Basics of gene therapy
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BASICS OF GENE THERAPY
By dr.Reena SINGH (MD)
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GENE ON CHROMOSOME region of DNA that controls
hereditary characteristic.
Corresponds to a sequence used in production of specefic protein or RNA.
ORIGIN :1920s
Term gene derived from greek word gennan meaning to produce.
Willard Johannsen
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HOW DO WE LOCATE GENES? Position on p or q arm based on
distinctive pattern of light & dark bands .
Designated by two digits representing a region and a band.
Number increases with distance from the centromere.
Eg14q21 rep 21 on long arm of chr 14
14q21 is closer to centromere than 14q22.
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CONTD.“cen” eg cen 16p
indicates that gene is close to centromere.
“ter” stands for terminal and means that gene is close to end of p/q arm .
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STRUCTURE OF A GENE
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WHAT IS GENE THERAPY???
Inserting genes into cells to treat diseases
Newly introduced genes encode proteins and correct the deficiency.
Primarily involves genetic manipulations to correct diseases.
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WHAT CAUSES PROTEIN DYSFUNCTION?????
Mutation in gene
…change in codon…
change in amino acid…
change in protein conformation…
..change in protein function..
genetic disorders
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APPROACHES:1) NORMAL GENE INSERTED TO
COMPENSATE FOR A NON FUNCTIONAL GENE
2) ABNORMAL GENE TRADED FOR A NORMAL GENE
3) ABNORMAL GENE REPAIRED THROUGH SELECTIVE REVERSE MUTATION
4) CHANGE THE REGULATION OF GENE PAIRS
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BROAD CLASSIFICATION SOMATIC CELL
GENE THERAPY : insertion of fully functional and expressible gene into a target somatic cell.
GERM CELL GENE THERAPY: reproductive cells of an organism constitute germ cell line.
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ILLUSTRATION:
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OR:EX VIVO GENE THERAPY
Involves transfer of genes in
cultured cells( like bone
marrow cells)which are then
reintroduced into the patient.
IN VIVO GENE THERAPYDirect delivery of genes
into the cells of a particular tissue
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EX VIVO GENE THERAPY Only selected tissues,which can be
cultured in lab Technique:isolation of abnormal cells,
….introduction of therapeutic gene,,,cell culture,select stable transformants…..transplant in patient.
Involves patients own cells and returned back to patient after correction.
No adverse immune response Efficient only when therapeutic gene is
stably incorporated and expressed.
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ILLUSTRATION:
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BONE MARROW CELLS Contains totipotent embryonic stem
cells. These can divide and differentiate
into various cell types(rbc,platelet,macrophage,osteoclast,B &T lymphocytes)
Widely used for many genetic diseases…(sickle cell anemia,scid,thalassemia.)
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HUMAN ARTIFICIAL CHROMOSOME Synthetic chromosome that can
replicate with other chromosomes,besides
encoding a human protein.
The problems arising with retroviruses as vectors can be overcome.
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IN VIVO GENE THERAPY Direct delivery of therapeutic gene Many tissues can be used
(liver,muscle,skin,spleen,lung,brain ,blood cells)
Gene delivery can be done by both viral and non viral vectors
Success depends on Efficiency of uptake of therapeutic gene Intracellular degradation of gene and
uptake by nucleus Expression capability of gene
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HOW DOES IT WORK???
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RETROVIRUS: Created double stranded DNA copies from
RNA genome The retrovirus goes through reverse
transcription using reverse transcriptase and RNA
the double stranded viral genome integrates into the human genome using integrase integrase inserts the gene anywhere
because it has no specific site May cause insertional mutagenesis
One gene disrupts another gene’s code (disrupted cell division causes cancer from uncontrolled cell division)
vectors used are derived from the human immunodeficiency virus (HIV) and are being evaluated for safety
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USING HERPES SIMPLEX VIRUS
Double stranded DNA viruses that infect neurons
Ex. Herpes simplex virus type 1
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NON VIRAL OPTIONS: Direct introduction of therapeutic DNA
Requires a lot of DNA Creation of artificial lipid sphere with aqueous
core, liposome Carries therapeutic DNA through membrane
Chemically linking DNA to molecule that will bind to special cell receptors
DNA is engulfed by cell membrane Less effective
Trying to introduce a 47th chromosome Exist alongside the 46 others Could carry a lot of information But how to get the big molecule through
membranes?
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BEGINNING 1980S
Bacterial cell…
gene inserted
….transcription and translation…
protein…
..introduction of protein into human cells
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FIRST GENE THERAPY SEPTEMBER 14th 1990 ASHANTI DESILVA SCID DOCTORS REMOVED WBCs and
inserted the missing gene into the wbc and put in blood stream
Worked only few months
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CONT.. Led by BLAESE & ANDERSON at THE
NATIONAL INSTITUTE OF HEALTH,USA…
First and most publicised
For treating SEVERE COMBINED IMMUNODEFICIENCY..the patients of which lack the enzyme ADENOSINE DEAMINASE.
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SEVERE COMBINED IMMUNODEFICIENCY INHERITED AS X LINKED OR AUTOSOMAL RESSESSIVE.
