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Genetic Manipulations
An essential step in the study or utilization of a gene is the preparation of large numbers of the
DNA molecules of interest and the ability of expressing it in different organisms. A DNA fragment of
interest is linked through standard 3’->5’ phosphodiester bonds to a vector DNA molecule, which
can replicate when introduced into a host cell. Two types of plasmid vectors are commonly used E.
coli plasmid vectors and bacteriophage lambda vectors.
Plasmids
Plasmids are circular, double-stranded DNA (dsDNA) molecules that are separate from a cell’s
chromosomal DNA. Plasmids occur naturally in bacteria, yeast, and some eukaryotic cells. They
range from a few thousand bp to 100 kilobases. Plasmid DNA is duplicated before every cell
division. , and at least one plasmid copy is segregated to the daughter cell.
Plasmids used in recombinant DNA technology are optimized to be used as vectors, they’re 3 kb
and have only some essential genes for their use in cloning: a replication origin, a drug-resistance
gene, and a region in which exogenous DNA fragments can be inserted.
The replication origin (ORI) is a 100 bp seq that must be present in order for them to replicate.
Host-cell enzymes bind to ORI, initiating replication of the circular plasmid. Once DNA replication is
initiated at ORI, it continues around the circular plasmid regardless of its nucleotide sequence, so
any DNA sequence inserted into such a plasmid is replicated along with the rest of the plasmid
DNA.
Plasmids are integrated into cells in a process called transformation. Only 1:10000 cells from the
population will incorporate the plasmid, so it’s necessary to select those that did it from those that
did not. A plasmid vector must contain a selectable gene, most commonly a drug resistance gene
encoding an enzyme that inactivates a specific antibiotic, for example beta-lactamase, which
inactivates ampicilin. By growing the cells in ampicilin we can easily select those that incorporated
the plasmid, since only this subpopulation will survive the treatment and will multiply, with all the
daughter cells having the gene of interest.
Restriction enzymes and DNA ligases are bacterial enzymes that recognize specific 4-8 bp
sequences called restriction sites and then cleave both DNA strands at this site. Many are short
inverted repeat sequences.
At room temperature, the ss sticky ends can base-pair bond with complementary DNA from other
sources. A DNA ligase – the same enzyme that catalyzes ligation of okazaki fragments during
replication – catalyzes formation of 3’->5’ phosphodiester bonds between restriction fragments
during the time the sticky ends are transiently base-paired.
Protein expression systems
Low abundance proteins can be expressed at high levels in E. coli through use of specially
designed expression vectors. The vector is similar to the above depicted one, but has also a strong
promoter region, which initiates transcription many times per minute. The lac promoter, for example,
activates a gene that metabolizes lactose (or its analog IPTG). When lacZ gene is replaced by a
different one, as G-CSF, it will be activated in the same conditions, overexpressing the gene of
interest.
Proteins that are extensively modified during or following their synthesis, such as glycoproteins to
which carbohydrate groups are added. E. Coli lacks enzymes that catalyzes such reactions.
Eukaryotic expression vectors permit addition of appropariate post-translational modificatrions to
expressed proteins.
Cell Transfection
Transfection is the introduction of foreign DNA into eukaryotic or prokaryotic cells, such as animal
or bacterial cells.
Cells exposed to such a DNA precipitate take up the DNA and transport it to the nucleus, where it
can be transcribed for several days a phenomenon called transient expression. In a smaller
fraction of cells (0.1% or less), the foreign DNA becomes stably integrated into the cell genome
and is transferred to progeny cells at cell division just as any other cell gene is. These stably
transformed cells can be isolated if the transfected DNA contains a selectable marker, such as
resistance to a drug that inhibits the growth of normal cells.
For most applications of transfection, it is sufficient if the transfected gene is only transiently
expressed. Since the DNA introduced in the transfection process is usually not inserted into the
nuclear genome, the foreign DNA is lost at the latest when the cells undergo mitosis.
These methods include direct microinjection of DNA into the cell nucleus, incorporation of DNA into
lipid vesicles (liposomes) that fuse with the plasma membrane, and exposure of cells to a brief
electric pulse that transiently opens pores in the plasma membrane (electroporation). Calcium
phosphate. The calcium phosphate and DNA form co-precipitates on the surface of the target cells.
The high concentration of the DNA on the plasma membrane may increase the efficiency of
transfection.
Expression in living cells.
