medicine Dr.A.Sab .Sabith itha a Rani ni Dr. r.C. Jyot othsn - - PowerPoint PPT Presentation

GENE THERAPY -genes as the medicine

By By Dr.A.Sab .Sabith itha a Rani ni Dr. r.C. Jyot

• thsn

sna Lt.E.M.S .Sunith itha Dr.M.Madhavi Madhavi Dr.K. Shailaja laja Dr.Raf afat at Yasmee een

Refresher Course in Life Sciences Academic Staff College (ASC), Osmania University (August 5th-28th, 2013) Group Project

• n

 Definiton: an experimental technique for correcting defective

genes that are responsible for disease development

 The most common form of gene therapy involves inserting a

normal gene to replace an abnormal gene

 Other approaches used:

• Replacing a mutated gene that causes disease with a healthy

copy of the gene.

• Inactivating, or “knocking out,” a mutated gene that is

functioning improperly.

• Introducing a new gene into the body to help fight a disease.

WHAT IS GENE THERAPY ?

 Gene therapy is the insertion of genes into an

individual cells and tissues to treat a disease in which a defective mutant allele is replaced with a functional one

 DNA is used as a therapeutic agent  Genetic diseases, hematological disorders,

acquired immunodeficiency syndromes, cancers are mainly treated

 Researchers are studying gene therapy for a number

• f diseases, such as
• Severe combined immuno-deficiencies (SCID)
• Hemophilia
• Parkinson's disease
• Cancer
• HIV

Diseases for applying gene therapy

Disease Defect Target cell Severe combined Adenosine deaminase 4 Bone marrow cells or immunodeficiency T-lymphocytes Hemophilia Factor VIII, Factor IX deficiency Liver, muscle. Cystic fibrosis Loss of CFTR gene Airspaces in the lung Hemoglobulinpathies  or  globulin gene Bone-marrow cells 1-antitrypsin deficiency 1-antitrypsin Lung or liver cells Cancer Many causes Many cell types Neurological diseases Parkinson’s, Alzheimers Direct injection into the brain Cardiovascular Restenosis, arteriosclerosis Vascular endothelium Infectious diseases AIDS, hepatitis B T cells, macrophages, Liver cirrhosis Fibrogenesis Hepatocyte growth factor Autoimmune disease Lupus, diabetes MHC, 2-microglobulin

GERM LINE GENE THERAPY

• In germ line gene therapy, germ cells, i.e., sperm or eggs, are

modified by the introduction of functional genes, which are integrated into their genomes.

• Result in permanent changes.
• This would allow the therapy to be heritable and passed on to

later generations.

• Potential for offering a permanent therapeutic effect for all

who inherit the target gene.

• Possibility of eliminating some diseases from a particular

family.

• Also raises controversy:
• Some people view this type of therapy as unnatural, and

liken it to "playing God”.

• Others have concerns about the technical aspects.

SOMATIC GENE THERAPY

• The therapeutic genes are transferred into

the somatic cells, or body, of a patient.

• Affects only the targeted cells in the patient, and

is not passed to future generations.

• Short-lived because the cells of most tissues

ultimately die and are replaced by new cells.

• Appropriate and acceptable for many disorders,

including cystic fibrosis, muscular dystrophy, cancer, and certain infectious diseases.

Types of somatic gene therapy

• 1. The genetic material is transferred directly into the

body of the patient

• 2. More or less random process;

small ability to control; less manipulations

• 3. Only available option for tissues

that can not be grown in vitro;

• r if grown cells can not be transferred back

In vivo gene therapy

• 1. The genetic material is first transferred

into the cells grown in vitro

• 2. Controlled process;

transfected cells are selected and expanded; more manipulations

• 3. Cells are usually autologous;

they are then returned back to the patient

Ex vivo gene therapy

Gene therapy utilizes the delivery of DNA into cells, which can be accomplished by Vectors Two major methods of gene transfer Viral vectors

• Retrovirus
• Adenovirus
• Adeno-associated virus
• Herpes simplex virus

Non-viral vectors

• Naked DNA/Plasmid
• Liposomes

Mec Mechan hanism ism of

• f Gen

Gene T e Ther herapy

Viral vectors

• Virus replicate by inserting their DNA into a

host cell as part of their replication cycle.

• Gene therapy uses this by removing viral

DNA and using the virus as a vehicle to deliver the therapeutic DNA.

