Gene therapy:

to correct a genetic defect by transferring of a

functional normal copy of the gene into cells.

Examples of diseases caused by genetic defect

SCID( severe combined immunodeficiency)

Ornithine transcarbamylase (OTC deficiency)

Hemophilia (blood coagulation factors VIII or IX)

Muscular dystrophy

Cystic fibrosis
Sickle cell anemia
Genetic Defects that are Candidates for Gene
Therapy
 Applications of gene therapy

Genetic disorder (deficiency): OTC

Cancer
Genetic predisposition
Mutation in oncogene or tumor suppressor
gene

Autoimmunity diseases: rheumatoid

arthritis
Delivery of counteracting gene

Diseases involve several genes and the

 Factors to be considered in Gene therapy

How to deliver genes to specific cells, tissue and whole

animals? (methods of delivery)

How much and how long the introduced gene will be

expressed?

The site and dose of gene delivery

Is there any adverse immunological consequence of

both delivery vehicle (Virus) and the gene in animals?

Is there any toxic effects?

 Introduction of Genes Into Animals

METHODS MAJOR LIMITATIONS

Calcium Phosphate
DEAE Dextran Low Efficiency
Cationic Lipids, Liposomes

Direct DNA Injections Low Efficiency

Electroporation Transient expression

Virus:

Virus are obligate intracellular parasites

Very efficient at transferring viral DNA into host cells
Specific target cells: depending on the viral attachment proteins (capsid or
glycoproteins)
Gene replacement: non-essential genes of virus are deleted and exogenous
genes are inserted
 The Ideal Vector for Gene Transfer

High concentration of virus allowing many cells to be infected or transduced

Convenience and reproducibility of production

Ability to transduce dividing and non-dividing cells

Ability to integrate into a site-specific location in the host chromosome, or to be

successfully maintained as stable episome

A transcriptional unit that can respond to manipulation of its regulatory elements

Ability to target the desired type of cell

No components that elicit an immune response

 Generation of viral vector for gene therapy

Replication-competent virus

Replication-defective virus
Amplicon: doesnt encode structural proteins

Cant replicate beyond the first cycle of infection

Elements needed to generate amplicon

Transfer Vector: plasmid (promoter, gene of interest, ori,
packaging signal)
Packaging vector (cosmid or cell lines): provide the viral
structural proteins for packaging of transfer vector
Helper virus (packaging of transfer vector): deleted
Packaging signal sequence
 pac ori
Amplicon
Amplicon VectorSystem
Plasmid

CotransfectionwithHSV1bacmidlacking
packagingsequences("helpergenome")

Plasmidisreplicatedintoconcatemers,cutinto
genomeunitlengthmolecules&packagedinto
viralparticlesbythehelpergenome;thehelper Helperfree
itselfcannotbepackaged amplicons
Adenoviral vectors:
Double-stranded DNA viruses, usually cause benign respiratory disease; serotypes 2
and 5 are used as vectors.
Can infect dividing and non-dividing cells, can be produced at high titers.
Replication-deficient adenovirus vectors can be generated by replacing the E1 or E3
gene, which is essential for replication.
The recombinant vectors are then replicated in cells that express the products of the E1
or E3 gene and can be generated in very high concentrations.
Cells infected with recombinant adenovirus can express the therapeutic gene, but
because essential genes for replication are deleted, the vector cant replicate.
Adenovirus vectors

Advantages
High titers
Both dividing and non-dividing cells
Wide tissue tropism
Easily modify tissue tropism
Disadvantages
Transient expression ( not good for genetic diseases)
Highly immunogenic
High titers of virus can be toxic
More suitable for cancer immunotherapy
Adeno-associated virus

To produce an AAV vector, the rep and cap genes are replaced with a transgene.

The total length of the insert cannot exceed 4.7 kb, the length of the wild type genome.

Production of the recombinant vector requires that rep and cap are provided in trans along
with the helper virus gene products.

The current method is to cotransfect two plasmids, one for the vector and another
for rep and cap into cells infected with adenovirus.

This method is cumbersome, low yielding and prone to contamination with adenovirus and
wild type AAV.

Interest in AAV vectors is due to their integration into the host genome allowing prolonged
gene expression.
Adeno-associated virus vectors:
Advantages:
All viral genes removed
Safe
Transduction of dividing and non-dividing cells (Liver, Muscles, Neurons
etc)
Stable expression
Disadvantages:
Small genome limits size of foreign DNA
Labor intensive production
Status of genome not fully elucidated
 Retrovirus:

The recombinant retroviruses such as the Moloney murine leukemia virus have the
ability to integrate into the host genome in a stable fashion.

