applications. Most used molecules are cationic lipids and polymers which comprise more than 50% of the transfection agents available and used. Anionic plasma membrane attaches to the complex and gets absorbed well by electrostatic interactions in mammalian membrane. As compared to anionic liposomes and other agents, thelipoplexes yield high efficiency of transfection. Though nuclear transport of these complexes is not well understood but is being studied. There are a number of factors which determine success of delivery of the DNA such as,supramolecular structures, cell membrane and DNA interaction, chemical structure, intracellular localization and internalization, and DNA release.

19.2 Methods to characterize structure and mechanism of the reagents

In order to understand potential mechanism of transfection, we need to identify and decipher the structure of the DNA-cationic lipoplex complex. Light scattering techniques are used in order to understand the colloidal properties; ethidium bromide is used to analyze DNA condensation using fluorescence. Phase behavior of the lipid is studied by differential scanning calorimetry (DSC) while the topography of condensed matter is studied using electron and scanning probe microscopy. Nuclear magnetic resonance (NMR) and electron spin resonance (ESR) determines the local structural and kinetic features of the polymer part or segment. High resolution methods like small angle X ray scattering in combination with electron microscopy is used to study the DNA-lipid complex. Laser scanning confocal

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microscopy analyses release ofDNA while quantitative structure-activity relationships can be used to studymolecular gene delivery.

19.3 Mechanism of transfection of cationic lipids and polymers

Nano-sized lipoplex or polyplex complexes are formed by combining cationic lipids and nucleic acid in suitable buffer system. Electrostatic interactions bind the lipid complex and the cell surface together being of opposite polarity. Once it enters the cell, endosomal low pH leads to release of the nucleic acid from the lipoplex into the cytoplasm. This cytoplasmic transport is required for bringing the transfected material in the perinuclear region. DNA then enters the nucleus by one of the following two mechanisms; (1) passive transfer during cell division when nuclear membrane ruptures,and(2) via nuclear pores by active transport. Nuclear uptake has been a kind of barrier to gene delivery. Cationic lipids destabilize the membrane of the endosomes which leads to initial DNA release in cytoplasm. For efficient transfection, dioleoylphosphatidylethanolamine (DOPE) or similar kind of fusogenic peptides are required. The flip flop of transbilayer phospholipids that are negatively charged leads to charged neutral ion formation which releases DNA from the complex by weakening the electrostatic interactions. Endosomal escape has been attributed to the destabilization of endosomes (detergent like) which cannot be done by cationic lipids as they lack hydrophobic domain.

19.4 Formation, structure and stability of lipoplex

19.4.1 Structure of cationic lipids:

Large amount of DNA and lipids can formlipoplex by interacting with each other. Lipoplex is basically composed of cationic lipids and neutral lipids (helper lipids). Neutral lipids used are usually DOPE and cholesterol whereas cationic lipids consist of amphiphillic molecules having positive polar head connected with hydrophobic domain with a connector. At neutral pH as well as physiological temperature DOPE is in the form of hexagonal inverted phase (HII) structures. Along with the cationic lipid, it forms a bilayer. When negatively charged molecules interact with the cationic lipids, phase change occurs, which leads to destabilization of the cellular membrane and non bilayer structure and thus cytoplasmic delivery of DNA.

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The cationic liposomes containing cholesterol were found to be more stable thus enabling intact DNA to reach target site by facilitating transfection and protecting DNA from degradation. Hydrocarbon chains are of various types among which the two most common types are C8:0 and C18:1. The connector or linkers used are usually carbamate or amide variety which is biodegradable and chemically stable. For transfection efficiency the major parameters for cationic head group are size and charge.Lipopolyamines were developed in order to bind with compact DNA molecules. Acid labile molecules yield enhanced transfection than acid resistance analog. Disulfide cationic lipids were formed to increase intracellular release of DNA from cationic liposomes and to reduce the cytotoxicity. The disulfide bond gets reduced in the presence of strong reducing environment inside the cell thus collapsing carrier-DNA complex toenhancethe release of DNA.

19.4.2 Self assembly and formulation of lipoplex:

Preparation of liposomes can be done by lipid hydration, dehydration- rehydration ethanolic injection, reverse phase evaporation etc. geometry of the lipoplex is greatly determined by the molecular shape of the lipid molecules for example large headgroups and smaller hydrocarbon tail would prefer cone like structure. Cylindrical ones have equal size of hydrocarbon chain and headgroup. The one with smaller headgroups form inverted phases. Roughly the concentration, temperature, mixing kinetics and environment affectsthe lipoplex formation. Liposome formation is an entropy driven process which has two steps. The first one is exothermic and reversible process where electrostatic interactions take place and the other is the endothermic, slow irreversible process which involves fusion of the liposomes and their rearrangements.

