Gene Transfer by

Animal Viruses &

Introduction of Gene
into Cell
วาสนา ศิรริ ังษี
25 สิงหาคม
2552
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Objectives: after finish this
class, student should be able
to explain these topics;
 How virus acts as a genetic vector.
 Various procedures in performing gene
transfer (non-viral & viral vector).
 Types of viral vectors.
 Viral vector construction.
 Advantages and disadvantages of specific
viral vector.
 Viral vector application in medicine.
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Viruses as vectors
 Viruses are obligate intra-cellular parasites
which infect cells, often with great specificity
to a particular cell type.
They tend to be very efficient at transfecting
their own DNA into the host cell, which is
expressed to produced new viral particles.
By replacing their non-essential genes with
foreign genes of interest, the recombinant
viral vectors can transduce the cell type it
would normally infect.

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Gene transfer into eukaryotic
cells
Transduction: is the process by which DNA is transferred
to cell or bacterium by a virus/bacteriophage using natural
process of viral infection.
 Common techniques are the use of viral vectors.

 Transfection: is a process to introduce naked DNA into cell

culture by various procedures.
 The mostly used procedure is that cells are treated with
calcium phosphate or DEAE-dextran that increase cell
permeability.
 Other procedures are
--lipofection (DNA is bound to positively charged lipids;
cationic liposome) that are capable of fusing with the lipid
bilayer of the cell membrane.
--electroporation 4
 Electroporation
Electroporation, or electropermeabilization, is a significant increase in the
electrical conductivity and permeability of the cell plasma membrane caused
by an externally applied electrical field.

Cuvette

ลักษณะของผนั งเซลล์: ปกติ ปล่อยกระแสไฟฟ้ า (รูพรุน) หลัง

หยุดกระแสไฟฟ้ า กลับมาปกติ
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Gene transfer (con.)
 Microinjection: microinject directly DNA into the cell’s
nucleus. The nuclei of oocytes and eggs are well suited
for this approach.
 High energy bombardment: DNA is coated on the gold
particle and is shot directly into the cell.

 Molecular conjugates: DNA is bound to protein or

synthetic ligands. Targeting proteins include
asialoglycoprotein, transferin, and polymeric IgA.
Delivery technique is similar to those for liposomes.
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Types of viral vectors
 RNA virus vectors
Retrovirus, Lentivirus
 DNA virus vectors
Adeno-associated virus (AAV; Human Parvovirus),

Adenovirus,
Herpes simplex virus,
Poxvirus (vaccinia virus, canarypox virus)

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Comparison of genetic
vectors
Vector

Non-viral
Insert size (kb) Persistence

Unlimited Transient
Advantages

No viral sequence,
Disadvantages

Low-efficiency entry,
large capacity transient persistence

Retrovirus 1-7.5 Stable Effective cell entry, Target cell division

stable integration required

Lentivirus 1-8 Stable Transduce deviding & Biosafety profile

nondividing cell uncertain

AAV 4 (±5%) Stable Efficient cell entry Limitation on

Replication defective packaging size

Adenovirus 2-35 Transient Effective in vivo Transient persistence

gene delivery

HSV Unknown Stable in Neuronotropic Complex virus

(genome 150kb) neuron large capacity

Poxvirus 25 Stable Cheap, heat-stable, Low efficiency

stimulate HMI&CMI
recombinant vector
production
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RNA Viruses: (1)
Retroviral

vector
Retroviruses are a class of enveloped viruses containing
a duplicated ssRNA genome.
 Following infection, the viral genome is reverse
transcribed into ds DNA, which integrates into the host
genome and is expressed as proteins.
 The viral genome is approximately 10kb, containing at
least three genes: gag (coding for core proteins), pol
(coding for enzymes; reverse transcriptase, protease,
integrase) & env (coding for the viral envelope protein).
 At each end of the genome are long terminal repeats
(LTRs) which include promoter/enhancer regions &
sequences involved with integration and sequences
required for packaging the viral DNA.

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Retroviral vectors are most frequently based upon the
Moloney murine leukaemia virus (Mo-MLV), which is
an amphotrophic virus (infect mouse & human cells),
enabling vector development in mouse models, &
human cells, enabling human treatment.
A requirement for retroviral integration and expression
of viral gene is that the target cells should be dividing.
This limit gene therapy to proliferating cells in vivo or ex
vivo, whereby cells are removed from the body, treated
to stimulate replication and then transduced with the
retroviral vector, before returning to the patient.

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 Ψ+

A 5’ LTR gag pol env 3’ LTR

Ψ+
B 5’ LTR Exogenous gene (s) 3’ LTR

Map of typical simple retrovirus and retroviral vectors.

A: the retrovirus has an LTR at each end, Ψ: Psi, the encapsidation
sequence, and region encoding the Gag, Pol, and Env polyproteins.
B: retroviral vectors containing the LTR and Ψ+ regions of the retrovirus
with the exogenous gene sequences cloned in between.