• Human gene therapy utilizes number of

viruses like retrovirus, adenovirus, adeno-associated virus, lentivirus, herpes simplex virus etc

Retr etrovir viruses uses

• Retroviruses are RNA viruses which possess

a reverse transcriptase function

• Following infection (transduction) reverse

transcriptase transcribes the viral RNA genome resulting CDNA copy and integrate into the human genome

• DNA transfer is very efficient and stable,
• ffering the possibility of a permanent cure for

a disease.

• Most promising vehicles for gene delivery and

about 60% of clinical protocols utilize retroviral vectors.

*The maximum size of DNA insert is 8kb *Can only transduce dividing cells, therefore limits potential target cells

Lentiv Lentivir iruses uses ( ( e.g. HIV) e.g. HIV)

• Lentivirus are RNA virus, includes HIV

virus.

• These are complex retroviruses that infect

macrophages and lymphocytes

• Unlike retroviruses, lentiviruses are able to

transduce nondividing cells and integrate into host cell chromosomes

• Now considerable efforts are being devoted to

making lentivirus vectors for gene therapy. *Maximum insert size 7-7.5 kb *May cause intentional mutagenesis

Adeno Adenovir viruses uses

• Adenovirus are Double stranded DNA virus

that cause respiratory, intestinal and eye infections in humans

• Second most popular delivery system in

gene therapy

• These are Human viruses, infecting dividing

and non-dividing cells

• Large viruses and potential for accepting

large insert size > 30 kb *Extensive unwanted immunological responses *Pre-existing host immunity

• Group of small, single stranded DNA viruses
• Productive infection only with co-infection by

helper virus, such as an adenovirus or herpes simplex virus.

• AAV vectors can accommodate inserts up to 4.5

kb, but have long-term gene expression

• Provide high degree of safety: because 96% of

parental AAV genome is deleted with the gene

• f interest

Adeno Adeno-As Associa sociated ted Vir iruses uses (AAVs)

• Complex double stranded DNA vectors
• Infects central nervous system (CNS) and can

establish lifelong latent infections in neurons

• Have comparatively large insert size capacity (>20

kb) but are nonintegrating and long-term expression of transferred genes is not possible

• Used for delivering genes into neurons for the

treatment of neurological diseases (Parkinson's disease and CNS tumors) Her Herpes pes simple simplex x Vir iruse uses s (HSV)

applications are expected to be in delivering genes into neurons for the treatment of neurological diseases, such as Parkinson's disease and for.

• Spherical vesicles composed of synthetic lipid

bilayers which mimic the structure of biological membranes.

• The DNA to be transferred is packaged in

vitro with the liposomes and transferred to target tissue in vivo

• Lipid coating allows the DNA to survive in vivo,

bind to cells and endocytosed into the cells.

• These are the popular vehicles for gene transfer
• Unlike viral vectors, DNA/lipid complexes are

easy to prepare and there is no limit to the size

• f DNA that is transferred.

* Efficiency of gene transfer is low * Introduced DNA is not designed to integrate into chromosomal DNA.

Lipo Liposome

• mes-Non

Non vir iral al vect ector

• rs

Direct injection/particle bombardment

• DNA can be injected directly with a syringe and

needle into a specific tissues

• Particle bombardment (‘gene gun’) : DNA is

coated on to metal pellets and fired from a special gun into cells.

• Successful gene transfer into number of

different tissues can be obtained

• Direct injection is simple and comparatively

safe. However, there is poor efficiency of gene transfer and a low level of stable integration

1970s and earlier:

• In 1972 Friedmann and Roblin authored a paper in Science titled

"Gene therapy for human genetic disease?".

Mature T-cells GT September 14, 1990.

• Ashanti DeSilva; advanced stage of SCID; 4 yr old;
• Cynthia Cutshall January 31, 1991
• Ashanthi De Silva - A rare genetic disease

called severe combined immunodeficiency (SCID)

• Defective adenosine deaminase gene

results in deficiency of ADA protein

• It plays important role in deamination

reaction

• Lack of healthy immune system

Professor William French Anderson

First Approved Gene Therapy

X-linked SCID (bubble disease)

"bubble boy" disease, named after David Vetter, a Texan who lived out his 12 years in a plastic, germ-free bubble. Gene therapy trial for X-linked SCID successed in 2000; 8 of 10 patients significantly improved and live normal life. More severe than ADA-SCID, as X-SCIDs have no B-, T-, NK cells

• It is a significant achievement because viral vectors are too big

to get across the blood–brain barrier.

