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GENE THERAPY
INTRODUCTION
GENE is limited portion of genomic DNA (or
RNA for some viruses) responsible for a
concrete and specific function
GENE is inherited part of the genome that has
an influence on a phenotypic character
genes
regulatory Protein-coding RNA-coding
Structural
genes
STRUCTURE OF DNA
MOLECULE
Formation of the double
spiral is due to
hydrogen bonds facing
each other bases.
Adenine interacts with
thymine forming a
"weak link" (two
hydrogen bonds), and
guanine with cytosine
form a "strong link"
(three hydrogen bonds).
SCHEME OF EUKARYOTIC GENE
REGULATORY GENES
replication genes containing sites that are
responsible for the beginning and end of DNA
replication
recombination genes containing specific
recombination sites recognized by
recombination enzymes
segregation genes that determine the
interaction of chromosomes with the spindle
during meiosis and mitosis
genes containing sites of joining (target sites)
for proteins, hormones and other molecules
GENE THERAPY
CONCEPTION
The most radical way to deal with all kinds of diseases
caused by changes in the genetic content of cells is
correction or destruction of most of the genetic causes of
disease rather than its consequences
The causes of genetic diseases:
• mutation in the germ-line cells, which is inherited in hereditary
diseases,
• somatic mutation that causes, for ex., cancer,
• foreign genetic material appearing in the cell, such as a virus
infection.
Way of dealing with genetic changes:
artificial introduction in the affected cells of new genetic information,
designed to improve the one with which the disease is associated
HEREDITARY
DISEASES
due to irregularities in the storage,
transfer and implementation of
genetic information
Hereditary diseases
genetic
monogenic
A-dominating
A-recessive
X-bonded
Y-bonded
poligenic
chromosomic
multifactorial
Change of chromosomes’ number
monosomy
trisomy
chromosome
rearrangements
GENERAL SCHEME OF
GENETIC DISEASES
mutant allele → modified primary product →
chain of biochemical processes in the cell body
→ organs → organism
RESULTS of gene mutation
* Abnormal protein synthesis;
* Excess production of the gene product;
* Lack of production of primary products;
* Development of a reduced amount of normal primary product.
TYPES OF HEREDITY
Autosomal-dominant
•
Disease in each generation
•
Both sexes are affected equally
•
The disease is more severe in homozygotes than heterozygotes
•
Probability of birth of affected child if one of the parents is sick is 50%
Huntington's disease, polydactyly, vitiligo, breast cancer of type I
Autosomal-recessive inheritance
• The sick child is born from healthy parents
• Sick brothers and sisters
• Both sexes are affected equally
• If both spouses are sick, then all children will be sick
Metabolic diseases - cystic fibrosis, phenylketonuria,
hemochromatosis, etc.
X-bonded inheritance
• Affects only boys on the mother (depending on heredity)
• The sick man does not transmit disease, but his daughters are
carriers
• Female-carriers married with sick male 50% of sons and 50% of
daughters are sick
Haemophilia, color blindness, lack of G-6-PD, mucopolysaccharidosis
type II
TREATMENT OF HEREDITARY
DISEASES BY NON-GENETIC WAY
Symptomatic treatment of manifestations of the disease (diet for
phenylketonuria)
Replacement therapy (the introduction of the blood clotting protein in
hemophilia)
Organ and tissue transplantation
Surgical intervention
Etc.
MULTIPLE EXPENSIVE
Symptomatic treatment
isn’t efficient!
GENE THERAPY
Fetal gene therapy:
foreign DNA is introduced into the zygote or embryo at
an early stage of development, it is expected that the
introduced material gets into all cells of the recipient
(and even in the germ cells, thus ensuring the
transmission of the next generation).
Somatic gene therapy:
genetic material is introduced into somatic cells only
and is not transmitted to sex cells.
• positive
• negative
METHODS OF DELIVERY OF
GENETIC MATERIAL
• Technology in vivo is systemic delivery
through the blood
• Technology in situ is the introduction of a
local genetic information using vectors
• Technology ex vivo is transplantation of
own cells with the use of blastocysts,
fibroblasts, stem cells
EX VIVO
The advantage of ex vivo gene therapy is that you can
fully describe the obtained transformed cells before
transplantation into the body, you can get numerous
clones of these cells and to select clones with high
levels of expression of the desired gene, and the
deletion of clones with dangerous transformation,
which could harm body.
DELIVERY SYSTEMS (EX VIVO)
Fibroblasts
Blastocytes
Astrocytes
Stem cells (hemapoetic,
bone-marrow, fetal)
POSITIVE GENE THERAPY
… is aimed at restoring the function of a gene whose
expression may be inadequate or missing entirely. In
most cases, different methods of positive gene
therapy are aimed at correcting the damaged gene.
1.
2.
Correction of the gene at the level of
chromosomal DNA
Extrachromosomal expression of injected gene
CORRECTION OF THE GENE AT THE
LEVEL OF CHROMOSOMAL DNA
Targeting is embedding design gene therapy to a
specific location of the genome
Replenish gene therapy is introduction of healthy
genes while maintaining a defected gene in a cell
The use of single-stranded oligonucleotides:
creation of additional hydrogen bonds causes the
ability to inhibit transcription by overlapping of sites of
transcription factor binding
Etc.
EXTRACHROMOSOMAL
EXPRESSION OF INJECTED GENE
Creating of genetic construction:
1. Isolation of a therapeutic gene in the context of regulatory sequences. At
present, this step is much simpler due to using polymerase chain reaction
(PCR).
2. Selection or creation of the vector DNA, satisfying the goal.
3. Selection or artificial synthesis of regulatory elements.
4. Ligation of DNA fragments (DNA vectors, gene regulatory elements,
marker sites) in the correct sequence and orientation.
