How vectors work to transfer genetic material
Gene therapy
utilizes the delivery of DNA into cells, which can be accomplished by
several methods, summarized below. The two major classes of methods are
those that use recombinant viruses (sometimes called biological
nanoparticles or viral vectors) and those that use naked DNA or DNA
complexes (non-viral methods).
Viruses
All viruses
bind to 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,
hacking 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 insert their genome into the host's
cytoplasm, but do not actually enter the cell. Others penetrate the cell
membrane disguised as protein molecules and enter the cell.
There are two main types of virus infection: lytic and lysogenic.
Shortly after inserting its DNA, viruses of the lytic cycle quickly
produce more viruses, burst from the cell and infect more cells.
Lysogenic viruses integrate their DNA into the DNA of the host cell and
may live in the body for many years before responding to a trigger. The
virus reproduces as the cell does and does not inflict bodily harm until
it is triggered. The trigger releases the DNA from that of the host and
employs it to create new viruses.
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, namely reverse transcriptase and integrase,
into the cell. This RNA molecule from the retrovirus must produce a DNA
copy from its RNA molecule before it can be integrated into 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 has been inserted, it
can be said that the host cell has been modified to contain new genes.
If this host cell divides later, its descendants will all contain the
new genes. Sometimes the genes of the retrovirus do not express their
information immediately.
One of the problems of gene therapy using retroviruses is that
the integrase enzyme can insert the genetic material of the virus into
any arbitrary position in the genome of the host; it randomly inserts
the genetic material into a chromosome. 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 or by including certain sequences such as the beta-globin locus control region to direct the site of integration to specific chromosomal sites.
Gene therapy trials using retroviral vectors to treat X-linked severe combined immunodeficiency
(X-SCID) represent the most successful application of gene therapy to
date. More than twenty patients have been treated in France and Britain,
with a high rate of immune system reconstitution observed. Similar
trials were restricted or halted in the USA when leukemia was reported in patients treated in the French X-SCID gene therapy trial.
To date, four children in the French trial and one in the British trial
have developed leukemia as a result of insertional mutagenesis by the
retroviral vector. All but one of these children responded well to
conventional anti-leukemia treatment. Gene therapy trials to treat SCID
due to deficiency of the Adenosine Deaminase (ADA) enzyme (one form of SCID) continue with relative success in the USA, Britain, Ireland, Italy and Japan.
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.
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 been promoted for
treating cancer and indeed the first gene therapy product to be
licensed to treat cancer, Gendicine, is an adenovirus. Gendicine, an adenoviral p53-based
gene therapy was approved by the Chinese food and drug regulators in
2003 for treatment of head and neck cancer. Advexin, a similar gene
therapy approach from Introgen, was turned down by the US Food and Drug Administration (FDA) in 2008.
Concerns about the safety of adenovirus vectors were raised after the 1999 death of Jesse Gelsinger
while participating in a gene therapy trial. Since then, work using
adenovirus vectors has focused on genetically crippled versions of the
virus.
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 resistant 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. 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, and many
concerns from the medical community would be alleviated. 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.
Replication-competent vectors
A
replication-competent vector called ONYX-015 is used in replicating
tumor cells. It was found that in the absence of the E1B-55Kd viral
protein, adenovirus caused very rapid apoptosis of infected, p53(+)
cells, and this results in dramatically reduced virus progeny and no
subsequent spread. Apoptosis was mainly the result of the ability of EIA
to inactivate p300. In p53(-) cells, deletion of E1B 55kd has no
consequence in terms of apoptosis, and viral replication is similar to
that of wild-type virus, resulting in massive killing of cells.
A replication-defective vector deletes some essential genes.
These deleted genes are still necessary in the body so they are replaced
with either a helper virus or a DNA molecule.
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.
Herpes simplex virus
The herpes simplex virus
is a human neurotropic virus. This is mostly examined for gene
transfer in the nervous system. The wild type HSV-1 virus is able to
infect neurons and evade the host immune response, but may still become
reactivated and produce a lytic cycle of viral replication. Therefore,
it is typical to use mutant strains of HSV-1 that are deficient in their
ability to replicate. Though the latent virus is not transcriptionally
apparent, it does possess neuron specific promoters that can continue
to function normally. Antibodies to HSV-1 are common in humans, however complications due to herpes infection are somewhat rare.
Caution for rare cases of encephalitis must be taken and this provides
some rationale to using HSV-2 as a viral vector as it generally has
tropism for neuronal cells innervating the urogenital area of the body
and could then spare the host of severe pathology in the brain.
Non-viral methods
Non-viral
methods present certain advantages over viral methods, with 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 have yielded molecules and techniques with
transfection efficiencies similar to those of viruses.
Injection of naked DNA
This is the simplest method of non-viral transfection. Clinical trials 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. Cellular uptake of
naked DNA is generally inefficient. Research efforts focusing on
improving the efficiency of naked DNA uptake have yielded several novel
methods, such as electroporation, sonoporation, and the use of a "gene gun", which shoots DNA coated gold particles into the cell using high pressure gas.
Physical methods to enhance delivery
Electroporation
Electroporation
is a method that uses short pulses of high voltage to carry DNA across
the cell membrane. This shock is thought to cause temporary formation of
pores in the cell membrane, allowing DNA molecules to pass through.
Electroporation is generally efficient and works across a broad range of
cell types. However, a high rate of cell death following
electroporation has limited its use, including clinical applications.
More recently a newer method of electroporation, termed
electron-avalanche transfection, has been used in gene therapy
experiments. By using a high-voltage plasma discharge, DNA was
efficiently delivered following very short (microsecond) pulses.