They contain a reverse transcriptase that allows integration into the host
genome.

These virus are capable of infecting their target cells and delivering their viral
payload, but then fail to continue the typical lytic pathway that leads to cell lysis
and death.

Certain retrovirus are acutely oncogenic because they carry particular genes
(oncogene) that promote host cell division.

Retroviral vectors can either be replication-competent or replication-defective

Most retrovirus do not kill the host but produce progeny virions over an indefinite
period.
Retroviral Genome

U3 is the HIV-1 promoter.

R region marks the starting point of transcription,

U5 region is critical for reverse transcription.

packaging signal () The packaging signal, as in many other virus

species, allows RNA genome encapsidation during virion assembly in the
cytoplasm.

The polypurine tract (PPT), these PPT element is a key element for
reverse transcription
Retroviral Genome
Splice donor (SD)
and acceptor (SA)
sites

The ends of the LTRs subsequently participate in integration of the provirus into the
host genome.

Once the provirus has been integrated, the LTR on the 5 end serves as the
promoter for the entire retroviral genome, while the LTR at the 3 end provides for
nascent viral RNA polyadenylation.

The transcript begins, at the beginning of R, is capped, and proceeds through U5

and the rest of the provirus, usually terminating by the addition of a poly A tract
just after the R sequence in the 3' LTR.
Retroviral
vector
Moloney murine leukemia virus (MuLV)
Generation of replication defective retroviral vector
Transfer plasmid vector:
Gene of interest
Long terminal repeats(LTR): promoter, polyA, integration, replication and reverse
transcription
Primer binding site (PBS) (origin of replication)
RNA packaging signal
Poly purine tract (important for replication)
Packaging vector
Cell line stably transfected with plasmid constructs containing Gag/pol and Env
 Characteristics of retroviral vector

Advantages
Integration: permanent expression
Pseudotyped virus

Disadvantages
Only infecting dividing cells
Insertional mutagenesis (tumor formation)
Activate oncogenes
Inhibit tumor suppressor genes

Expression is reduced by inflammatory interferons acting on viral

LTRs, as the retroviral DNA integrates, viral LTR promoters are
inactivated.
Possibility of random integration of vector DNA into the host
chromosome.
 Belong to the retrovirus family but can infect both dividing and non-dividing cells.

They are more complicated than retroviruses, containing an additional six proteins, tat,
rev, vpr, vpu, nef and vif.

Human immunodeficiency virus (HIV) has been disabled and developed as a vector for
in vivo gene delivery.

Low cellular immune response, thus good possibility for in vivo gene delivery with
sustained expression over six months.

No potent antibody response.

Lentiviral Vectors:
Belong to infect both dividing and non-dividing cells.
Their genome is much more complex than simple retroviruses, containing an
additional six genes; tat, rev, vpr, vpu, nef and vif.
lentiviruses are enveloped, however they additionally comprise a pre-integration
complex surrounded by the capsid shell.

The pre-integration complex is composed of the viral integrase, Vpr and the matrix
protein (a product of the gag gene) and mediates the targeting of virions to the
nucleus of the cell, where the complex is able to enter the nucleus through nuclear
membrane pores.

Each copy of their genome is 10 kb in length and is flanked by two LTRs, containing a
viral packaging signal on the right side

Low cellular immune response, thus good possibility for in vivo gene delivery with
sustained expression over six months.

No potent antibody response.

Infectious viruses have three main genes coding region..
5-gag-pol-env-3.
Two regulatory genes, tat and rev.
Additional accessory genes depending on the virus (e.g., for HIV-1: vif, vpr, vpu,
nef)- involved in regulation of synthesis and processing viral RNA and other
replicative functions.

The Long terminal repeat (LTR) is U3, R and the U5 region.

 Reverse transcriptase also has RNaseH activity for destruction of the RNA-template.

Integrase binds both the viral cDNA generated by reverse transcriptase and the host
DNA.

Integrase processes the LTR before inserting the viral genome into the host DNA.

Tat acts as a trans-activator during transcription to enhance initiation and elongation.

The Rev responsive element acts post-transcriptionally, regulating mRNA splicing and
transport to the cytoplasm
Lentivectors are usually obtained following a three-plasmid co-transfection in 293T cells
Splice donor (SD)
and acceptor (SA)
sites