DISORDER OF T LYMPHOCYTES MAINLY AND B TO A LESSER EXTENT
DEFECT IN GENE LOCATED ON CHROMOSOME 20 ,CODE FOR ADENOSINE DEAMINASE
DEOXYADENOSINE AND ITS METABOLITES ACCUMULATE,DESTROY T LYMPH.
LEADS TO IMMUNE DEFICIENCY
PATIENTS SUFFER ,INFECTIONS..DIE YOUNG.
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TECHNIQUE OF THERAPY FOR ADENOSINE DEAMINASE DEFICIENCY
CHILD WITH SCID
ISOLATION OF LYMPHOCYTES
TRANSFECTION WITH VIRAL
VECTORS
LYMPHOCYTES WITH VIRAL
DNA AND ADA GENECELL CULTURE
TO VERIFY EXPRESSION OF
ADA GENE IN PATIENT
INFUSE EXPRESSIVE
CELLS TO PATIENT
SYNTHESIS OF ADA
CORRECTION OF SCID
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GENE THERAPY STRATEGIES FOR CANCER
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GENE MUTATIONS LEAD TO CANCER
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TUMOR NECROSIS FACTOR GENE THERAPY Tumor necrosis factor is a protein produced
by macrophages,provide defence against cancer cells
brought by enhancing cancer fighting ability of tumor infiltrating lymphocytes.
Tumor infiltrating lymphocytes transfected with tnf has been used for malignant melanoma
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SUICIDE GENE THERAPY
Gene encoding thymidine kinase often referred to as suicide gene
Thymidine kinase phosphorylates nucleosides to form nucleotides,used for synthesising DNA during cell division.
Gancyclovir bears close resemblance to certain nucleosides eg thymidine.
Mistakingly TK phosphorylates gancyclovir to form TRIPHOSPHATE-GCV.
Inhibition of DNA polymerase by triphosphate-gcv.
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CONT..
Elongation of DNA molecule abruptly stops at a point containing false nucleotide of gancyclovir.
Triphosphate-gcv can enter and kill neighbouring cancer cells also…BYSTANDER EFFECT.
Cancer cells cannot multiply and therefore die . This type of approach is also called PRODRUG
ACTIVATION GENE THERAPY.
Used for glioblastoma with limited success.
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GENE REPLACEMENT THERAPY
P 53 TUMOR SUPPRESSOR GENE PROTEIN IT ENCODES BINDS WITH DNA AND
INHIBITS REPLICATION
TUMOR CELLS OF SEVERAL TISSUES WERE FOUND TO HAVE MUTATED P53 ,SYNTHESISING ALTERED PROTEINS
ALTERED PROTEINS CANNOT INHIBIT DNA REPLICATION
REPLACE DAMAGED P53 BY NORMAL GENE BY EMPLOYING ADENOVIRUS VECTOR SYSTEM.
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ANTIGENE AND ANTISENSE THERAPY CERTAIN DISORDERS LIKE CANCER, VIRAL AND
PARASITIC INFECTIONS RESULT IN OVERPRODUCTION OF NORMAL CELLS.
BLOCKING TRANSCRIPTION USING SINGLE STRANDED NUCLEOTIDE SEQUENCE(antigene oligonucleotide) THAT HYBRIDISES WITH SPECEFIC GENE ..ANTIGENE THERAPY
INHIBITION OF TRANSLATION SIMILARLY IS ANTISENSE THERAPY
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TREAT MITOCHONDRIAL DNA DISEASES…HEALTHY BABIES…
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PROBLEMS THAT ARISEShort lived:hard to
rapidly integrate therapeutic DNA into genome and rapidly dividing nature of cells prevent therapy for long time
Immune response
Viral vectors themselves can create probem inflammation,toxic immune response,can cause disease
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Hard to treat multigenic disorders eg heart disease,high blood pressure,arthritis,diabetes etc coz we need to introduce more than one gene
May intoduce a tumor if integrated into a tumor suppressor gene bcoz of insertional mutagenesis
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PROB CONT.. One does not have control over where the
gene will be inserted into genome.
Location of a gene in the genome is of importance for the degree of expression of the gene and for the regulation of gene
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RECENT DEVELOPMENTS Genes get into brain using
liposomes coated in polymer call polyethylene glycol potential for treating
Parkinson’s disease RNA interference or gene
silencing to treat Huntington’s siRNAs used to degrade RNA
of particular sequence abnormal protein wont be
produced Create tiny liposomes that
can carry therapeutic DNA through pores of nuclear membrane
Sickle cell successfully treated in mice
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GENE BANKS---A NOVEL CONCEPT
Gene banks are the centres for the storage of individual’s DNAfor future use to diagnose diseases.
DNA is usually isolated from white blood cells.
For the risk assesment of any disease,it is advisable to have DNAs from close relatives of at least 2 generations at least.
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CURRENT STATUS:
FDA hasn’t approved any human gene therapy product for saleReasons:In 1999, 18-year-old Jesse Gelsinger died from multiple organ failure 4 days after treatment for omithine transcarboxylase deficiency.
Death was triggered by severe immune response to adenovirus carrier
January 2003, halt to using retrovirus vectors in blood stem cells because children developed leukemia-like condition after successful treatment for X-linked severe combined immunodeficiency disease
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THANK YOU!!!!