The frog oocyte system is a cellular equivalent of the in vitro translation system in that
mRNA appropriately generated from cloned cDNA is injected directly into fertilized frog
eggs. The frog produces unusually large eggs, which permit direct manual injection of a
relatively large quantity of mRNA. The injected mRNA is translated by the intrinsic
metabolic machinery within the egg and processed to form functional proteins. Thus, the
function of the produced protein, if known or suspected, can be tested. This system
allows only a brief, transient expression of the exogenously injected mRNA, and again,
the quantity that can be generated is very small. Nevertheless, the system has been used
widely in neurobiology because many expressed membrane proteins, such as receptors
and channel proteins, are integrated into the oocyte membrane and their functions can be
studied by sophisticated tools, such as patch clamping.
Perhaps one of the most commonly used mammalian cell types for transient expression is COS-1,
transformed African green monkey kidney cells. When transfected by cDNAs subcloned into
appropriate plasmid vectors that include a suitable transcriptional control element upstream of the
cloned cDNA, COS-1 cells generate a gene product which can then be tested for its known or
suspected function. Expression is transient, with peak activity between 1 and 4 days after
transfection. Unless some mechanism is used to select the transfected cells, no more than 10 to
20% of the cells in the culture express the exogenous gene. While the prokaryotic overexpression
system can produce preparative quantities of cloned gene products, the lack of post-translational
processing limits its usefulness when the protein requires such modifications for functional activity.
Transfection to mammalian cells
Viruses
A virus is a small parasite that cannot reproduce itself. Once it infects a susceptible cell, a virus can
direct the cell machinery to produce more viruses. They have both RNA or DNA as their genetic
material that can be single or double stranded. The virus consists of a nucleic acid and an outer
shell of protein.
The nucleic acid of a virion is enclosed within a protein coat called capsid, composed of multiple
copies of one or a few proteins. In each virus, clefts are observed which interact with cell surface
receptors, attaching it to the cell membrane. In some viruses, the nucleocapsid is covered by an
external membrane which consists of a normal phospholipids bilayer but also contains
glycoproteins (encoded by the virus genome) that also interact with cell receptors, that determine
the virus host. Then the virus DNA crosses the membrane to the cytoplasm in different ways,
sometimes accompanied by inner viral proteins. The capsid can enter the cell or remain outside it.
The genome of most DNA-containing viruses is transported to the cell nucleus. Inside the cell, the
viral DNA interacts with the hosts machinery for transcribing DNA into mRNA, The viral mRNA is
then translated into viral proteins by host-cell ribosomes, tRNA, and translation factors. The viral
genome, which encondes from 4 to 200 proteins, doesn’t encode for transcription and translation
proteins.
Most viral proteins are: special enzymes needed for viral replication, inhibitory factors that stop
host-cell DNA, RNA and protein synthesis, and structural proteins used in the construction of new
virions. After the synthesis of hundres to thousands of new virions, cells rupture suddenly or
gradually, releasing all the virions to the medium. The lytic cycle of a virus comprises those events:
adsorption, penetration, replication and release.
The enveloped viruses are more complex. Within the nuclecapsid are viral enzymes for
synthesizing viral mRNA and replicating the viral genome. The envelope around is a phospholipids
bilayer containing copies of a transmembrane glycoproteins. After entering a cell, the DNA can
become integrated to the host cell genome, and is there replicated as part of the cell’s DNA. This
association is called lysogeny. Under certain conditions, the prophage DNA is activated, leading to
its excision from the host chromosome and entrance into the lytic cycle.
Viral vectors for gene therapy: the art of turning infectious agents into
vehicles of therapeutics. Mark A. Kay1, Joseph C Glorioso2 & Luigi Naldini3
The viral life cycle can be divided into two temporally distinct phases: infection and
replication. Infection results in the introduction of the viral genome into the cell. This
leads to an early phase of gene expression characterized by the appearance of viral
regulatory products, followed by a late phase, when structural genes are expressed
and assembly of new viral particles occurs. In the case of gene therapy vectors, the
viral particles encapsulate a modified genome carrying a therapeutic gene cassette in
place of the viral genome. Transduction is defined as the abortive (non-replicative or
dead-end) infection that introduces functional genetic information expressed from
the recombinant vectors into the target cell.