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19.4.3 Structure of lipoplex:

Different structures of lipoplexes are revealed by X-ray diffraction studies. The planar lipoplexes have two types, the one with cationic membranes sandwiching the DNA layer and the other is an inverted hexagonal structure.The formation of hexagonal phase in lipoplex after the interaction of anionic lipid is important in order to translocate the nucleic acids into the cytoplasm.DOTAP/DOTMA/cholesterol increases the transgene expression by four folds.Liposome mediated gene delivery does not only depend on the cationic liposome formulation but also on its interaction with the cell which changes its final structural organization.

19.4.4 Charge and stability of lipoplex:

Ratio of anionic DNA phosphodiester bonds and cationic charges together form anionic, neutral or cationic complex. High transfection efficiency is seen for positively charged lipoplexes. The negatively charged proteoglycan of the cell surface interacts with the cations of the lipoplex by electrostatic interactions. If the molecules get neutralized, they aggregate into large molecular assemblies which might reduce gene expression.Thus high positive:negative ratio increases the transfection efficiency remarkably. These are usually purified because if not then free liposomes can compete with lipoplexes for binding to the cell. Charged lipoplexes are found to be more stable at low ionic strength. Polyethylene glycol like hydrophilic polymers can be used to coat uncharged particles and thus provide them stability and prevent self aggregation.

In the natural scenario, before the liposomes get inside the cell, they are degraded and DNA is released breaking the lipoplex structure in presence of the serum.PEGylation prevents the degradation but affects transfection efficiency. The DNA becomes transcriptionally active when it is released in the cytoplasm.DNA is removed from the lipoplex and hexagonally packed DNA polyamine particles are formed in bulk in the cytosol.F-actin and other negatively charged molecules might also help in dissociation of the lipoplex DNA complex.

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Lecture 20

Lipoplexes and polyplexes (part II)

20.1 Structure-mechanism relationship of lipoplex

Lipoplexes are liquid crystalline globules as studied by fluorescence microscopy. Lipoplex after entering the cell by endocytosis cause DNA to remain trap

in the endosome. Charge of cationic liposome vector σM governs the transfection

efficiency. As σMdecreases, the transfection efficiency increases. When the σMis low,

then the lipoplexcontaining DNA remains intact. Whereas when σM is high, the DNA is released in the cell. When liposomes contain DOPE, the transfection efficiency is considerably increased. Mixed phospholipids have anionic lipids and cholesterol

along with DOPE to promote HII phase organization.

20.2 Formation, structure and stability of polyplexes

20.2.1 Structure of cationic polymers

Cationic polymers like histones are naturally present in the biological system. Polyethylenimine (PEI), cationic dendrimers, 2-dimethylaminoethyl methacrylate (pDMAEM), and chitosan are some of the synthetic cationic polymers. Most widely used polymers for gene delivery includes poly L-lysine (PLL) and PEI, wherein PEI efficiently delivers the transgene and leads to a permanent expression specifically only in the target region.Cationic polymers form a complex with DNA and condense it to a small size, which is an important parameter for in vivogene transfer.

Cationic polymers unlike cationic lipids lack hydrophobic domain so cannot fuse with endosomal membrane. Polylysine and polyarginine are first generation cationic polymers. They possess a weak endosomal escape and transfection capability, their transfection requires simultaneous co-transfection of endosome lytic agents such as inactivated adenovirus. Second generation polymers include PEI and polyamidoaminedendrimers(PMAM), which acts as proton sponge causing endosome disruption.

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The PLLs are biodegradable in nature which enables its use for in vivo application such as gene transfer. Howeverit binds to plasma proteins and is rapidly removed from circulation. To avoid, PLLs are usually coated with PEG which enhance their transfection efficiency and half life.Other modifications which can be made to improve the transfection efficiency include a targeting ligand coupled with PEG and introduction of histidine residues to create proton sponge effect. The chitosan, carbohydrate based polymer, can also condense DNA into small size to form a stable particles which can be used as a transfection reagent. Chitosan polyplexes are more effective for long termexpression. Starburst polyamidoaminedendrimers are spherically branch shaped structures used for gene transfer,its efficiency depends on shape, size, primary amine groups, and cell type. Fractured dendrimersare formed by heat degradation and are more flexible then dendrimers. Fractured dendrimers have better transfection ability.

