Viruses
All
viruses attack their hosts and introduce their genetic material
into the host cell as part of their replication cycle. This genetic
material contains basic 'instructions' of how to produce more copies
of these viruses, hijacking the body's normal production machinery
to serve the needs of the virus. The host cell will carry out these
instructions and produce additional copies of the virus, leading to
more and more cells becoming infected. Some types of viruses
actually physically insert their genes into the host's genome (it is
the defining feature of
retroviruses, the family of viruses that includes HIV, the virus
that causes AIDS). This incorporates the genes of that virus among
the genes of the host cell for the life span of that cell.
Doctors and molecular biologists realized
that viruses like this could be used as vehicles to carry 'good'
genes into a human cell. First, a scientist would remove the genes
in the virus that cause disease. Then they would replace those genes
with genes encoding the desired effect (for instance, insulin
production in the case of diabetics). This procedure must be done in
such a way that the genes which allow the virus to insert its genome
into its host's genome are left intact. This can be confusing, and
requires significant research and understanding of the virus' genes
in order to know the function of each. An example:
A virus is found which replicates by
inserting its genes into the host cell's genome. This virus has two
genes- A and B. Gene A encodes a protein which allows this virus to
insert itself into the host's genome. Gene B causes the disease this
virus is associated with. Gene C is the "normal" or "desirable" gene
we want in the place of gene B. Thus, by re-engineering the virus so
that gene B is replaced by gene C, while allowing gene A to properly
function, this virus could introduce your 'good gene'- gene C into
the host cell's genome without causing any disease.
All this is clearly an oversimplification,
and numerous problems exist that prevent gene therapy using viral
vectors, such as: trouble preventing undesired effects, ensuring the
virus will infect the correct target cell in the body, and ensuring
that the inserted gene doesn't disrupt any vital genes already in
the genome. However, this basic mode of gene introduction currently
shows much promise and doctors and scientists are working hard to
fix any potential problems that could exist.
Retroviruses
The genetic material in retroviruses is in
the form of
RNA molecules, while the genetic material of their hosts is in
the form of DNA. When a retrovirus infects a host cell, it will
introduce its RNA together with some enzymes into the cell. This RNA
molecule from the retrovirus must produce a DNA copy from its RNA
molecule before it can be considered for part of the genetic
material of the host cell. The process of producing a DNA copy from
an RNA molecule is termed
reverse transcription. It is carried out by one of the enzymes
carried in the virus, called
reverse transcriptase. After this DNA copy is produced and is
free in the
nucleus of the host cell, it must be incorporated into the
genome of the host cell. That is, it must be inserted into the large
DNA molecules in the cell (the chromosomes). This process is done by
another enzyme carried in the virus called
integrase.
Now that the genetic material of the virus
is incorporated and has become part of the genetic material of the
host cell, we can say that the host cell is now modified to contain
a new gene. If this host cell divides later, its descendants will
all contain the new genes.
One of the problems of gene therapy using
retroviruses is that the integrase enzyme can insert the genetic
material of the virus in any arbitrary position in the genome of the
host. If genetic material happens to be inserted in the middle of
one of the original genes of the host cell, this gene will be
disrupted (insertional
mutagenesis). If the gene happens to be one regulating cell
division, uncontrolled cell division (i.e.,
cancer) can occur. This problem has recently begun to be
addressed by utilizing
zinc finger nucleases[1] or by including certain
sequences such as the
beta-globin locus control region[6] to direct the
site of integration to specific chromosomal sites.
Gene therapy trials to treat
severe combined immunodeficiency (SCID) were halted or
restricted in the USA when
leukemia was reported in three of eleven patients treated in the
French Therapy X-linked SCID (XSCID) gene therapy trial. Ten XSCID
patients treated in England have not presented leukemia to date and
have had similar success in immune reconstitution. Gene therapy
trials to treat SCID due to deficiency of the Adenosine Deaminase
(ADA) enzyme continue with relative success in the USA, Italy and
Japan.
