Spreading Knowledge is a great Legacy. MMG

1. VIRAL DELIVERY SYSTEMS
FOR GENE THERAPY

2. General concerns
The Food and Drug Administration (FDA) has not yet approved
any human gene therapy product for sale.
Four major problems with gene therapy:
2) Immune response. It reduces gene therapy effectiveness and makes
repetitive rounds of gene therapy useless
3) Problems with viral vectors . Toxicity, immune and inflammatory
responses, also fears that viral vector may recover disease-causing ability
4) Multigene disorders. Most commonly occurring disorders, such as
heart disease, Alzheimer's disease, arthritis, and diabetes, are caused by
the combined effects of variations in many genes.
1) Short-lived nature of gene therapy. Very hard to achieve any long-
term benefits without integration and even with it.

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

4. Methods of gene delivery
(therapeutic constructs)
-- Injection of naked DNA into tumor by simple needle and syringe
-- DNA transfer by liposomes
(delivered by the intravascular, intratracheal,
intraperitoneal or intracolonic routes)
-- DNA coated on the surface of gold pellets
which are air-propelled into the epidermis
(gene-gun), mainly non applicable to cancer
-- Biological vehicles (vectors) such as viruses and bacteria.
Viruses are genetically engineered
so as not to replicate once inside the host.
They are currently the most efficient means of gene transfer.

5. 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

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

7. Retrovirus based vectors

8. Retroviruses
• Probably the most studied group of viruses
in molecular biology!!!
• Enveloped, positive-strand RNA viruses
• Unique morphology and replication
• Replicate through a DNA intermediate by
reverse transcriptase (RT)

9. • Retroviral vectors are based on Moloney murine leukemia
virus (Mo-MLV) which is capable of infecting both mouse and
human cells.
• The viral genes, gag, pol and env, are replaced with the
transgene of interest and expressed on plasmids in the
packaging cell line.
• Because the non-essential genes lack the packaging
sequence, they are not included in the virion particle.
• To prevent recombination resulting in replication
competent retroviruses, all regions of homology with the
vector backbone is removed.

10. • Transcription could be under the control of LTRs or
enhancer promoter elements might be engineered within
the transgene.
• The chimeric genome is then introduced into a
packaging cell, which produces all of the viral proteins,
such as the products of the gag, pol and env genes, but
these have been separated from the LTRs and the
packaging sequence.
• Only the chimeric genomes are assembled to generate
a retroviral vector.
• The culture medium in which these packaging cells have
been grown is then applied to the target cells, resulting
in transfer of the transgene.

13. Retroviral vectors are able
to infect dividing cells only
Good for cancer gene therapy
Nevertheless, retroviruses are most often used vectors
for common disease gene therapy
Every therapeutic construct should include safety features
In dividing cells nuclear membranes are broken down,
so viral genome can enter and integrate into the chromosome
Preintegration complex of retroviruses
non able to penetrate nuclear membrane.
Infection of dividing cells only

14. A critical limitation of retroviral vectors is their inability to
infect nondividing cells, such as those that make up
muscle, brain, lung and liver tissue.
The cells from the target tissue are removed, grown in vitro
and infected with the recombinant vector, the target cells
producing the foreign protein are then transplanted back
into the animal (ex vivo gene therapy).
Problems with expression being shut off, prolonged
expression is difficult to attain.
Expression is reduced by inflammatory interferons acting
on viral LTRs
Possibility of random integration of vector DNA into the
host chromosome.
Retroviral vectors- Limitations

15. Lentiviral vectors
• Lentiviruses are retroviruses that can infect both
dividing and nondividing cells
• Preintegration complex of lentiviruses can get through
the intact membrane of the nucleus of the target cell.
• Able to infect nondividing or terminally differentiated
cells such as neurons, macrophages, hematopoietic
stem cell, retinal photoreceptors, and muscle and liver
cells
• Example of lentiviruses: HIV-1 (infects T-helper cells) – AIDS.

16. • They are more complicated than retroviruses, containing
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.

19. 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 can’t replicate.

20. Adenovirus particle structure:
• Nonenveloped particle
• Contains linear double
stranded DNA
• Does not integrate into the
host genome
• Replicates as an episomal
element in the nucleus

22. • Adenoviral vectors can infect cells in vivo, causing them to
express high levels of the transgene. However, expression
lasts for only a short time (5-10 days post-infection).
• Immune response is the reason behind the short-term
expression.
• Immune reaction is potent, eliciting both the cell-killing
“cellular” response and the antibody producing “humoral “
response.
• Humoral response results in generation of antibodies to
adenoviral proteins and prevents any subsequent infection if a
second injection of the recombinant adenovirus is given.
Adenoviral vectors- Limitations

23. Adenoviral vectors:
Advantages:
Higher titer
Efficient transduction of nondividing cells
in vitro and in vivo
Disadvantages:
Toxicity
Immunological response
Prior exposure

24. Adeno-associated viral vectors:
• AAV is a simple, non-pathogenic, single stranded DNA virus
dependent on the helper virus (usually adenovirus) to
replicate.
• It has two genes (cap and rep), sandwiched between
inverted terminal repeats that define the beginning and the end
of the virus and contain the packaging sequence.
• The cap gene encodes viral capsid proteins and the rep
gene product is involved in viral replication and integration.
• It can infect a variety of cell types and in the presence of the
rep gene product, the viral DNA can integrate preferentially
into human chromosome 19.

25. AAV vectors:

26. • 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.

27. Adeno-associated virus vectors:
Advantages:
All viral genes removed
Safe
Transduction of nondividing cells
Stable expression
Disadvantages:
Small genome limits size of foreign DNA
Labor intensive production
Status of genome not fully elucidated

28. Herpes Simplex Virus
• Belong to the alphaherpesvirus subfamily of herpesviruses
• Double stranded DNA enveloped virus with a genome of around 150 kb
• The genome of HSV-1 and HSV-2 share 50 - 70% homology.
• They also share several cross-reactive epitopes with each other. There is also
antigenic cross-reaction with VZV.
• Man is the only natural host for HSV.
• Structure and Composition
– Spherical iscoahedron, 150-200 nm
– Double-stranded DNA, linear
– More than 35 proteins
– Enveloped
– Replication from nucleus (budding)
– Features
• Encode many enzymes
• Establish latent infections
• Lifelong persistence
• Significant cause of death in immunocompromised hosts
• Some can cause cancers

29. • Nuclear escape
• Viral proteins induce budding of the
capsid through both nuclear
membranes
• Thus, capsid escapes into the
cytoplasm
• Viral proteins associate with the
cellular vesicles
• These proteins have affinity for the
capsid proteins and cause the vesicle
to wrap around the virus, providing it
with an double-layered envelope
• Virus traverses the ER then
Golgi prior to release from the
cell
• The outer membrane fuses with the
plasma membrane
• This permits the virus to leave the
cell while retaining the inner
membrane

30. • Advantages
– High titers (108-109 particles/ml)
– Ability to produce gutless vectors
• Disadvantages
– Potential titre drops when more genes added
– Cytotoxicity