Different approaches for designing and optimizing chemical structures are being formulated. First one is based on the biological barriers for gene transfer that includes rational designing of chemical structure. Second approach is its structure activity relationship by modifyingits chemical structure. Molecular weight, branching, and surface charges are considered for designing of polymer.

20.2.2 Formulation of polyplexes

Polyplex formulation effects transfection efficiency and stability. It is a kinetically controlled process, where successive addition of components, polymer and DNA effectsthe polyplex size and its transfection efficiency.PEI interacts electrostatically with DNA and RNA to form a stable complexwhen condensed. Polymer characteristics like molecular weight, density of charges and the composition of complex influence the process of complex formation and condensation. For instance, low molecular weight and lower charge density decreases condensation. The medium composition also affects the complexformation process. Ionic strength of saline solution controls the size of complex formed, higher the concentration larger the complex and less its binding efficiency.Sodium phosphate is a better carrier when compared to NaCl or phosphate buffered saline (PBS). Higher the ratio of positive to negative charge on the cationic polymer, better is the compaction process. At charge

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ratio1, DNA is bound completely with the complexwhile at neutral state it aggregates and shows low solubility.

20.2.3 Structure of polyplexes

DNA-PEI condensates are spherical, globular or rod like in the presence of polycations. Toroid shaped structures are formed when unmodified polylysine condenses DNA. The nature of complex formed depends on the degree of branching, as linear structures have lower rate of transfection compared to branched structures. For example PEIs having high branching form small structure with high transfection ability but are toxic. More branching with secondary and tertiary amine groups enhance transfection efficiency and decrease toxicity.

20.3 Charge and stability of polyplexes

Solubility of polyplexes increases on increasing the number of polycationic charge because of the formation of hydrophilic cationic core around the polyplex. At neutral charge polyplexes based on PEI and DNA show no solubility. The final structure and transfection capacity depends on the storage conditions employed for polyplexes. For instance, storage for 3 weeks improves the transfection of polyplexes by highly branched PEI derivative due to stronger electrostatic interaction. Salt causes a charge shielding effect when used at high concentration and thus decreases binding between DNA and polymer used. Polymer size is an important factor in determining transfection efficiency and is also considered for biocompatibility and extravasation when used for targeting cells outside vasculature. The size factor is itself based on the structure and the complex nature of polyplexes. Fully complexed structures are condensed completely. Condensation rate is high for high molecular weight, longer, and branched chain polymer. Completely condensed structures are protected from degradation in extracellular environment and are easily taken up by the cell through electrostatic interaction. Large PEI-DNA polyplexes consist of aggregates of smaller units. As time increases positively charged polyplexes aggregates more rapidly. Surface charge, molecular weight and ionic strength of the medium also influence the aggregation. The components which shield the charge, decrease interaction and hence aggregation, thus inhibiting rapid elimination by the reticulo-endothelial system. The N/P ratio controls the aggregation of complexes. Hydrophobic interaction and van-der

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waal forces cause aggregation in complexes with low N/P value while high N/P ratio decreases aggregation because of electrostatic repulsion between positive charge surface present on the complex.

20.4 Structure –mechanism relationship in polyplexes

Cationic polymers lack hydrophobic group and are completely soluble in water, unlike cationic lipid. These can be easily synthesized with different modifications and of different length. Hence various structure function relationship studies can be done based on the different stages of transfection.

Figure 20.1Some of the cationic lipids used in the gene therapy:

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20.5 Cell binding and uptake

Polyplexes enter the cell and interacts with anionic proteoglycans by a transmembrane protein called syndecans. The presence of cationic surface charge is essential for binding and cellular uptake. The type of cell and polymer used, determines the internalization of the complex and thus its transfection efficiency. Linear PEI-polyplexes uses a clathrin-coated pit pathway for transfection in African green monkey kidney (COS-7) and hepatocellular carcinoma cells (HUH-7). Both lipid raft and clathrin dependent pathway are used by branched polyplexes such as in HeLa cells. But the lipid based pathway is much more efficient in transfection.

20.6 Escape from endosome

PEI based polyplexes and PMAM have high transfection efficiency as they behave as proton sponge. Proton sponge hypothesis states that only few nitrogen atoms are protonated at normal physiological pH. When the pH gets lowered such as in endosomes, more number of nitrogen atoms get protonated and a gradient is created that causes influx of chloride ion. The increase chloride ion influx causes water influx and thus endosome swells and gets ruptured. Proton sponge activity is absent in polymers with buffer capacity at pH 5. Polymers based on PEI and PMAM buffers the interior of endosomes leading to decreased acidification, high chloride content and increased volume. Protonable amine groups if removed decreases transfection efficiency.

