b.

Adenoviral and additional vector-

based viruses
• Adenoviruses are relatively large structures, without sheaths,
containing double-stranded DNA in their genetic material. Their
genome is much larger (around 35 kb) and more complex than those
of retroviruses. In most cases, only a small portion of this genome is
removed when building an adenovirus-based vector. After cell
infection, adenoviral DNA becomes localized in the nucleus, but does
not integrate into the host cell's DNA. Usually, infection based on wild
type adenoviruses is associated with, at worst, mild clinical symptoms
in humans.
Some advantages and disadvantages of adenovirus
characteristics as a potential vector for gene therapy

• The advantage:
• Adenoviruses are able to transfer genes to non-separators
• They are easily propagated in large numbers.
• High levels of gene expression are usually noted
• They are relatively stable viruses.
Disadvantages:
• Adenoviruses are very immunogenic in humans
• The duration of expression of transferred genes can be
• varies, and is usually temporary
• Permissive cell infections with wild type adenoviruses usually cause
cell lysis
• Adenoviruses display broad selectivity in the types of cells they can
infect
• The main advantages relate to their ability to infect cells that do not
divide efficiently and expression that is usually observed from a large
number of desired gene products. However, the failure that adenoviral-
based DNA to integrate into host cells generally means that its
resistance and, therefore, the duration of gene expression, is limited.
• Adenovirus-based vectors, carrying various gene markers (ie genes
whose expression products are easily detected), have been given to
animals. Marker gene expression has been recorded in various tissues,
including the heart, liver, muscles, bone marrow, central nervous system
and endothelial cells. The duration of expression marker genes ranges
from 2-3 weeks to several months.
• While short-term high-level gene expression may be appropriate for
some gene therapy applications, it would be less useful for treatment,
for example, genetic diseases, where long-term gene expression is
needed.
• However, adenovirus triggers a strong immune response, which limits
the effectiveness of repetitive administration.
• The death of a gene therapy trial in 1999, as mentioned earlier, was
apparently caused by a severe and unexpected infection reaction to
the adenoviral vector used.
• Additional viruses that may be used as virus vectors are adeno viruses and
herpes viruses.
• Adeno-related viruses are very small single-stranded DNA viruses that contain
only two genomes. It does not have the ability to replicate independently and
can do so only in the presence of co-infected adenoviruses (or other selected
viruses).
• The herpes simplex virus represents another potential vector system that
receives increasing attention. Because the herpes simplex virus is a neurotropic
virus, it might prove specifically useful in sending genes to peripheral and central
nervous system neurons. After infection, the herpes simplex virus usually
remains latent in non-dividing neurons, with the genome remaining in an
integrated form.
• An additional virus that has recently gained attention as a possible
vector is the sindbis virus.
• The Sindbis virus is simple, strong, capable of infecting cells that do
not divide and generally supports high levels of gene expression.
However, it displays a wide range of hosts and, therefore, lacks the
inherent targeting characteristics of the innate city of an ideal virus
vector.
• Recently, a new recombinant Sindbis virus, which shows the changes
in a specified cell city, has been produced. The scientists entered the
nucleotide sequence code for the IgG binding domain of Staphyloccus
aureus into the E2 virus gene.
• Sindbis virus generation that is engineered can be targeted to bind to
certain cell types. (A Simplified depiction of the virus, displaying
surface protein E2. (B) Genetic engineering facilitates disruption of
the E2 gene by combining the IgG binding domain of protein A. (c)
Incubation of the engineered virus particles with most types of
monoclonal antibodies results in effective antibody immobilization
Thus, virus vectors that are engineered must be targeted to all specific
cell types only by pre-incubation with monoclonal antibodies that
selectively bind to surface antigens that are uniquely associated with
target cells.
• Disruption of the E2 gene makes its protein product unable to bind
laminin (thereby destroying natural viral tropism). However, its
protein domain allows chimaeric E2 products to bind to monoclonal
antibodies. This altered virus might prove to be a useful generic or
‘null’ vector, potentially potentially specifically targeted to whatever
cell type is desired. This will only require pre-incubating the virus with
increased monoclonal antibodies against surface antigens that are
unique to the proposed target cell population.
c. Virus vector creation
• The creation of virus vectors for therapeutic purposes involves the
proper distribution of initial viruses in animal cell lines, virus recovery,
concentration, purification and formulation. Making alternative virus
vectors is likely to follow a similar approach.
Figure 14.7 Making large-scale adenoviral vectors for use in
clinical protocols based on gene therapy. See text for details
d. Vector non-viral
• Although the current virus-mediated gene delivery system dominates,
a large number of clinical trials are currently using non-viral gene-
based delivery methods.
• General benefits cited with respect to non-viral delivery systems
include:
• low / not immunogenic;
• the absence of integration of therapeutic genes into the host
chromosome (this eliminates the potential to disrupt the essential
host gene or activate the host oncogene).
• Bare plasmid DNA This research path was first opened in 1990, when
it was shown that naked plasmid DNA was expressed in mouse muscle
cells after i.m. injection. The plasmid DNA in question places the β-
galactosidase gene as a reporter. The subsequent expression of β-
galactosidase activity can last for anything from a few months to the
rest of animal life. Recorded transfection rates are low (1-2 percent of
muscle fibers assimilated with DNA), and DNA is not integrated into
the host cell chromosomes.
• Modern systems that are not virus-based usually require complexing /
packaging genes of interest (present, together with appropriate
promoters, etc., in circular plasmids) with additional molecules,
especially various lipids or polypeptides. These generally display
positive charges and, therefore, interact with negatively charged DNA
molecules. The function of the carrier molecule is to stabilize DNA,
protect it from, for example, serum nucleases and ideally modulate
interactions with biological systems (eg helping to target DNA to
certain cell types, or away from other cell types).
• The most commonly used polymers are cationic lipids and polysine
chains. Cationic lipids can gather in a water-based system to form
vesicles / liposomes, which in turn will interact spontaneously with
DNA.
• To increase the delivery of new DNA into cells, DNA must be
protected from damage and entry into cells must be facilitated. For
this purpose new molecules, lipoplexes and polyplexes, have been
created that have the ability to protect DNA from unwanted damage
during the transfection process.
• Whereas lipoplexes / polyplexes generally protect plasmids from
serum nuclei, overall the positive charge characteristics of these
structures lead to non-specific interactions with cells (both blood cells
and blood vessel endothelial cells) and serum proteins. Also, following
i.v. injection, DNA complexes like that in practice tend to accumulate
in the lungs and liver. Complex DNA targeting for certain cell types
also poses considerable technical challenges (most of which are not
met). Approaches, such as the incorporation of antibodies directed
against certain cell surface antigens can provide a path in the future
to achieve the targeting of these cells.
• There are two potential routes by which plasmid DNA can reach the
nucleus:
• direct nuclear entry as a consequence of damage to the nuclear
membrane associated with mitosis;
• transportation through nuclear pores, which can occur through
passive diffusion or energy needed by the transportation process.