Viruses are highly evolved biological machines that efficiently gain access to host cells and exploit
the cellular machinery to facilitate their replication. Ideal virus-based vectors for most gene-therapy
applications harness the viral infection pathway but avoid the subsequent expression of viral genes
that leads to replication and toxicity. This is achieved by deleting all, or some, of the coding regions
from the viral genome, but leaving intact those sequences that are required in cis for functions such
as packaging the vector genome into the virus capsid or the integration of vector DNA into the host
chromatin. The terminal repeats, are short non-coding DNA sequence found at each end of the
viral genome, which contains elements required for the replication and packaging of the viral DNA.
The expression cassette of choice is then cloned into the viral backbone in place of those
sequences that were deleted. The deleted genes encoding proteins that are involved in replication
or capsid envelope proteins are included in a separate packaging construct to provide helper
functions in trans. The packaging cells into which the vector genome and packaging construct are
co-transfected then produce the recombinant vector particles.
Generic strategy for engineering a virus into a vector. The helper DNA contains
genes essential for viral replication placed in a heterologous/unrelated DNA context
that can be delivered as a plasmid, helper virus or stably inserted into the host
chromosomal DNA of the packaging cell. The helper DNA can be delivered as a single
molecule or in some cases split into different DNA molecules for safety reasons (see
text). The helper DNA lacks the packaging domain ( ) so it itself or its RNA cannot be
packaged into a viral particle. The helper DNA of some vectors also lacks additional
transfer functions, to increase safety. The vector DNA contains the therapeutic
expression cassette and non-coding viral cis-acting elements that include a
packaging domain. Some vectors contain viral genes that are relatively inactivated
(not transcriptionally active at the same level as in a wild-type infection) due to the
absence of other viral genes. The viral proteins required for replication of the vector
DNA are produced, leading to the synthesis of many copies of the vector genome
(RNA or DNA, depending on the type of vector). Viral structural proteins recognize
the vector (psi plus) but not the helper (psi negative) nucleic acid to result in
packaging of the vector genome into a particle.
For gene therapy to be successful, an appropriate amount of a therapeutic gene
must be delivered into the target tissue without substantial toxicity. Each viral vector
system is characterized by an inherent set of properties that affect its suitability for
specific gene therapy applications. For some disorders, long-term expression from a
relatively small proportion of cells would be sufficient (for example, genetic
disorders), whereas other pathologies might require high, but transient, gene
expression. For example, gene therapies designed to interfere with a viral infectious
process or inhibit the growth of cancer cells by reconstitution of inactivated tumor
suppressor genes may require gene transfer into a large fraction of the abnormal
cells.
As the expression of viral genes is responsible for most pathological and immunological
consequences of viral infection, gene transduction by recombinant vectors is often well tolerated.
Problems that may be observed with gene transfer vectors include acute toxicity from the infusion
of foreign materials, cellular immune responses directed against the transduced cells, humoral
immune responses against the therapeutic gene product and the potential for insertional
mutagenesis by certain integrating vectors.
VIRAL VECTORS FOR GENE DELIVERY TO THE NERVOUS SYSTEM
The brain is a complex organ with discrete and intricate interconnections between various types of
neurons, glia and other cells. It therefore presents a complicated target for the genetic manipulation
of specific sets of cells for biological investigation, or for altering gene expression for therapeutic
intervention.
DNA fate and regulation of transgene expression
Fate of transgenes. Most virus vectors deliver genes into the nucleus of the target cell.
Exceptions to this rule include poliovirus replicons, pox virus and the alphaviruses (Sindbis and
Semliki Forest virus), which replicate and/or express genes transiently in the cytoplasm.
Within the nucleus, viral DNA can have several fates: (i) maintenance as a non-replicating
extrachromosomal element; (ii) integration into the host-cell genome; or (iii) replication as an
extrachromosomal element. In non-dividing cells such as neurons, viral DNA can be
maintained as a stable element in all of these states. These elements are maintained for
months in neurons.
Gene regulation. It has been problematic to control the level, duration and specificity of
transgene expression mediated by virus vectors. Typically, strong viral, cellular or hybrid
promoters are used, which give high-level expression in most cells. Viral promoters can be
inactivated by the host cell over time, at least in part by methylation, but they can also remain
active for years. Achieving long-term physiological levels of expression might require the use
of mammalian promoters that are normally active in the cells of interest. Robust neuronal
promoters include synapsin 1, neuron-specific enolase108, tyrosine hydroxylase,
Targeting infectivity. Virions present ligands that bind to HSPG, integrins and/or receptors on the
cell surface. Capsid virions typically undergo receptor-mediated endocytosis, and as the
endosomal compartment acidifies, the capsid breaks down. The DNA or RNA is then released into
the cytoplasm and transported to the nucleus, in association with nucleus-seeking proteins.