20.7 Dissociation of polyplexes

After the endosomal escape polyplex must reach nucleus and also DNA need to dissociate from carrier. Thus polyplexes having low molecular weight and tending to dissociate rapidly have better transfection ability as compared to high molecular weight polyplexes. PEI based polyplexes shows slow dissociation from DNA when analyzed using fluorescence microscopy. Reducible PLL polymers have been made that can be degraded by intracellular environment causing fast releases of DNA and its enhanced expression.

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20.8 Nuclear import

Nuclear import through passive process occurs when nuclear membrane disassembles during cell division. Transfection efficiency depends on cell cycle state. Such as linear PEI transfection does not depend on cell cycle state while branched chain polyplex does. PEI has the ability to facilitate nuclear translocation of DNA because microinjected PEI-DNA polyplexes have higher transfection ability when compared to naked DNA or lipoplexes.

20.9 Summary

Lipoplexes and polyplexes can be optimally designed to be used as an efficient non-viral vectors for gene therapy and disease control. There is a need for development of an ideal vector for gene delivery, which could be tailored according to the need considering its in vivo use, stability, and easy transport across membrane. Lipoplexes and polyplexes are such molecules which could be used as vector.

Lipoplexes transformed into hexagonal H11 phase have high transfection activity when compared to those in lamellar L phase. DOPE containing complexes aggregate in blood and thus cannot be used for systemic application but are good for cell culture work. Serum resistant lipoplexes and polyplexes are being formulated which are more stable in blood. Completely condensed DNA with the polymer has advantages of being protected from degradation, enhanced binding ability and improved endocytosis. But the problem lies with the dissociation of DNA from carrier inside cell. Thus there is a need of rational designing of vectors with efficient transfection ability.

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Lecture 21

Naked DNA

The simplest way of a non-viral gene delivery system is by using a naked vector DNA. Direct injection of DNA into a cell or tissue produces high levels of gene expression. Though it leads to gene expression but level of expression is much lower than with either viral or liposomal vectors. It is unsuitable for systemic administration due to the presence of serum nuclease. Therefore its application is limited to muscle cells and skin. However the efficiency of DNA injection in the muscle injection is variable. In spite of huge amount of research, the efficiency of DNA injection in muscle has not proved efficient enough to go for the clinical level. Several researchers have shown that the naked DNA can be efficiently delivered to cells by various means such as electroporation and intravenous injection. It is particularly applied to cancer tissues where the DNA can be injected either directly into the tumor or can be injected into muscle in order to express tumor antigens that might function as a cancer vaccine. It can also be used to treat genetic diseases in the tissues which are available for direct injection such as skin.

21.1 Progress in naked DNA based gene delivery

Naked DNA is generally introduced into a muscle cell by transfection in vivo which allows the enhance uptake of DNA into muscle and skin. Intravenous delivery of plasmid DNA results in a high gene transfer to liver cells. Plasmid DNA (pDNA) delivery to tail vein is another simple and effective way of transfecting liver cells in rodents. Effective transfection of skeletal muscle cells in rodents, canines, and monkey is achieved via intravenous delivery of plasmid DNA. Many commercial pDNA expression vectors allows high-level sustained expression of DNA into different organs. The mechanism of intravenous DNA delivery is thought to involve active pDNA uptake by a targeted cell. Naked DNA delivery has now entered into many clinical trials, for eg in treatment of peripheral arterial occlusion disease. Small interfering RNA can be delivered very efficiently by intravenous route and results in silencing of targeted gene expression.

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21.2 Mechanism of action of naked DNA

The plasmid DNA after injection or electroporation crosses the cell membrane. The DNA needs to go inside the nucleus in order to transcribe the mRNA by using RNA polymerase II which further migrates to cytoplasm for protein synthesis. The transfer of the DNA to nucleus is facilitated by nuclear pore.

21.3 Electroporation of plasmid DNA into muscles and skin

DNA can be efficiently introduced into muscle and skin in vivo by electroporation. Electroporation of DNA has been known to increase the transduction efficiency by 10 fold higher as compare to normal DNA injection. It is still not clear whether the efficiency of electroporation is more because of more expression level or with more cells transfected at the same time. Diseases like anemia and muscular dystrophy is successfully targeted by using naked DNA.

21.4 Intravenous injection of plasmid DNA

Intravenous administration of gene in the form of plasmid DNA is highly efficient way to transfer the DNA to the target site. The intravenous injection distributes the gene to each and every cell of the body within no time because of the involvement of circulatory system. Intravenous delivery may be systemic in case required for the whole body or local for specific tissue or organ system. Intravenous delivery of cationic-DNA complex results in efficient gene expression in the endothelial lining of the blood vessels and to hepatocytes. The expression of transgene to the hepatocytes following the intravenous injection is mostly directed by the portal system.

