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Gene therapy involves the genetic modification of a patient’s cells to
achieve a positive therapeutic outcome. There are two types of gene therapy as indicated
below.
A. GERM-LINE GENE THERAPY
1.
Germ-line gene therapy involves the genetic modification of a gamete, zygote, or
a preimplantation embryo that produces a permanent modification that can be
transmitted to other generations.
2.
Germ-line gene therapy is not a realistic option at present for purely technical rea-
sons, but there are also serious ethical questions that have resulted in this type of
gene therapy being prohibited by law in many countries.
B. SOMATIC CELL GENE THERAPY. Somatic cell gene therapy involves the genetic mod-
ification of specific somatic cells or tissues of a particular patient. All current gene
therapy clinical trials and protocols are for somatic cell gene therapy. There are a num-
ber of novel approaches to somatic cell gene therapy which include the following:
1.
Gene augmentation
a.
This approach involves the insertion of a functioning copy of a gene into a
diseased somatic cell or tissue to express a curative protein.
b.
This approach is used to treat a loss-of-function mutation.
2.
Inhibition of gene expression
a.
This approach involves the insertion of microRNA (miRNA) or small inter-
fering RNA (siRNA); antisense RNA; or ribozyme into a diseased somatic cell
of tissue to block expression of a certain mutated gene.
b.
This approach is used to treat a gain-of-function mutation.
c.
miRNA can inhibit the translation of a complementary mRNA by forming
dsRNA which either physically blocks mRNA translation or is degraded by RNA-
Induced Silencing Complex, or RISC.
d.
Antisense RNA can inhibit the translation of a complementary mRNA by form-
ing dsRNA which either physically blocks mRNA translation or is degraded
by RNAse H. Antisense RNA therapy has rather unpredictable results, where
the translation of the target gene may be silenced efficiently; nonspecific
effects; or little-to-no effect.
e.
Ribozyme (e.g., hammerhead, hairpin, human delta virus, Varkud satellite) is
an RNA molecule that has enzymatic activity to degrade mRNA. A ribozyme
can be designed to bind to a complementary mRNA and then degrade the
mRNA.
3.
Direct killing of disease cells.
This approach involves the insertion of a prodrug
gene into a diseased somatic cell or tissue to express a curative drug.
4.
Assisted killing of disease cells.
This approach involves the insertion of a for-
eign antigen gene into the diseased somatic cell or tissue to stimulate an immune
response against the diseased cells.
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CHAPTER 16
Ex Vivo and In Vivo Gene Therapy
A. EX VIVO GENE THERAPY involves the removal a patient’s cells (e.g., hematopoietic
cells, skin cells), insertion of a therapeutic gene, amplification of the genetically modi-
fied cells in culture, and return of the genetically modified cells to the patient. Ex vivo
gene therapy is only applicable to cells that can be easily removed from and returned
to the patient.
B. IN VIVO GENE THERAPY involves the insertion of a therapeutic gene directly into a
patient’s tissue (e.g., muscle) or directly into the general circulation but designed so
that the therapeutic gene is directed to only the desired cell or tissue.
Integration into Host Cell Chromosomes or as Episomes
A. HOST CELL CHROMOSOMES
1.
The insertion of the therapeutic gene into the host cell chromosomes will result in
the replication of the therapeutic gene whenever the host cell divides. This results
in long-term expression of the therapeutic gene but with serious risks.
2.
Because the insertion of the therapeutic gene into the host cell chromosomes is
a random event, a number of less-than advantageous possibilities exist which in-
clude as follows:
a.
The therapeutic gene may never be expressed, expressed at low levels, or ex-
pressed for a short time and then silenced.
b.
The therapeutic gene may insert within the DNA sequence of a normal gene
and inactivate the normal gene.
c.
The therapeutic gene may activate a nearby proto-oncogene leading to cancer
(Note: this occurred in two children who underwent gene therapy for severe
combined immunodeficiency and later develop a novel type of T-cell
leukemia).
B. EPISOMES
1.
The insertion of the therapeutic gene as extrachromosomal episomes will not re-
sult in the replication of the therapeutic gene whenever the host cell divides. This
results in short-term expression of the therapeutic gene because the episomes will
be diluted out as the cell population grows.
2.
This means that repeated treatments may be necessary, but the safety of a self-
limiting process will likely make this a mainstay therapy tool.
Viral Vectors Used in Gene Therapy.
Most protocols for human gene therapy employ
the use of a wide variety of mammalian viruses as vectors which are designed to include a
therapeutic gene. These vectors include the following:
A. ONCORETROVIRAL VECTORS
1.
The oncoretroviruses are RNA viruses that deliver a nucleoprotein complex into
the cytoplasm of the cell.
2.
The viral RNA genome is transcribed using reverse transcriptase into cDNA.
3.
The cDNA is then used as a template to make a second strand forming dsDNA.
4.
The dsDNA is inserted into the host cell chromosomes.
5.
