Introduction of Gene Therapy
-The concept of correcting or alleviating the symptoms of disease by the
delivery of genes whose expression limits the impact of the disease has
been termed gene therapy.
-Advanced development in gene transfer methods has made clinical gene
therapy possible for the treatment of diverse types of diseases, including
metabolic, cardiovascular, and autoimmune diseases, as well as cancer.
For example, clinical trials for gene therapy of cancer, cystic fibrosis, and
arthritis have been initiated in recent years.
-Gene therapy takes advantage of recent advances in many areas of
molecular and cell biology, including the identification of new
therapeutic genes, improvement in both viral and nonviral gene delivery
systems, better understanding of gene regulation, and improvement in
cell isolation and transplantation.
Construction of Adenovirus Vectors
-Adenovirus was first discovered in 1953 as an agent causing upper
respiratory tract infections in human.
-In 1962, certain adenovirus species have been found to be oncogenic
and induce tumors in rodent. This has attracted a tremendous surge of
interest in the study of the molecular biology of human adenoviruses.
-Adenoviruses are widespread in nature with linear double-stranded
DNA molecules of approx 36kb and around 100 different serotypes have
been isolated. The human adenovirus comprise 43 distinct serotypes
which cause a variety of ailments such as respiratory, ocular,
gastrintestinal and urinary tract diseases.
-Most of the current knowledge stems from studies of the human
subtypes 2 and 5, which belong to the non-oncogenic subgroup.
-After attachment of the virion to the cell, the virial DNA is transported
to the nucleus where early transcription takes place. Human cells are
permissive and therefore productively infected by adenoviruses, which
replicated within the infected cells.
-Infected cells produce 1000-10,000 plaque-forming units (PFU), and the
virus remains concentrated within the cell long after yields have reached
maximal levels, making collection and concentration of virus extremely
easy.
-For a number of reasons, adenoviruses are attracting increasing attention
as potential mammalian cell expression vectors and recombinant
vaccines
a. Viral particles are relatively stable.
b. Viral genome does not undergo rearrangement at a high rate.
c. Insertions of foreign genes are generally maintained without change
through successive rounds of viral replication.
d. Relative easy to manipulate by recombinant DNA techniques.
e. Virus replicates efficiently in permissive cells.
Structure of Adenovirus
-Adenoviruses are non-enveloped viruses that replicate and assemble
their virions in the nucleus of the infected cells.
-The adenovrius consists of two structural complexes:
capsid: outer icosahedral shell is composed of 252 capsomers,
including 240 hexons and 12 pentons
core: internal body comprising the nucleocapsid and the core shell
-Adenoviruses are icosahedral in shape, i.e. they have 20 triangular facets.
The diameter of the icosahedral-shaped capsid varies from 65 to 80 nm
depending on the serotype.
-The adenoviruses have noncovalently attached protein, known as the
fiber. Each penton complex consists of a fiber which is responsible to
cellular attachment. The fiber serves as the attachment organ when the
virion binds to its receptor on the surface of the host cell. There are about
105 of such receptors on each HeLa cell.
-Aednovirus particles contain 87% protein and 13% ds DNA. The virion
mass is about 1.7 x 105 Kd, in which the genome is a single linear
molecule of ds DNA of MW 2 x 104 Kd.
-The genome is covalently linked at each 5’ end to individual 55 kd
terminal proteins, which serve as the origin of transcription, and they
associate with each other to circularlise the DNA upon lysis of the virion.
Biological Properties of Adenovirus
-Two modes of entry of adenovirus into cells: receptor-mediated
endocytosis and direct entry. Most virus particles are taken up via
receptor-mediated endocytosis. Typically, 10 - 20 minutes after infection
virus particles are found close to the nuclear membrane, where they are
uncoated and the core pass into the nucleus through nuclear pores.
-The replication of adenovirus DNA takes place within the nucleoplasma
and is not associated with the nuclear membrane.
-The adenovirus genome is functionally divided into 2 major non
contiguous overlapping regions, early and late, based on the time of
transcription after infection (Figure 1).
Early genes: Correspond to events occuring before the onset of viral
DNA replication. Most of the early gene products are involoved with
regulation of viral and cellular synthetic activities, but some also
encode viral structure proteins.
Late genes: Correspond to the period after initiation of DNA
replication and encode the numerous components of the virion
pentamers, hexamers, and fibers. The switch from early to late gene
expression takes place about 7 hours after infection.
-There are 6 distinct early regions, E1A, E1B, E2A, E2B, E3, and E4,
and one late region with 5 coding units (L1 - L5) in adenovirus genome.
Each early and late region appears to contain a cassette of gene coding
for polypeptides with related functions.
-Once the viral DNA is inside the nucleus, transcription is initiated from
the viral E1 promoters. This is the only viral region that can be
transcribed with the aid of viral-encoded transcripiton factor.
E1A: one of the protein coded from this region can activate
transcription of the other early regions and amplify viral gene
expression.
E1B: proteins coded from this region can stop the cellular protein
synthesis and are essential to transform primary culture.
E2A/B: coded proteins are involoved in viral replication, including
viral DNA polymerase, the terminal protein and DNA binding proteins.