21.5 Plasmid loss during intravenous administration

Injection of plasmid DNA to a particular organ induces a local damage to the cells and sometime expression of the transgene gets vanished because of the cell death. Moreover the pathological condition occurs and restores after few days because the injections are always transient. Intravenous injection causes the increase in cell cycle that leads to loss of non-integrated DNA. In addition the replication of cells also causes loss of plasmid DNA. Many a time the inactivity of the tissue specific promoter present in the plasmid also causes loss of gene expression. Another very

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important way of losing the plasmid DNA is by the immune response produced following the expression of the protein from the transgene.

Some facts:

The mammalian DNA contains CpG sequences that are methylated while the bacterial DNA is not methylated. Therefore the plasmid DNA originated from bacterial origin shows an immune response after entering into the mammalian system. Minimizing the CpG sequence in a plasmid construct decreases the immune response and increases the transgene expression.

In general cells are originated from three lineages, ectoderm, mesoderm, and endoderm. The integrity of all the three types is lost when grown in cell culture flask in vitro.

21.6 Small interfering RNA (siRNA)

RNA interferences (RNAi) is a powerful tool to knockdown the expression of targeted gene. The mechanism of RNAi is mediated by dsRNA molecules having sequence identity with that of the targeted mRNA. The same phenomenon can be repeated in mammalian cells by using 21-25bp small interfering RNA (siRNA). The use of siRNA is to subvert the immune response that is otherwise mediated by dsRNA. The transfection of siRNA to the susceptible cells reduces the protein expression in the targeted tissue by 90%. Many targeted proteins are down regulated by use of siRNA technique including lung, heart, and liver. The delivery of siRNA is far complicated than the plasmid DNA because of its small size and possibility of its leakage while injection through intravenous route. The duration of siRNA in the tissue is still an issue. Its half life time and bioavailability in the tissue is responsible to modulate its activity.

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21.7 Prospects in naked DNA based gene delivery

Non viral gene transfer will become more important as better delivery methods become available. Tail vein injections in rodents will become a widely used technique for rapidly testing expression vectors and gene therapy approaches. RNA interference will become a major element in the gene therapy field.

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Lecture 22

Transposons

22.1 Introduction

For non-viral gene delivery to mammalian and human cells DNA based transposon vectors offer a varied mechanism. These DNA based transposon vectors work either by “cut-and-paste” or by “copy-and-paste” mechanism.Transposase enzyme helps in integrating a transgene(s) which is present in transposon DNA into chromosomal DNA. Sleeping beauty and piggyBac are the two most commonly used transposon system used for genetic modification of mammalian and human cells.Ease and relatively low cost of producing sufficient amounts which is required to meet the entire patient population, less storage problems, less innate immunogenicity, and easy co-delivery of multiple genes unlike viral vectors are some of the main advantages of transposon vectors.Transposons have shown good results in genetic modification of cell types of various clinical grades such as induced pluripotent stem cells, human T lymphocytes and stem cells. Integration of transposon DNA cargo by user-selected and site-directed genomic integration is the on-going research which is focused on manipulating transposon systems thereby improving safety and efficacy of transgene delivery.

22.2 Transposonsas gene delivery systems:

Transposons or mobile genetic elements as “jumping genes” which are responsible for mosaicism in maize (corn) were first described by Barbara McClintock. Transposons are found in all eukaryotes. In humans around 47% of the genome is derived from transposons. Class I (copy and paste) and Class II (cut and paste) mechanisms are the two means by which transposons work in eukaryotes.

In Class I mechanism a copy of transposon itself is made via an RNA intermediate and hence they are also known as “retrotransposons”. In Class II DNA transposons, the enzyme transposase excises the transposon which gets relocated to a new locus by creating double stranded breaks in situ.In this mechanism, inverted terminal repeat sequences (IRs) are recognized by the enzyme transposase.