The dsDNA gains access to the host cell chromosomes only during mitosis when
the nuclear envelope disintegrates. This means that oncoretroviral vectors can be
used only in dividing cells.
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GENE THERAPY
B. ADENOVIRAL VECTORS
1.
The adenoviruses are dsDNA viruses that deliver their DNA genome into the nu-
cleus of the cell. The dsDNA remains in the nucleus as an episome.
2.
The viral DNA gains access to the nucleus in both dividing and nondividing cells.
3.
The major problem with adenoviral vectors is the occurrence of unwanted immune
reactions in the patient (e.g., a gene therapy trial for ornithine transcarbamylase
deficiency resulted in the death of the patient 2 days after receiving an intrahep-
atic injection of an adenoviral vectors containing a therapeutic gene).
4.
In addition, episomes result in the short-term expression of the therapeutic gene
(e.g., a gene therapy trial for cystic fibrosis resulted in a decline in expression of
the therapeutic gene after 2 weeks and no expression after 4 weeks).
C. ADENO-ASSOCIATED VIRUS VECTORS (AVAs)
1.
The AVAs are single-stranded DNA viruses that rely on coinfection by an aden-
ovirus or herpes helper virus to replicate.
2.
The DNA gets inserted into a specific site at host cell chromosome 19q13.3 which
is a highly desirable property because it eliminates many of less than advantageous
possibilities connected to integration into host cell chromosomes (see IIIA2 above).
3.
The major problem with AVAs is that they are small and can accommodate inserts
of only
4.5 kb.
D. LENTIVIRUS VECTORS
1.
The lentiviruses are RNA viruses that deliver a nucleoprotein complex into the cy-
toplasm of the cell.
2.
The viral RNA genome is transcribed using reverse transcriptase into cDNA.
3.
The cDNA is then used as a template to make a second strand forming dsDNA.
4.
dsDNA is inserted into the host cell chromosomes.
5.
Unlike oncoretroviral vectors, the dsDNA gains access to the host cell chromo-
somes in nondividing cells.
6.
The most popular lentivirus used to construct lentivirus vectors is the human im-
munodeficiency virus which understandably causes great concern for inadver-
tently generating a competent virus.
E.
HERPES SIMPLEX VIRUS VECTORS
1.
The herpes simplex viruses are dsDNA viruses that are tropic for the central nerv-
ous system and can establish a lifelong latent infection in sensory ganglia.
2.
The dsDNA remains in the nucleus as an episome.
Nonviral Vectors Used in Gene Therapy.
The use of nonviral vectors in gene ther-
apy eliminates many of the safety concerns involved with viral vectors. However, nonviral
vectors have a low efficiency of gene transfer.
A. LIPOSOMES
1.
Liposomes are synthetic lipid vesicles.
2.
Cationic liposomes form with DNA bound on the outside of the liposome and an-
ionic liposomes form with DNA bound in the inside of the liposome.
3.
The liposomes bind to the cell membrane and then allow the DNA to enter the
cytoplasm of the cell.
4.
Most gene therapy protocols use cationic liposomes.
5.
The DNA inserted into the cell remains as an episome.
B. DIRECT INJECTION
1.
DNA can be injected directly with a needle and syringe (e.g., in muscle tissue for
gene therapy of Duchenne muscular dystrophy).
2.
The DNA inserted into the cell remains as an episome.
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CHAPTER 16
C. RECEPTOR-MEDIATED ENDOCYTOSIS
1.
The DNA is coupled to a targeting molecule (e.g., asialoglycoprotein or transfer-
rin) that binds to specific cell membrane receptors (e.g., asialoglycoprotein recep-
tors on hepatocytes or transferrin receptors on hematopoietic cells).
2.
The binding to a specific cell membrane receptor induces receptor-mediated endo-
cytosis and transfer of DNA into the cytoplasm of the cell.
3.
In general, substances internalized by receptor-mediated endocytosis are directed
to endolysosomes for degradation. Therefore, the vector must be designed with
some mechanism to allow for escape from lysosomal degradation (e.g., cotransfer
with an adenovirus which disrupts the endolysosome and allows escape).
4.
The DNA inserted into the cell remains as an episome.
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3rd Position
(3’ end)
U
C
A
G
U
C
A
G
U
C
A
G
U
C
A
G
2nd Position
Cys
Cys
STOP
Trp
Arg
Arg
Arg
Arg
Ser
Ser
Arg
Arg
Gly
Gly
Gly
Gly
G
Tyr
Tyr
STOP
STOP
His
His
Gln
Gln
Asn
Asn
Lys
Lys
Asp
Asp
Glu
Glu
A
Ser
Ser
Ser
Ser
Pro
Pro
Pro
Pro
Thr
Thr
Thr
Thr
Ala
Ala
Ala
Ala
C
Phe
Phe
Leu
Leu
Leu
Leu
Leu
Leu
Ile
Ile
Ile
Met
Val
Val
Val
Val
U
U
C
A
G
1st Position
(5’ end)
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