Vectors lacking the genes encoding the retroviral proteins

are replication defective and shouldn’t be capable of
repeated replication after transduction of target cells.
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 Production of retroviral vector

1 2

(Virus
No packaging signal contains
gene of
interest)

Mouse embryo fibroblast cells

1. Production of packaging cell: cell that expresses viral proteins but
lacks packaging signal.
2. Transfection of “LTR + gene of interest + packaging signal” to
packaging cell.

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 Retroviral vector life cycle
Recombinant vector plasmid (dsDNA)
Transfect into packaging (producer cell)

Production Integrated or episomal DNA (dsDNA) in packaging cell

of viral
vector Transcription
Recombinant viral genome (ssRNA)
Translation
Package into virion & release virus vectors from producer cell
Enter target cell
Reverse transcribe Integrate
Production
of gene Provirus (dsDNA) in target cell
product Transcription
Transcripts (ssRNA)
Translation
Gene product (Protein of interest)
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Limitation of retroviral vector:

Some retroviruses contain proto-oncogenes, which

when mutated can cause cancers, however, in the
production of vectors these are removed.
Retroviruses can also transform cells by integrating near
to a cellular proto-oncogene and driving inappropriate
expression from the LTR, or by disrupting a tumour
suppresser gene. This event, termed insertional
mutagenesis, though extremely rare could still occur
when retroviruses are used as vectors.

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(2) Lentivirus vectors
 Lentiviruses are a subclass of retroviruses
which are able to infect both proliferating &
non-proliferating cells.
 They are more complicated than simple
retroviruses, containing basic gag-pol-env
genes and additional 6 proteins, tat, rev, vpr,
vpu, nef & vif. The lentiviral vectors used are
derived from the HIV & are being evaluated
for safety, with a view to removing some of
the non-essential regulatory genes.

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 Applications
 Because of the long and stable insertion of gene into the host
genome, retroviral vectors were the earliest vectors applied to
gene therapy.
 The first human gene therapy protocol involved removal of
peripheral blood cells from 2 children with adenosine deaminase
(ADA) deficiency, in vitro transduction with a retroviral vector
encoding ADA, and reintroduction of the cells into children.
 A further development of ADA gene therapy was transduction of
hematopoietic stem cells with an ADA-expressing retroviral vector,
resulting in expression in multiple hematopoietic lineages and
clinical improvement in the patients.
 Because of ability to stably transduce nondividing cells, lentiviral
vectors have proved particular interesting for application in the
nervous system (e.g., Parkinson’s disease).
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 DNA viruses: (1) Adeno-
Associated Viruses (AAV)
 Adeno-associated viruses (AAV) are defective non-
pathogenic human parvoviruses, dependant on a
helper virus, usually adenovirus, to proliferate.
 They are capable of infecting both dividing & non
dividing cells.
 They can integrate into a specific point of the host
genome (human chromosome 19) at a high
frequency.
 The wild type genome is a ssDNA molecule,
consisting of 2 genes; rep, coding for proteins which
control viral replication, structural gene expression &
integration into the host genome, and cap, which
codes for capsid structural proteins.

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AAV genome

 At either end of the genome is a 145 bp terminal repeat (TR),

containing a promoter.
 When used as a vector, the rep & cap genes are replaced by
the transgene and its associated regulatory sequences.
 The total length of the insert cannot greatly exceed 4.7 kb, the
length of the wild type genome.
 Production of the recombinant vector requires that rep & cap are
provided in trans, along with helper virus gene products (E1a, E1b,
E2a, E4 & VA RNA from the adenovirus genome).

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AAV life cycle

Absence of helper
Adenovirus

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Advantage:
- AAV vectors integration into the host genome
allowing prolonged transgene expression.
- Gene transfer into vascular epithelial cells, striated
muscle & hepatic cells, with prolonged expression
when the transgene is not derived from a different
species.
- Neutralizing antibody to the AAV capsid may be
detectable, but does not prevent re-administration of
the vector or shut down promoter activity.

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 (AAV)
Gene Therapy for
cystic fibrosis

Produce normal mucus

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(2) Adenovirus
 Adenoviruses are non-enveloped viruses containing a
linear ds DNA genome. While there are over 40
serotypes of adenovirus, most of which cause human
respiratory tract infections.
 Subgroup C serotypes 2 or 5 are predominantly used as
vectors.
 The life cycle does not normally involve integration into
the host genome.
 Replicate as episomal elements in the nucleus of the
host cell & consequently there is no risk of insertional
mutagenesis.
 The wild type adenovirus genome is approx. 35 kb of
which up to 30 kb can be replaced with foreign DNA.

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Adenovirus vector construction
Adenovirus genome contains early genes (E1a, E1b, E2, E3 & E4), which have
regulatory functions, and a late genes L1-L5, which codes for structural proteins.

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 Adenoviral vestors are very efficient at transducing
target cells in vitro & vivo, & can be produced at high
titer (>1011 /ml).
 The initial delivery of large amounts of DNA packaged
within adenovirus proteins, the majority of which will be
degraded & presented to the immune system may still
cause problems for clinical trials.
 The development of vectors containing fewer genes,
in the "gutless" vectors which contain no viral coding
sequences, has resulted in prolonged in vivo transgene
expression in liver tissue.