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22.3Sleeping beauty (SB) transposon:

The genome of salmonid fish was reconstructed to make sleeping beauty (SB) transposon with the help of molecular phylogenetic data. SB transposon belongs to Tc1/mariner superfamily of transposons. Both the sides of sleeping beauty transposon are flanked by 230bp of inverted terminal repeat sequences (IRs) which have non direct repeats (DRs) in between them. It is one of the leading non-viral vectors for gene therapy.SB consists of two components: (i) a gene-expression cassette present in a transposon & (ii) a source for transposase enzyme. Sustained transgene can be achieved by transposing the expression of cassette from a plasmid into the genome. Treatment of epidermolysisbullosa, glioblastomamultiforme, sickle cell anemia and B-cell lymphoma have been done by using SB transposon. In rats, pulmonary hypertension and jaundice have been treated with the help of SB transposon.Various improvements that have been done for the use of SB transposon in clinics are:

(i) Efficacy: to achieve appropriate expression of therapeutic or reporter genes and SB transposase more significance is given to efficiency of transposition and engineered cassettes. (ii) Delivery: the method and routes that are being used for plasmid delivery as well as for cell culture conditions in which gene transfer is done ex vivoincells which are therapeutic. (iii) Safety: the time period of the transposition reaction and insertion preferences of SB transposes in cells which are transduced.

22.4piggyBactransposon:

It was isolated from cabbage looper moth Trichoplusiani. The significant feature of this transposon is the precise excision of the transposon from the donor site without any footprints leaving behind. This feature of piggyBac transposon makes it more attractive feature for cellular reprogramming. When the transposon is excised from the donor site, there is a creation of complimentary TTAA overhangs which can undergo simple ligation thereby regenerating the donor site without involving the process of DNA synthesis during transposition.

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Table 22.1 Comparison of sleeping beauty and piggyBac properties, TSS (transcriptional start sites):

sleeping beauty piggyBac

Capacity of cargo ~ 10 kB > 100 kB

Foot print Mutation of insertion site No mutation upon excision

Titration for optimal Yes Yes activity

Site preference integration Random Increased preference for genes and TSS

Modification of ‘N’ & ‘C’ 50% or more reduction in No reduction in efficiency terminal and their effect efficacy

Engineering the sites to Yes Yes bias integration

Hyper active versions SB100X (most active SB hyPBase version)

There are two types of gene delivery: (i) “cis” transposon mediated gene delivery (ii) “trans” transposon mediated gene delivery. The transposase enzyme is carried by the same plasmid having the same backbone of the transposon. The same vector background is used to carry transposaseand the transposon which carry the gene of interest (GOI). This is known as “cis” delivery. In “trans” delivery a separate circular plasmid carries the enzyme transposase.The transposase and transposon are delivered either in “cis” or in “trans” form in gene therapy purposes. But transposition efficiency is more in the case of “cis” configuration.

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22.5 Advantages of transposon in gene delivery system:

(i) Low cost as compared to viral vectors

The extensive uses of clinical grade viral vectors are now being constrained due to introduction of transposons because they are very expensive to manufacture. Viral vectors have limitation of less GMP certified production facilities. Production of clinical Good Manufacturing Practice (cGMP)involves lot of time, standardization of cell culture conditions, testing of microbial contamination, presence of viral particles having the ability to replicate, validity of sequence and their functioning. Limited shelf life of viral stocks is also one of the major drawbacks. In contrast, cGMP grade transposonplasmids can be manufactured more quickly. Scaling up of production is easy and in a much shorter time period upgradation and certification of existing facilities can be done. For gene delivery system the use of transposons decreases both the time and cost of production.

(ii) Delivery of large and multiple transgenes

For delivering multiple transgenes, retroviral and lentiviral vectors have been successfully used but these systems can carry a limited cargo of up to 8kb which is being limited by the packaging capacity of their capsid envelope.In earlier reports it has been found out that the efficiency of sleeping beauty system has reduced beyond transposon size of 10kB. On the other hand, piggyBac system is utilized to modify primary human lymphocytes with 15 kB transposon having an initial transfection efficiency of 20% which has increased upon selection and expansion to 90%. Mobilization of large transposons like 100kb the piggyBac system is used in mouse embryonic stem (ES) cells. The significance of an increased cargo capacity is that it helps in delivering multiple transgenes to the same cell. As for example, efficient modification of human cells to express three subunit functional sodium channel using piggyBac system which helped in retaining its electro-physiological properties after 35 passages.

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(iii) Less immunogenicity

In the year 1999, the death of a patient on receiving liver targeted adenoviral gene therapy for partial deficiency of ornithine transcarbamylase was due to immunogenicity.Within four days of administration of vector there was cytokine rush in the body which resulted in multiple organ failure. Various attempts have been made to reduce the immunogenicity of viral vectors by removing all the endogenous viral genes but even though such viral vectors are found to be potentially immunogenic. This has been proved by long term inflammation of rat brains which have been injected with adenoviral vectors that are replication deficient.TNF and IFN-α are inflammatory mediators which are produced when Toll-like receptor (TLR)-9 recognizes DNA with unmethylatedCpGdinucleotides in the endosome. DNA- dependentactivator of interferon (IFN)-regulatory factors (DAI), RNA polymerase III (Pol III), absent in melanoma 2 (AIM2), leucine-rich repeat (inFlightless I) interacting protein-1 (Lrrfip1), DExD/H box helicases (DHX9 and DHX36) and IFN-inducible protein IFI-16are other mechanisms of innate immune sensing of naked DNA.To initiate immune response to the delivered DNA, these molecules use independent and overlapping signaling pathways.