Compared to electroporation, the technique resulted in greatly increased
efficiency and less cellular damage.
Gene gun
The use of particle bombardment, or the gene gun,
is another physical method of DNA transfection. In this technique, DNA
is coated onto gold particles and loaded into a device which generates a
force to achieve penetration of the DNA into the cells, leaving the
gold behind on a "stopping" disk.
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 cells.
Magnetofection
In a method termed magnetofection,
DNA is complexed to magnetic particles, and a magnet is placed
underneath the tissue culture dish to bring DNA complexes into contact
with a cell monolayer.
Hydrodynamic delivery
Hydrodynamic delivery involves rapid injection of a high volume of a solution into vasculature (such as into the inferior vena cava, bile duct, or tail vein). The solution contains molecules that are to be inserted into cells, such as DNA plasmids or siRNA,
and transfer of these molecules into cells is assisted by the elevated
hydrostatic pressure caused by the high volume of injected solution.
Chemical methods to enhance delivery
Oligonucleotides
The
use of synthetic oligonucleotides in gene therapy is to deactivate 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 molecules of RNA called siRNA to signal the cell 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. Additionally, single stranded DNA
oligonucleotides have been used to direct a single base change within a
mutant gene. The oligonucleotide is designed to anneal with
complementarity to the target gene with the exception of a central base,
the target base, which serves as the template base for repair. This
technique is referred to as oligonucleotide mediated gene repair,
targeted gene repair, or targeted nucleotide alteration.
Lipoplexes
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.
Polymersomes
Polymersomes are synthetic versions of liposomes (vesicles with a lipid bilayer), made of amphiphilic block copolymers. They can encapsulate either hydrophilic or hydrophobic
contents and can be used to deliver cargo such as DNA, proteins, or
drugs to cells. Advantages of polymersomes over liposomes include
greater stability, mechanical strength, blood circulation time, and
storage capacity.
Polyplexes
Complexes
of polymers with DNA are called polyplexes. Most polyplexes consist of
cationic polymers and their fabrication is based on self-assembly by
ionic interactions. One important difference between the methods of
action of polyplexes and lipoplexes is that polyplexes cannot directly
release their DNA load into the cytoplasm. As a result, co-transfection
with endosome-lytic agents such as inactivated adenovirus was required
to facilitate nanoparticle escape from the endocytic vesicle made during
particle uptake. However, a better understanding of the mechanisms by
which DNA can escape from endolysosomal pathway, i.e. proton sponge
effect,
has triggered new polymer synthesis strategies such as incorporation of
protonable residues in polymer backbone and has revitalized research on
polycation-based systems.
Due to their low toxicity, high loading capacity, and ease of
fabrication, polycationic nanocarriers demonstrate great promise
compared to their rivals such as viral vectors which show high
immunogenicity and potential carcinogenicity, and lipid-based vectors
which cause dose dependence toxicity. Polyethyleneimine and chitosan
are among the polymeric carriers that have been extensively studied for
development of gene delivery therapeutics. Other polycationic carriers
such as poly(beta-amino esters) and polyphosphoramidate
are being added to the library of potential gene carriers. In addition
to the variety of polymers and copolymers, the ease of controlling the
size, shape, surface chemistry of these polymeric nano-carriers gives
them an edge in targeting capability and taking advantage of enhanced
permeability and retention effect.
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 complementarity 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 some 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.
Producing dendrimers has historically been a slow and expensive
process consisting of numerous slow reactions, an obstacle that severely
curtailed their commercial development. The Michigan-based company
Dendritic Nanotechnologies discovered a method to produce dendrimers
using kinetically driven chemistry, a process that not only reduced cost
by a magnitude of three, but also cut reaction time from over a month
to several days. These new "Priostar" dendrimers can be specifically
constructed to carry a DNA or RNA payload that transfects cells at a
high efficiency with little or no toxicity.
Inorganic nanoparticles
Inorganic nanoparticles, such as gold, silica, iron oxide (ex. magnetofection) and calcium phosphates have been shown to be capable of gene delivery.
Some of the benefits of inorganic vectors is in their storage
stability, low manufacturing cost and often time, low immunogenicity,
and resistance to microbial attack. Nanosized materials less than 100 nm
have been shown to efficiently trap the DNA or RNA and allows its escape from the endosome without degradation. Inorganics have also been shown to exhibit improved in vitro transfection for attached cell lines due to their increased density and preferential location on the base of the culture dish. Quantum dots
have also been used successfully and permits the coupling of gene
therapy with a stable fluorescence marker. Engineered organic
nanoparticles are also under development, which could be used for
co-delivery of genes and therapeutic agents.
Cell-penetrating peptides
Cell-penetrating peptides (CPPs), also known as peptide transduction domains (PTDs), are short peptides
(< 40 amino acids) that efficiently pass through cell membranes
while being covalently or non-covalently bound to various molecules,
thus facilitating these molecules’ entry into cells. Cell entry occurs
primarily by endocytosis but other entry mechanisms also exist. Examples of cargo molecules of CPPs include nucleic acids, liposomes, and drugs of low molecular weight.
CPP cargo can be directed into specific cell organelles by incorporating localization sequences into CPP sequences. For example, nuclear localization sequences are commonly used to guide CPP cargo into the nucleus. For guidance into mitochondria, a mitochondrial targeting sequence can be used; this method is used in protofection (a technique that allows for foreign mitochondrial DNA to be inserted into cells' mitochondria).
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.