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Until recently, the mechanism by which the
adenovirus targeted the host cell was poorly
understood.
Tissue specific expression was only possible by
using specific cellular promoter/enhancers.

e.g. - the myosin light chain 1 promoter

- the smooth muscle cell SM22a promoter

or by direct delivery to a local area.

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 Applications
 To date, >170 clinical trials with Ad vectors have
been performed in humans.
 Monogenic diseases (an inherited disease controlled by a
single pair of gene) – Ex. Cystic fibrosis
 Cancer- Ex. Delivery of a wide range of cancer therapeutic
genes, including a) genes that encode immunostimulatory
molecule such as IL-2; b) tumor suppressor genes such as p53;
c) suicide genes such as HSV-Thymidine Kinase gene.
 Vaccine – vaccine delivery vector against HIV, Ebola virus,
and Plasmodium.

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(3) Herpes simplex virus
 Herpes simplex virus type 1 (HSV-1) is a
human neurotropic virus.
 HSV-1 as a vector for gene transfer to the
nervous system.
 The wild type HSV-1 virus is able to infect
neurons & either proceed into a lytic life cycle
or persist as an intranuclear episome in a
latent state.

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The viral genome is a linear ds DNA molecule of
152 kb. There are two unique regions, long &
short (termed UL & US) which are linked by
internal repeat sequences (IRL & IRS). At the
non-linker end of the unique regions are terminal
repeats (TRL & TRS).

TRL UL IRL IRS US TRS

There are up to 84 genes of which about half are

not essential for growth in cell culture. Once these
non essential genes have been deleted, 40-50 kb
of foreign DNA can be accommodated within the
virus.
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Structure of HSV-1 genome and viral vector capacities

Comparison between different viral vector capacities

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HSV-1 as efficient gene
transfer vector
 HSV-1 has a large ds DNA genome containing many
non-essential genes which can be deleted without
reduction of virus yield, providing space for insertion of
either large single gene or multiple transgenes.
 Virus possesses a natural mechanism for maintenance
of the viral genome as an episome in nucleus of
latently infected nerve cells.
 Virus possesses a novel promotor system that is active
during latency.
 Virus has a broad host range.
 HIV-1 is able to transduce both dividing and non-
dividing cells.
 Even highly deficient virus recombinant can be
propagated to the high titers (>1010 PFU/mL) required
for in vivo gene therapy application.
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Applications
 Neurobiology- gene transfer to the peripheral nervous
system for treatment of drug-induced peripheral
neuropathies.
 Oncology- Oncolytic HSV-1 vectors have been tested
for tumor-specific delivery of antineoplastic gene products,
including immunoregulatory molecule, pro-drug converting
enzymes, and angiogenesis inhibitors.
 Vaccine – Gene to be expressed from an HSV vector
was the HBV surface antigen, with the idea of using the
recombinant virus as a vaccine for HBV.

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(4) Poxviruses
 In 1982, it was suggested that since vaccinia
virus had been widely used and proven safe
(against smallpox), it could be used as a
vector for carrying foreign antigens and
thereby immunizing against other pathogens.
 To date, hundreds of different vaccinia virus
recombinants have been described.

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Advantages of recombinant vaccinia virus vector vaccines:
• cheap to manufacture and administer,
• stable - do not require refrigeration, stimulate both HMI & CMI

Disadvantages:
• Vaccinia can lead to death in immune deficient individuals.
• To overcome this disadvantage, avian poxviruses (fowlpox
and canarypox) are now being used as vectors.
• These viruses can infect mammalian cells but cannot produce
new infectious virus in mammalian hosts.

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Basic strategy for Vaccinia
virus
 vector
1. Plasmid construction: Create a gene construct
under the vaccinia promoter control. The foreign
hybrid gene can be inserted in a non-essential
site in the vaccinia genome:
e.g. the thymidine kinase (TK) gene is the most popular
site of insertion because the virus does not need TK for
replication.
 2. Transfect a gene construct and a wild-type
vaccinia virus into a eukaryotic tissue culture cell.
 3. Viral vector selection:
TK (-) virus is less pathogenic and bromodeoxyuridine
(BUDR) resistant.
TK (+) wild type virus is BUDR sensitive.
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 Vaccinia virus vector
construction Foreign gene

Plasmid construction

Transfection into cells

Production of Vaccinia
virus carrying foreign
gene which is BUDR
Resistant (TK-).
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Application
 Live vector vaccine
- HIV-1 vaccine (Prime-Boost protocol)
(Phase III trial in Thailand: start Oct 2003, 16,000 volunteers)

“ALVAC, canarypox vector vaccine”

(ALVAC at weeks 0, 4, 12, 24 boost by AIDSVAX B/E a gp120
protein vaccine at weeks 12, 24 follow-up 3 yrs after
vaccination)

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Assignment:

Write 1 page essay of up to date the most

advanced;
-virus vector vaccine or
-virus vector for gene replacement therapy.
References should be included.

Deadline: September 16, 2009.

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