Enveloped virions undergo membrane fusion at the cell surface or in endosomes, depending on
the nature of the envelope. Capsids might be transported along the cytoskeleton, followed by
association with the nuclear membrane and DNA delivery into the nucleus through the nuclear pore.
Neurotropic viruses, such as HSV, pseudorabies virus and poliovirus, have evolved mechanisms
for uptake by nerve termini, followed by rapid retrograde transport to the cell body and subsequent
anterograde transport. For capsid virions, tropism can be redirected by introducing receptor ligands
into capsid proteins, by exchanging capsid proteins between serotypes, or by using bi-functional
antibodies that bind both to the virus and to target molecules on the cell surface. Importantly, these
strategies can be used either to restrict or to broaden the range of cells infected.
Delivery modalities
Delivery modalities can be grouped into those that attempt to achieve widespread gene delivery
throughout the brain (global), and those that target specific cell populations within the brain (focal).
Gene delivery can be achieved by direct injection of vector, or implantation of transduced cells into
the brain parenchyma, ventricles or vasculature, with different types of vectors, modes of injection
and cell vehicles designed to hit selected targets. Also promoters can give affinity to specific cell
subpopulations.
Generally, diffusion is limited, and transduction is restricted to cells within a few millimetres of the
injection site. However, neuronal cell bodies some distance from the injected area can also be
transduced by anterograde or retrograde transport of the vector within their processes.
Methods for global delivery include injection into the carotid artery, with promotion of entry across
the blood–brain barrier through osmotic shock59, pharmacological agents60, transferrin receptormediated targeting48, and the use of migratory cells producing gene products or vectors that can
spread throughout the brain. Injection into the ventricles for transport of vectors and gene products
through the cerebrospinal fluid61-63 usually results in periventricular delivery62.
The advantages of cell vehicles for virus delivery include the ability to carry out transduction ex vivo,
where conditions can be optimized and cell types can be selected for specific properties and gene
expression. Transduced cell types used for this purpose in the brain include astrocytes1, 64,
macrophages64, fibroblasts65 and neural precursor cells.
a | In direct vector delivery, a suspension of virus vector (green circles) is injected
stereotactically into the target area of the brain parenchyma, typically 10 5–108 transducing
units in a volume of 1–2 l in rodents. Depending on the vector type, it can be taken up by
cells at the injection site, or can diffuse away from the site of injection. Virus can also be
taken up and transported anterogradely or retrogradely to cell bodies through neuronal
processes projecting into the region from distant sites (arrowhead). Most viruses show limited
diffusion within the brain, with cells within a few millimetres of the injection site likely to be
infected with multiple particles. b | Cell vehicles can also be used to spread the vector or
transgene product over a wider distribution. Genetically modified cells (small green double
circles) are injected into the brain parenchyma, typically 104–105 cells in a volume of 1–2 l in
rodents. Some cell types can migrate away (wavy lines) from the injection site, releasing the
vector or product (green circles) along their path, and in the case of neural precursor cells,
might incorporate into the cytoarchitecture.
Different virus have differ in their characteristics: They infect different cell types differentially (even
with distinguishing differences such as cones and rods, Purkinje cells but not granulocytes, etc),
astrocytes, oligodendroglia, microglia, etc. They have different efficiency for retrograde transport.
Different transgene capacity. Differently activate the immune system (which is sometimes positive,
as in tumor therapy)
The spectrum of vectors used in basic research applications surpasses the few being used in
clinical trials, and comprises those with simple capsid virions — nucleic acid genome encased in a
proteinaceous shell — of which the most widely used are recombinant adenovirus and adenoassociated virus (AAV), and viruses with enveloped virions (in which the capsid is surrounded by a
lipid bilayer envelope;), which include RETROVIRUS/lentivirus, alphavirus, and herpes virus. some
cells are intrinsically more susceptible to infection with certain vectors. This is best determined by
direct comparison of several vectors expressing a reporter protein, such as green fluorescent
protein (GFP).
The transgene capacity also varies widely, with AAV having the smallest capacity (4.5 kb) and
herpes simplex virus (HSV) amplicons the largest (150 kb). Gutted versions of vectors such as
adenovirus, AAV, retrovirus/lentivirus and HSV amplicons tend to be less toxic, as they express no
viral genes. Further, expression patterns following viral delivery to the central nervous system
(CNS) can be altered by targeting infection through modification of the surface of the virions, or by
using different promoters to drive transgene expression.