Adenoviruses
Adenoviruses are viruses that carry their genetic material in
the form of double-stranded DNA. They cause respiratory (especially
the common cold), intestinal, and eye infections in humans. When
these viruses infect a host cell, they introduce their DNA molecule
into the host. The genetic material of the adenoviruses is not
incorporated into the host cell's genetic material. The DNA molecule
is left free in the nucleus of the host cell, and the instructions
in this extra DNA molecule are
transcribed just like any other gene. The only difference is
that these extra genes are not replicated when the cell is about to
undergo cell division so the descendants of that cell will not have
the extra gene. As a result, treatment with the adenovirus will
require readministration in a growing cell population although the
absence of integration into the host cell's genome should prevent
the type of cancer seen in the SCID trials. This vector system has
shown real promise in treating cancer and indeed the first gene
therapy product to be licensed is an adenovirus to treat cancer.
Adeno-associated
viruses
Adeno-associated viruses, from the
parvovirus family, are small viruses with a genome of single
stranded DNA. These viruses can insert genetic material at a
specific site on chromosome 19. There are a few disadvantages to
using AAV, including the small amount of DNA it can carry (low
capacity) and the difficulty in producing it. This type of virus is
being used, however, because it is
non-pathogenic (most people carry this harmless virus). In
contrast to adenoviruses, most people treated with AAV will not
build an immune response to remove the virus and the cells that have
been successfully treated with it. Several trials with AAV are
on-going or in preparation, mainly trying to treat muscle and eye
diseases; the two tissues where the virus seems particularly useful.
However, clinical trials have also been initiated where AAV vectors
are used to deliver genes to the brain. This is possible because AAV
viruses can infect non-dividing (quiescent) cells, such as neurons
in which their genomes be expressed for a long time. In recent human
trials, CD8+ immune cells have recognized the AAV infected cells as
compromised and killed these cells accordingly. This action appears
to be triggered by part of the capsid or outer coat of the type 2
virus. Recent studies have shown that humans will likely react in
the same way against the new serotype 8 AAV as well.
Envelope protein
pseudotyping of viral vectors
The viral vectors described above have
natural host cell populations that they infect most efficiently.
Retroviruses have limited natural host cell ranges, and although
adenovirus and adeno-associated virus are able to infect a
relatively broader range of cells efficiently, some cell types are
refractory to infection by these viruses as well. Attachment to and
entry into a susceptible cell is mediated by the protein envelope on
the surface of a virus. Retroviruses and adeno-associated viruses
have a single protein coating their membrane, while adenoviruses are
coated with both an envelope protein and fibers that extend away
from the surface of the virus. The envelope proteins on each of
these viruses bind to cell-surface molecules such as heparin
sulfate, which localizes them upon the surface of the potential
host, as well as with the specific protein receptor that either
induces entry-promoting structural changes in the viral protein, or
localizes the virus in endosomes wherein acidification of the lumen
induces this refolding of the viral coat. In either case, entry into
potential host cells requires a favorable interaction between a
protein on the surface of the virus and a protein on the surface of
the cell. For the purposes of gene therapy, one might either want to
limit or expand the range of cells susceptible to transduction by a
gene therapy vector. To this end, many vectors have been developed
in which the endogenous viral envelope proteins have been replaced
by either envelope proteins from other viruses, or by chimeric
proteins. Such chimera would consist of those parts of the viral
protein necessary for incorporation into the virion as well as
sequences meant to interact with specific host cell proteins.
Viruses in which the envelope proteins have been replaced as
described are referred to as pseudotyped viruses. For example, the
most popular retroviral vector for use in gene therapy trials has
been the lentivirus Simian Immunodeficiency virus coated with the
envelope proteins, G-protein, from Vesicular Stomatitus virus. This
vector is referred to as VSV G-pseudotyped lentivirus, and infects
an almost universal set of cells. This tropism is characteristic of
the VSV G-protein with which this vector is coated. Many attempts
have been made to limit the tropism of viral vectors to one or a few
host cell populations. This advance would allow for the systemic
administration of a relatively small amount of vector. The potential
for off-target cell modification would be limited, as well as many
concerns from the medical community. Most attempts to limit tropism
have used chimeric envelope proteins bearing antibody fragments.