(iv) Less tendency for oncogenic mutations:

For insertion of genes in SupT1 and Jurkat cells, human immunodeficiency virus (HIV) is preferred. Though murine leukemia virus (MLV) derived vectors are used for stable gene transfer but they give more preference to transcriptional start sites (TSS) for integration. In French X-SCID gene therapy trial, the integrations near the promoter of the LMO2 proto-oncogene have been associated with leukemia. The genome mapping of sleeping beauty transposons in mammals have showed that it is more partial to transcriptional units and upstream regulatory sequences which varies between different cell types. In primary human cells and cell lines piggyBac has no significance of integration site and has no preferred sites in chromosome too. The preference sites for integration of piggyBac are RefSeq genes, near TSS and CpG enriched motifs. This may be due to nature of the state of the cell or type of the cell. To improve the safety of gene transfer, both sleeping beauty and piggyBacare engineered for site-directed gene delivery. Till date, transposons have not been used in humans although one clinical trial has been approved.

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22.6 Challenges of transposon as gene delivery system:

(i) Gene delivery efficiency is low

The efficiency of transposon system which is carried by naked DNA plasmids is limited by getting the plasmid into the cell by physical and chemical means. Transfection is easy in some primary cells and cell lines (e.g. HEK293, HeLa, Hepatocytes) and the transposition efficiency of transposons is quite high.Nucelofection and electroporation are some of the methods that are used for transfection is toxic to cells leading to cell death thereby reducing the efficiency of a stable transfection. Novel delivery methods like cell-penetrating peptides (CPP)- piggyBac fusions are being developed to overcome such difficulties.Though some researchers have encapsulated transposon system within viruses to use the virus in delivering the DNA to target site but the problem of immunogenicity of viruses still remains unsolved.

(ii) Integration of profile randomly

With respect to genomic elements transposons’ have random preference to sites when it comes to integrationof the gene. This leaves the transposed gene under the influence of the neighboring genomic region. The risk of possible genotoxicity increases when there is uncontrolled or not site-directed integration.

(iii) Silencing of the integrated transgene

When sleeping beauty is used in cultured cells silencing of gene has been observed.Whereas with piggyBac, silencing of transgene and modification of epigenetic transgene has not been studied well.

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22.7 Applications

Sleeping beauty and piggyBac transposon system have helped in correction of various diseases.

Table 22.2 List of diseases corrected with sleeping beauty and piggyBac:

Disease Transposon system

Hemophilia B sleeping beauty

Hemophilia A sleeping beauty

Tyrosinemia Type I sleeping beauty

JunctionalEpidermolysisBullosa sleeping beauty

Diabetes sleeping beauty

Mucoploysaccharides I & VII sleeping beauty

Huntington’s disease sleeping beauty

α1-antitrypsin deficiency piggyBac

(i) Genetic modifications of human T lymphocytes

For immunotherapeutic purposes peripheral blood and umbilical cord T cells have been successfully modified with both viral and non-viral gene delivery. This method has been used both for the treatment of viral infections and Epstein Barr virus (EBV) associated lymphoma post autologous bone marrow transplantation. The generation of clinical grade T cells with the use of viral vectors is expensive, time intensive and not free of risks. Modification of peripheral blood mononuclear cells with a CD19-specific chimeric antigen receptor (CAR) has been successfully done with the help of sleeping beauty system. Generation of CAR+ T cells from modified PBMCs help in keeping intact CD4+, CD8+, central memory and effector-effector cell phenotypes. Stable transgene expressions in human T lymphocytes have been standardized in piggyBac system. Multiple transgenes have been used to modify primary lymphocytes in order to redirect their specificity for CD19 which makes them

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resistant to off target the effects of chemothererapeutic drugs like rapamycin. Human epidermal growth factor receptor-2 specific CAR (HER2-CAR) have been successfully modified with cytotoxic T lymphocytes specific for Epstein Barr Virus (EBV). Food and Drug Administration (FDA) has approved the first clinical trial which involves transposon modified autologous T cells with a second generation CD19-specific CAR.