Adeno-associated virus. AAV SEROTYPE 2 infects neurons preferentially4, apparently through the
interaction of AAV2 capsid proteins with HEPARAN SULPHATE proteoglycan (HSPG) moieties on the
cell surface. AAV2 does not seem to infect all classes of neurons equally well, however, and even
in cells that are susceptible to infection, the choice of promoter to drive transgene expression is
crucial for achieving high levels of sustained expression5. For example, studies in the neural retina
indicate that AAV2 transduces rod photoreceptor cells to a greater extent than cones 6, 7.
Mammalian promoters that are normally expressed in the targeted cell type can, in some cases at
least, achieve more sustained transgene expression than viral promoters. Retrograde transport of
AAV2 through neuronal processes seems to be limited to spatially close regions, and uptake from
motor nerve terminals is inefficient8. The properties of AAV4 and AAV5 differ from those of AAV2;
AAV5 diffuses more widely, and AAV4 primarily transduces EPENDYMAL CELLS9. In the cerebellum,
AAV5 transduces PURKINJE CELLS, but not GRANULE CELLS, with high efficiency10. Although AAV
vectors are highly effective for gene delivery, and are non-toxic, they have a relatively small
transgene capacity (4–5 kb).
Recombinant adenoviruses
Adenovirus vectors based on serotype 5 undergo retrograde transport from nerve terminals. One
feature of the adenovirus capsid, which can be detrimental to long-term gene expression but
advantageous in tumour therapies, is its extremely effective adjuvant properties. These promote an
effective immune response against infected cells. First-generation recombinant adenovirus vectors
are generated by deleting the parts of the virus genome that are required for replication, and
replacing them with transgene expression cassettes.
Herpes simplex virus. HSV is neurotropic in vivo, but nevertheless infects a broad range of cell
types. This large DNA virus shows highly efficient retrograde and anterograde transport within the
nervous system, and can enter a benign state of latency within neurons. Replication-competent
HSV is passed selectively across synapses, but eventually causes death of the infected cells.
Recombinant HSV vectors, in which portions of the virus genome are deleted, have a large
transgene capacity (at least 50 kb), can enter an EPISOMAL state with prolonged expression of
transgenes from the latency-associated promoter, and have minimal toxicity, although low-level
expression of virus genes can occur.
Retroviruses. They are enveloped viruses possessing an RNA genome, and replicate via a
dsDNA intermediate. Retroviruses rely on the enzyme reverse transcriptase to perform the reverse
transcription of its genome from RNA into DNA, which can then be integrated into the host's
genome with an integrase enzyme. When retroviruses have integrated their genome into the germ
line, their genome is passed on to a following generation. The viral envelope glycoprotein dictates
the host range of retroviral particles through its interaction with receptors on target cells.
Vectors derived from some retroviruses, such as Moloney murine leukaemia virus (MoMLV), have
limited applications as vectors for the CNS owing to their inability to deliver genes to non-dividing
cells. These vectors are used extensively, however, for ex vivo infection of cultured cells followed
by transplantation, and for clonal analysis in brain development
Lentiviruses
Unlike retroviruses, they rely on active transport of the preinitiation complex through
the nucleopore by the nuclear import machinery of the target cell The lentiviral
strategy for nuclear targeting enables infection of non-dividing cells, an attractive
attribute for a gene therapy vector.
VSV-pseudotyped lentiviral vectors can be delivered directly in vivo. They efficiently
transduce the neurons and glial cells of the central nervous system (CNS) of rodents
and non-human primates.
The obligatory RNA step in the retroviral lifecycle (for example, reverse transcription)
poses great constraints on the viral genome and on its exploitation for gene transfer
purposes. The transgene expression cassette must be of limited size, without introns
and internal polyadenylation signals. Together with the exposure to loco-regional
differences in the structure and activity of chromatin consequent to random
integration, these factors combine to limit expression of the transduced genes.
Applications in neurobiology
The ability to use vectors to achieve gene delivery to neural cells provides an important tool for
addressing basic questions both in neurobiology and in the molecular aetiology of disease.
Neurons in particular have proven to be resistant to most non-viral means of transduction,
especially in vivo. Vectors that express reporter genes such as GFP and alkaline phosphatase
have been used to visualize the morphology of individual neurons and to tag synapses.