• This method has potential for treating Parkinson's diseaseI

Gene Therapy for Parkinson’s Disease

In 2003, University of California, Los Angeles research team inserted genes into the brain using liposomes

• In 2006, Scientists at the National Institutes of Health (Bethesda,

Maryland) have successfully treated metastatic melanoma in two patients using killer T cells to attack the cancer cells.

• In March 2006, an international group of scientists announced the

successful use of gene therapy to treat two adult patients for a disease affecting myeloid cells.

• In August 2011, two of three subjects were confirmed to have been

cured from chronic lymphocytic leukemia (CLL).

Gene Therapy for Cancer

• In May 2006 a team of scientists led by Dr. Luigi Naldini and Dr.

Brian Brown from the San Raffaele Telethon Institute for Gene Therapy in Milan, Italy developed a way to prevent the immune system from rejecting a newly delivered gene.

• In November 2006, Preston Nix from the University of

Pennsylvania School of Medicine reported a gene-based immunotherapy for the treatment of human immunodeficiency virus (HIV).

Gene Therapy for HIV

• In September 2010, it was announced that a patient in France had

been cured of beta-thalassemia.

• In 2012, FDA (Food and Drug Administration) approved the

clinical trials of use of gene therapy on thalassemia major patients in the US.

Gene Therapy for Thalassemia

• In May 2007, Moorfields Eye Hospital and University College

London's Institute of Ophthalmology announced the world's first gene therapy trial for inherited retinal disease.

• In July 2012, the European Medicines Agency approved of a gene

therapy treatment for the first time in Europe and the US.

• In March 2013, Researchers at the Memorial Sloan-Kettering

Cancer Center in New York, reported that three of five subjects who had acute lymphocytic leukemia (ALL) had been treated with genetically modified T cells .

• In July 2013, the Italian San Raffaele Telethon Institute for Gene

Therapy (HSR-TIGET) reported that six children with two severe hereditary diseases had been treated with a lentivirus.

Limitations With Gene Therapy?

 Short-lived nature of gene therapy-

patients will have to undergo multiple rounds of gene therapy.

 Immune response- risk of stimulating

the immune system that reduces gene therapy effectiveness

 Problems with viral vectors- viruses,

sometimes present potential problems like toxicity, immune and inflammatory responses.

Risks of Gene Therapy

 New gene might be inserted into wrong location in the

DNA (misfire)

 Immune system complications  Vector viruses can infect more than one type of cell,

causing another disease or a predisposition to cancer

 Over-expression of missing protein  DNA could accidentally be introduced into

reproductive cells (germ-line gene therapy)

Adverse Effects of Gene Therapy

 Vector induced oncogenesis  Germline transfer of transgenic DNA sequences  Developmental aberrations caused by expression

• f the transgenic proteins

 Without proper specificity, there could be

detrimental immunological effects.

Ethical problems

 Gene therapy for serious genetic diseases OK but for

• ther health problems?

 Somatic cell treatment stays with the individual, germ

cell treatment passes down the germ line (becomes immortal)

 Very costly. Who pays? Who is eligible?

Social Concerns

 Who will have access to your genetic information?  Who will own your genetic information?

Genetic counseling is the process by which patients or relatives, at risk of an inherited disorder, are advised of the consequences and nature of the disorder, the probability of developing or transmitting it, and the options open to them in management and family planning in order to prevent, avoid or ameliorate it. This complex process can be seen from diagnostic (the actual estimation of risk) and supportive aspects.

Conclusions

• Over the last two decades molecular genetic technologies have

been spectacularly successful in identifying and characterizing novel disease genes.

• It helps in devising novel diagnostic tests for inherited

disorders.

• Still the dream of successfully applying molecular genetic

technologies on a large scale to cure the disease has remained unfulfilled

• A new era was heralded when the first gene therapy trial for an

inherited disease began in 1990, but exciting though this prospect, reviews of clinical trials have shown that the initial enthusiasm were misplaced.

• Before gene therapy can become the strategy of choice in a wide

variety of clinical settings, improvements in the efficiency of gene transfer helped in targeting cells

• The problem of efficient gene transfer will require not only

further research to improve delivery systems and vector constructions but also a parallel effort to under- stand the biology

• f the target cells.
• The future technological improvement in the gene therapy will

revolutionize the practice of medicine and also curing the many

• f the diseases that have affected our society for years.
• Our genome is the blueprint of our body and the key to our

future is locked in our genome.

THANK YOU