BASICS OF GENE THERAPY

By dr.Reena SINGH (MD)

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

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.

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 .

STRUCTURE OF A GENE

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.

WHAT CAUSES PROTEIN DYSFUNCTION?????

Mutation in gene

…change in codon…

change in amino acid…

change in protein conformation…

..change in protein function..

genetic disorders

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

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.

ILLUSTRATION:

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

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.

ILLUSTRATION:

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.)

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.

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

HOW DOES IT WORK???

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

USING HERPES SIMPLEX VIRUS

Double stranded DNA viruses that infect neurons

Ex. Herpes simplex virus type 1

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?

BEGINNING 1980S

Bacterial cell…

gene inserted

….transcription and translation…

protein…

..introduction of protein into human cells

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

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.

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.

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

GENE THERAPY STRATEGIES FOR CANCER

GENE MUTATIONS LEAD TO CANCER

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

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.

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.

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.

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

TREAT MITOCHONDRIAL DNA DISEASES…HEALTHY BABIES…

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

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

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

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

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.

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

THANK YOU!!!!