(ii) Generation of induced pluripotentstem cells (iPSCs)

Induced pluripotent stem cells (iPSCs) are generated from a patient’s own differentiated somatic cells which hold a good prospect in the field of regenerative medicine. Retroviral vectors have been used successfully which involves delivery of reprogramming factors. But around 20% of the chimeric offspring developed tumors due to re-activation of the c-myc oncogene. These chimeric offsprings were obtained from germline transmission of clones which were retrovirally reprogrammed. It has been found out that the ectopic expression of the reprogrammed factors have lead to formation of tumors and skin dysplasia. One of the ways to prevent the use of viral delivery system is by delivering the programming factors as recombinant proteins or by repeated plasmid transfections. But both of these methods have been proved as inefficient and slow. Since transposons have higher gene delivery efficiency and have the ability to get excised from the cells after reprogramming and differentiation hence they make a good choice for generating iPSCs. Transfection of somatic cells with piggyBac transposons carrying reprogramming factors and transposase from which reprogrammed iPSCs are selected and propagated to obtain individual iPSCs clones. Re-expression of transposase is done to remove the reprogramming factors in order to generate transgene-free iPSCs. This whole process is followed by negative selection to identify iPSCs which are transgene-free.

Since piggyBac system do not cause “foot print” mutation, it is therefore suitable for undergoing precise excision. Whereas, sleeping beauty system leaves behind altered insertion sites due to improper excision. Transgene-free iPSCs have been generated from both mouse and human embryonic fibroblasts from piggyBac system and its efficiency is comparable to retroviral vectors. Successful reprogramming of murine tail tip fibroblasts into fully differentiated melanocytes is

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being done using piggyBac system. piggyBac reprogramming system is now found to be more stable and quicker than lentiviral system.

(iii) Genetic modification of stem cells

Human embryonic stem cells are genetically modified using transposons. Even to insert bacterial artificial chromosomes (BACs) in human embryonic stem cells transposons are being used. To genetically modify hematopoietic stem cells both sleeping beauty and piggyBac are used. For permanent (or reversible in case of piggyBac) genetic modifications of various types of stem cell transposons provide an effective mechanism for further use in gene therapy.

22.8 Existing hot topics and future prospects

(i) Generation of hyperactive transposon elements

In human cells similar activity level is shown by both SB100X and native piggyBacwhich is altogether 100 fold more than the native sleeping beauty. Two to three folds more activity is seen in hyperactive piggyBactransposase (hyPBase) than SB100X or native PB (piggyBac). On engineering hyperactive versions of transposase there has been increase in transposition activity.Import of amino acids from related transposases, scanning of alanine and site-directed mutagenesis are some of the strategies employed during generation of hyperactive transposon elements. ~ 100 folds higher activity is seen in SB100X transposase than the original sleepingbeauty transposon. SB100X employs a high throughput screen of mutant transposes which is obtained from DNA shuffling.

Hyperactive version of piggyBac(hyPBase) transposase has been engineered with a 17-fold increase in excision and 9-fold increase in integration than the original piggyBac. It has been found that in hyPBase there are 7 amino acid substitution but none of the substitutions are present in the catalytic domain of the transposase. Foot print mutation is present in hyPBase with a frequency of <5% comparable to the wild type transposase and there is no effect on genome integrity. In SB100X there is 50% reduction even if there is an addition of 24 kDa ZFN tag which did not show any change in transposition efficiency.

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(ii) Engineering of transposon systems for site-directed integration

Arbitrary insertion of transposon system has resulted in adverse conditions like leukemia. Expressions of critical genes are affected if transgenes are inserted at other genetic loci. Significance of site-directed integration: (i) improvement of gene expression, (ii) at the site of integration there is reduction of positional effects and (iii) improvement in safety. In most of the studies DNA-binding domains have been utilized to which transposase is fused to achieve site directed integration. For transposase modification piggyBac system is more suitable because the efficiency of system is not altered even by addition of domains to the tranposase. Integration of Gal4-piggyBac fusion transposase has biased the insertion near Gal4 sites in episomal plasmids and genome. A zinc finger protein (ZFP) is engineered into a chimeric transposase which is fused to native piggyBactransposase and has successfully biased the integration at genomic level.Transcription factor on DNA binding domains fused with piggyBactransposaseto label transcription factor binding sites in the cells genome.Transposase have the ability to integrate on its own without targeting the machinery thereby leading to off-target integration and this is one of the major problems faced during site-directed integration. Further engineering of transposase and transposon is required to overcome such limitations.

Transposons make a promising non-viral gene delivery system due to low cost and widespread applications than viral vectors and the property for site-directed integration of gene delivery.

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