Viral Vectors PDF

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:

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.


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.

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.

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.

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.

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.

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.

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 polyethylenimineDNA
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 viralDNA 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.

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.

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 phageintegrase
create hybrid recombination sites and require separate excisionase for its function.
Some phage SSR like those from , HXO22 require cofactors like IHF for
recombination whereas other SSR like those from phage R4, TP901 and C31 donot
require cofactors and are capable of stable integration.
C31 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. C31 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.

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

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 repressor, E.coliLexA repressor, the Ty3 tRNA binding domain or
designed Zn finger protein E2C for use in gene therapy.

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 EpsteinBarr 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 -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.

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.

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.

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.

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 laymans 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.

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,
Inammation
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
Specicity: 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.

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.

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.

Figure 18.2 Schematic representation of a DNA vaccine:

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 nave 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
nave 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.

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.

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

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).

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 -glucoronidase E7 Enhanced stability

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

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.

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

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.

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.

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.

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 M governs the transfection
efficiency. As Mdecreases, the transfection efficiency increases. When the Mis low,
then the lipoplexcontaining DNA remains intact. Whereas when M 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.

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

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

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:

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.

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.

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.

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

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.

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.

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.

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.

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.

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.

(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- 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.

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.

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
Huntingtons disease sleeping beauty
1-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

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 patients 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

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.

(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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NPTEL Biotechnology Gene Therapy

Module 3 Viral vectors

Joint initiative of IITs and IISc Funded by MHRD Page 1 of 55

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,

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

rev Regulator of expression of Structural gene expression

nef Negative regulatory factor Virus infectivity

Figure 15.1 Schematic diagram of lentivirus:

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Figure 15.2 Generation of lentiviral vector:

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Recombinant simian virus 40

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Figure 16.1 Replication of SV40 in the host cell:

16.1 SV40 as a vector

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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.

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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

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Non viral vectors

Therapeutic gene expression has emerged as a potent tool in modern medicine.

17.2 Traditional non viral methods of transgenesis

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17.3 Physical methods of transgenesis

a. Hydrodynamic pressure techniques: In this technique intravascular injection

17.4 Cationic polymer method of transgenesis

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c. Polyethylenimine: Polyethylenimine is a linear or branched polymer with many

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17.5 Safe integration of corrective genes to human genome

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Figure 17.1Site specific recombination by Crerecombinase:

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Figure 17.2 Mechanism of integration of therapeutic gene in human genome

Prokaryotic origin of SSRs limits their application in eukaryotic hosts.

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Figure 17.3 Rep protein mediated site specific recombination in Adeno-

The use of AAV is limited by certain level of immunogenicity it induces in

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17.6 Extra chromosomal replicating units in transgenesis

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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

Human Artificial Chromosomes (HAC) can be synthesized de novo by

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Because of its large size, microcell mediated chromosome transfer and

17.8 Maintenance of transgene expression in host system

With evolution eukaryotic systems have developed mechanisms to maintain

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17.9 Future prospects