E3: responsible for counteracting the immune system
E4: this region contains a cassette of genes whose products act to
shutdown endogenous host gene expression and upregulate
transcription from the E2 and late regions
-There are at least three regions of the viral genome that can accept
insertions or substitution of DNA to generate a helper independent virus.
These are in E1, in E3, and in short region between E4 and the end of the
genome.
-E1 is not required for viral replication in human 293 cells, and E3 is
nonessential for replication of adenovirus in cultured human cells.
-The most DNA that can be packaged in virions is approximately 105%
of the wild-type genome, for a capacity of about 2 kb of extra DNA. To
incorporate larger DNA segments, it is necessary to compensate by
deleting appropriate amounts of viral DNA.
-Approximately 3 kb can be deleted from E1 to generate vectors
restricted to growth in 293 cells and able to accept inserts of 5 kb.
Collapsing the two naturally occuring XbaI sites within E3 can result in
1.9 kb of accommodation ability. Combining E1 and E3 deletions in a
single vector should yield a capacity of 7 kb.
-E1 deletion must not extend into the E1 region containing the coding
sequences for protein IX (from 10 to 11 mu), since IX is a virion
structural component that is necessary for packaging full length viral
genomes, and deleting this gene results in a net decrase in capacity.
-If the generation of helper/dependent vectors is a suitable option, then,
except for the extreme terminal sequences (which must be retained to
allow DNA replication) and sequences near the left end (which are
needed in cis for packaging), theoretically, almost the entire genome
(approximate 35 kb) can be substituted with foreign DNA.
Basic steps for rescuing genes into the viral genome:
1. Gene is first inserted into a subsegment of the viral genome with
subsequent cloning into a bacterial plasmid.
2. The resulting chimeric construct is then cotransfected into
mammalian cells together with appropriately prepared viral DNA.
Usually, rescue is achieved by in vivo recombination in the
transfected cells.
3. Figure 2 shows the procedure for rescue of either E1 insertions or E3
insertions into the viral genome by cotransfection with appropriately
restricted viral DNA.
For E1 inserts:
*The first requirement is a bacterial plasmid containing the left end
of the genome and having an appropriate deleting E1 sequences and
a restriction site into which to clone a foreign gene.
*pXCX2 is a plasmid containing the left 16% of the Ad5 genome
cloned in pBR322, minus a deletion in E1 from 1.3 to 9.3 mu, and
having a unique XbaI cloning site for insertion of foreign DNA.
*The resulting construct is cotransfected into 293 cells along with
virion DNA, usually derived from dl309 virus, that has been cleaved
at the left end to eliminate or at least reduce infectivitiy of the
parental viral DNA. In vivo recombination results in rescue of the
cloned viral sequences into the left end of the genome.
For E3 inserts:
*The pFGdX1 is a plasmid containing the right 40% of the genome
form 59.5 to 100 mu with a deletion in E3 and a unique XbaI
restriction enzyme site for cloning.
The disadvantage of this DNA substitution for viral recombination:
There is often a background of infectious parental DNA, which can
be a serious problem if the desired vector replicates significantly
slower than wild-type virus.
Solutions:
I. Plasmid pFG140 is a circular dl309 genome with an insert at 3.7
mu of a small 2.2 kb DNA segment carrying ampicillin resistance
and a bacterial origin of replication that permits propagation in E.
Coli. Substitution of the 2.2 kb insert with a 4.4 kb DNA
segment, resulting in a plasmid designated as pJM17. Because an
insertion of 4.4 kb is too large to be packaged into infectious
virions, cotransfection with left-end sequences containing
substitutions or insertions that do not exceed the packaging
constraints select for recombination and rescue of the E1 inserts
into infectious virus.
II. pFG173 has a lethal deletion around the E3 region to select for
recombination with viral DNA spanning that part of the genome.
Neither of the cotransfected plasmids need be digested with
restriction enzymes to obtain in vivo recombination.
*The advantages of this approach are that the background of
infectious parental DNA is zero (when pFG173 is used) or low
(using pJM17) and, once the appropriate plasmids have been made,
purification and restriction of viral DNA can be avoided.
-Replicating viral DNA is usually first detected at about 6-8 hours post
infection. DNA synthesis reaches its maximum level about 19 hours post
infection. Approximately 100 viral genomes are produced by 24 hours
post infection, but only about 20% are packaged into virus particles.
Replication of cellular DNA is heavily reduced during adenovirus
infection.
-Adenoviral structure proteins are synthesized in the cytoplasma and
rapidly transported to the nucleus. The core proteins and viral DNA are
inserted into the preformed capsid. DNA replication and assembly of
virus-coded proteins are carried out in the nucleus. Surplus capsomers
proteins are often formed in paracrystalline arrays in the cell. Virions are
slowly released after the death of the cell.
-Adenoviruses rarely intergrate into host genome and therefore have little
chance to activate an oncogene or interrupt a tumour suppressor gene.
The recombinant adenovirus vector, once inside the nucleus, remains as a
nonreplicating extrachromosomal entity.
-If the virus does not become associated with the host cell genome, the
viral genome will be degraded or simply diluted out over several cell
generations, resulting in abortive transformation. This is the
disadvantage of adenovirus-mediated gene transfer. The duration of
expression from the mini-cassette replacing the E1 region in the
recombinant adenovirus vector is usually short (about 8 weeks). A
repeated administration may be necessary.