5. Cloning in the optimal cells and selection of recombinant clones.
6. Verification of the effectiveness of gene expression and the properties of
its products.
Targeted delivery of genetic construction to target the target cells, i.e.,
transfection (in the broad sense) or transduction (using viral vectors)
METHODS OF GENETIC
MATERIAL INTRODUCTION
Viral systems
Non-viral systems
DNA-protein
Adeno-assotiated viruses
Liposomes
DNA-protein-defected virus
Retroviruses
Herpesviruses
Adenoviruses
Physical administration
VIRAL
VECTORS
Adenoviral
Retroviral
BENEFITS
effectively transfere genes into
dividing and non-dividing cells
are not integrated into the
genome, provide high titers of
the recombinant virus and high
expression of introduced genes
DISADVANTAGES
cause non-specific inflammation
and antiviral cellular immune
response, resulting in
expression decreased to weeks
or months
BENEFITS
effective integration of foreign
DNA into the genome and
persistence of genetic changes
DISADVANTAGES
Can be introduced only into
dividing cells
can cause insertional mutations
give relatively low titers of the
recombinant virus, and the
expression of the inserted gene
is often reduced to very low
levels in a few months
RETROVIRAL GENOME
“PACKING LINES”
LACKS OF VIRAL
SYSTEMS
• Small amount of pseudoviral particles
• Penetration of retrovirus into the cell is strictly
conditional on receptor interaction
• Dependence on the position of the gene
expression in a cell
NONVIRAL DELIVERY
SYSTEMS
Requirements:
No degradation of DNA
The stability of DNA to endosomes
Long-term expression of genes in the DNA
STRATEGY 1 : creation of lipid-bonded complexes with DNA in
liposomes
STRATEGY 2: imitation of the virus
LIPOSOMES
Liposome-DNA complexes are
positively charged
Easily penetrate bilipid layer
But!
In the particles there is no
information directing them
straight to the core
SOLUTIONS:
Conjugation of liposomes to
antibodies or ligand (e.g.,
transferrin)
Conjugation of liposomes with
viral particles (such as Sendai
virus)
Creating a "superliposom"
(liposome-antibody complexvirus)
Creating a virus (viral particles
containing nucleic acid vector
molecule entirely enclosed in
liposomes of various structures)
IMITATION OF VIRAL
DELIVERY
Compex of DNA with polycation, ligand and endosomalitic
Binding with cell and
endocytosys
Cell specifity of
binding
Facilitates the release of
DNA complex in
endosomes and
cytoplasm
Direct injection of DNA constructs:
ballistic transfection
A - shotgun: 1 - powder charge, 2 - felt wad, 3 - shot;
Б - a powder accelerator by Klein and Stanford: 1 - propellant charge
2 – macro-carrier (analog of wad) 3 - volfram microparticles carrying
the inhtroduced DNA 4 lock aperture for stop microparticles:
B - accelerator by Kolesnikov: 1 - charge of mercury fulminate, 2 –
macro-carrier, 3 - a mixture of volfram and gold microparticles coated
with introduced DNA 4 - lock aperture stop to microparticles, 5, 6 mesh aperture for removal of the destroyed parts and disintegration of
macro-carrier conglomerate of microparticles respectively
Antisense RNA
Natural or artificially produced polyribonucleotide is complementary
to a specific mRNA which suppresses its biological activity through
the formation of a duplex with it, which prevents translation of the
mRNA on ribosomes
Antisense
oligonucleotides
Short antisense nucleotide sequence consisting of 15-40
units capable of blocking protein synthesis from the
corresponding mRNA due to the formation of duplex on the
portions of mRNA and create obstacles for ribosome
functioning
Unlike A.RNK:
are not part of the genome of the cell
constant supply of A. oligonukleotides into the cell is
required
Problems of implementation:
low permeability into the cell
low specificity binding with a target
Ribosimes
contain within themselves antisense sites and sites asking
for an enzymatic reaction
HIV
Cancer gene therapy
Potential cancer control strategy:
Increased immunogenicity of tumor cells by introducing genes coding for
these heterologous cell antigen (cytokine genes, genes coding for major
histocompatibility complex, lymphocyte ligands)
Promotion of immune system cells to enhance their antitumor activity, by
introducing genes into the tumor cells of cytokines.
Introduction into tumor cells genes killers are synthesized product, under
certain conditions, leading to the death of the tumor cells
Block oncogene expression by intracellular immunization by
administering to the cells constructs programming the synthesis of
antisense RNA or antibodies to oncoproteins
Introduction into tumor cells tumor suppressor genes (p53)
Protection of stem cells from the toxic effects of chemotherapy by
administering them drug resistance gene (MDR-1)
Locking mechanisms by which tumor cells escape immune destruction
system by introducing genes encoding antisense RNA against the IGF-1
Directional killing tumor cells by introducing genes encoding toxins
controlled promoter specifically expressed in tumor cells
Modification of genetic engineering
T-lymphocyte gene therapy for
cancer
Intake of T-lymphocytes infiltrating the tumor (TIL)
Modification using retroviral vectors
Growing TIL in culture growth factor Interleukin-2
Introduction back to the patient
+
1. Delivery of T lymphocytes directly to the tumor
2. No side effects
1. The complexity of the transformation
2. Low expression of cytokines
INCREASING OF
IMMUNE RESPONSE
B7 molecules are
expressed on the
membrane of
antigenprezenti-controlling
cells. Protein binding to
CD28 on the surface of
presenting T cell
proliferation provides top
T cell immune response
THANK YOU
FOR YOUR
ATTENTION!