1. 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 To this end, many vectors have been developed in which the endogenous viral envelope proteins have been replaced by either envelope proteins from other viruses, or by chimeric proteins.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 stomatitis virus. This vector is referred to as VSV G-pseudotyped lentivirus, and infects an almost universal set of cells.
2. Adenoviruses
Adenoviruses are viruses that carry their genetic material in the form of double-stranded DNA. They cause respiratory, intestinal, and eye infections in humans (especially the common cold). When these viruses infect a host cell, they introduce their DNA molecule into the host. The genetic material of the adenoviruses is not incorporated (transient) 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.
Eg: for cystic fibrosis , cardiac heart failure etc.
1.
Cis and trans-acting elements:
Replication-defective
vectors always contain a “transfer construct”. The transfer construct carries
the gene to be transduced or “transgene”. The transfer construct also carries
the sequences which are necessary for the general functioning of the viral
genome: packaging sequence, repeats for replication and, when needed, priming
of reverse transcription. These are denominated cis-acting elements, because
they need to be on the same piece of DNA as the viral genome and the gene of
interest. Trans-acting elements are viral elements, which can be encoded on a
different DNA molecule. For example, the viral structural proteins can be expressed
from a different genetic element than the viral genome
2.
Adeno-associated viruses
Adeno-associated viruses, from the parvovirus family, are small
viruses with a genome of single stranded DNA. The wild type AAV can insert
genetic material at a specific site on chromosome 19 with near 100% certainty.
But the recombinant AAV, which does not contain any viral genes and only the
therapeutic gene, does not integrate into the genome. Instead the recombinant
viral genome fuses at its ends via the ITR (inverted terminal repeats)
recombination to form circular, episomal forms which are predicted to be the
primary cause of the long term gene expression.
3.
Lipoplexes and polyplexes:
To improve
the delivery of the new DNA into the cell, the DNA must be protected from
damage and (positively charged). Initially, anionic and neutral lipids were
used for the construction of lipoplexes for synthetic vectors. However, in
spite of the facts that there is little toxicity associated with them, that
they are compatible with body fluids and that 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, were first used to condense
negatively charged DNA molecules so as to facilitate the encapsulation of DNA
into liposomes. Later it was found that the use of cationic lipids
significantly enhanced the stability of lipoplexes. Also as a result of their
charge, cationic liposomes interact with the cell membrane, endocytosis was widely
believed as the major route by which cells uptake lipoplexes. Endosomes are
formed as the results of endocytosis, however, if genes can not be released
into cytoplasm by breaking the membrane of endosome, they will be sent to
lysosomes where all DNA will be destroyed before they could achieve their
functions. It was also found that although cationic lipids themselves could
condense and encapsulate DNA into liposomes, the transfection efficiency is
very low due to the lack of ability in terms of “endosomal escaping”. However,
when helper lipids (usually electroneutral lipids, such as DOPE) were added to
form lipoplexes, much higher transfection efficiency was observed. Later on, it
was figured out that certain lipids have the ability to destabilize endosomal
membranes so as to facilitate the escape of DNA from endosome, therefore those
lipids are called fusogenic lipids. Although cationic liposomes have been
widely used as an alternative for gene delivery vectors, a dose dependent
toxicity of cationic lipids were also observed which could limit their
therapeutic usages.
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 lipoplexes 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
as does chitosan and
trimethylchitosan.
6.
Non –viral methods:
i.
Sonoporation
Sonoporation uses
ultrasonic frequencies to deliver DNA into cells. The process of acoustic
cavitation is thought to disrupt the cell membrane and allow DNA to move into
cell.
i.
Magnetofection
In a method
termed magnetofection,
DNA is complexed to a magnetic particles, and a magnet is placed underneath the
tissue culture dish to bring DNA complexes into contact with a cell monolayer.
ii.
Dendrimers
A dendrimer is a highly
branched macromolecule
with a spherical shape. The surface of the particle may be functionalized in
many ways and many of the properties of the resulting construct are determined
by its surface.
In particular it
is possible to construct a cationic dendrimer, i.e. one with a positive surface
charge. When in the presence of genetic material such as DNA or RNA, charge
complimentarity leads to a temporary association of the nucleic acid with the
cationic dendrimer. On reaching its destination the dendrimer-nucleic acid complex
is then taken into the cell via endocytosis.
In recent years
the benchmark for transfection agents has been cationic lipids. Limitations of
these competing reagents have been reported to include: the lack of ability to
transfect a number of cell types, the lack of robust active targeting
capabilities, incompatibility with animal models, and toxicity. Dendrimers
offer robust covalent construction and extreme control over molecule structure,
and therefore size. Together these give compelling advantages compared to
existing approaches.
7.
Hybrid methods
Due to
every method of gene transfer having shortcomings, there have 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.
8. Introduction of Suicide Genes Into Melanoma
Cells:
The
genes used in the suicide gene transfer strategy are those that, when
introduced into tumor cells, have the capacity to convert a nontoxic prodrug
into a toxin within the tumor cell.9,10 The most widely used gene in clinical
trials using this approach is the herpes simplex virus thymidine kinase (HSVtk)
gene. This gene is introduced into tumor cells, and patients are subsequently
given the drug ganciclovir. This drug is an acyclic nucleoside analogue that,
when phosphorylated by HSVtk, is incorporated into DNA (as ganciclovir- triphosphate)
resulting in the termination of DNA elongation during S-phase of transduced
tumor cells.
9. Targeted mutant p53 expression:
The TP53 gene is one of
the most studied genes in human cancer. In recent years, considerable interest
was focused on mutant p53, the abnormal protein product of TP53 somatic or
germline alleles with missense mutations that often accumulate in cancer cells.
There is now compelling experimental evidence that many mutations can exert
mutant-specific, gain-of-function effects by perturbing the regulation of
expression of multiple genes. This notion is supported by the observation that
targeted mutant p53 expression enhances the formation of specific cancers in
the mouse even in the absence of wild-type p53 expression. In addition,
clinical studies are producing a wealth of functional pathway data
demonstrating correlations between specific TP53 mutations and gene expression
patterns identified by transcriptome studies. These correlations imply that
alteration of p53 function is critical in shaping gene expression patterns in
cancers.
10. Stem cells:
Cancer
stem cells (CSCs) are defined by their ability to (i) fully recapitulate the
tumor of origin when transplanted into immunodeficient mouse hosts, and (ii)
self-renew, demonstrated by their ability to be serially transplanted.
1. Neural Stem Cells as Gene Delivery Vehicles:
May Provide New Strategies AgainstNeurodegenerative DiseaseThe NSCs are easily
transduced in vitro by common gene transfer methods, and they express the genes
of interest rapidly and for long periods of time. NSCs can be manipulated to
carry multiple copies or several transgenes that are driven by specific
promoters, for example. After ex vivo engineering, it is relatively easily
injected into the germinal zones of the central nervous system, where they are
able to reintegrate into the host cytoarchitecture. The engrafted NSCs can even
migrate to more distantly affected areas and adjust to different environments
due to their high plasticity.
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BITS Pilani
Tags:
Pharmacology