These vectors show great promise for the development of "magic
bullet" gene therapies.
Non-viral methods
Non-viral methods present certain advantages
over viral methods; simple large scale production and low host
immunogenicity being just two. Previously, low levels of
transfection and
expression of the gene held non-viral methods at a disadvantage,
however recent advances in vector technology has yielded molecules
and techniques with transfection efficiencies similar to that of
viruses.
Naked DNA
This is the simplest method of non-viral
transfection. Clinical trials have been carried out of intramuscular
injection of a
naked DNA plasmid have occurred with some success, however the
expression has been very low in comparison to other methods of
transfection. In addition to trials with plasmids, there have been
trials with naked
PCR product, which have had similar or greater success, however
this success does not compare to that of the other methods, leading
to research into more efficient methods for delivery of the naked
DNA such as
electroporation and the use of a
"gene gun", which shoots DNA coated gold particles into the cell
using high pressure gas.
Oligodeoxynucleotides
The use of synthetic oligodeoxynucleotides
in gene therapy is to inactivate the genes involved in the disease
process. There are several methods by which this is achieved. One
strategy uses
antisense specific to the target gene to disrupt the
transcription of the faulty gene. Another uses small catalytic
molecules of RNA called
siRNA to cleave specific unique sequences in the
mRNA transcript of the faulty gene, disrupting translation of
the faulty mRNA, and therefore expression of the gene. A further
strategy uses double stranded oligodeoxynucleotides as a decoy for
the transcription factors that are required to activate the
transcription of the target gene. The transcription factors bind to
the decoys instead of the promoter of the faulty gene which reduces
the transcription of the target gene, lowering expression.
Lipoplexes and
polyplexes
To improve the delivery of the new DNA into
the cell, the DNA must be protected from damage and its entry into
the cell must be facilitated. To this end new molecules, lipoplexes
and polyplexes, have been created that have the ability to protect
the DNA from undesirable degradation during the transfection
process.
Plasmid DNA can be covered with lipids in an
organized structure like a micelle or a liposome. When the organized
structure is complexed with DNA it is called a lipoplex. There are
three types of lipoplexes, anionic (negatively charged), neutral or
cationic (positively charged). Initially, anionic and neutral lipids
were used for the construction of lipoplexes for synthetic vectors.
However, although there is little toxicity associated with them,
they are compatible with body fluids and there was a possibility of
adapting them to be tissue specific, they are complicated and time
consuming to produce so attention was turned to the cationic
versions.
Cationic lipids, due to their positive charge, naturally complex
with the negatively charged DNA. Also as a result of their charge
they interact with the cell membrane,
endocytosis of the lipoplex occurs and the DNA is released into
the cytoplasm. The cationic lipids also protect against degradation
of the DNA by the cell.
The most common use of lipoplexes has been
in gene transfer into cancer cells, where the supplied genes have
activated tumor suppressor control genes in the cell and decrease
the activity of oncogenes. Recent studies have shown lipoplexes to
be useful in transfecting respiratory
epithelial cells, so they may be used for treatment of genetic
respiratory diseases such as cystic fibrosis.
Complexes of polymers with DNA are called
polyplexes. Most polyplexes consist of cationic polymers and their
production is regulated by ionic interactions. One large difference
between the methods of action of polyplexes and lipoplxes is that
polyplexes cannot release their DNA load into the cytoplasm, so to
this end, co-transfection with endosome-lytic agents (to lyse the
endosome that is made during endocytosis, the process by which the
polyplex enters the cell) such as inactivated adenovirus must occur.
However this isn't always the case, polymers such as
polyethylenimine have their own method of endosome disruption.
Hybrid methods
Due to every method of gene transfer having
shortcomings, there has been some hybrid methods developed that
combine two or more techniques. Virosomes are one example; they
combine liposomes with an inactivated HIV or influenza virus. This
has been shown to have more efficient gene transfer in respiratory
epithelial cells than either viral or liposomal methods alone. Other
methods involve mixing other viral vectors with cationic lipids or
hybridising viruses.
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