GENE THERAPY

BYSUNNY DHIMAN2ND SEM M-PHARMPHARMACOLOGYISCP MOGA (PUNJAB)

What is gene therapy? Why is it used?• Gene therapy is the application of genetic principles in

the treatment of human disease

• Gene therapy = Introduction of genetic material into normal cells in order to:– counteract the effect of a disease gene or– introduce a new function

• GT is used to correct a deficient phenotype so that sufficient amounts of a normal gene product are synthesized to improve a genetic disorder

• Can be applied as therapy for cancers, inherited disorders, infectious diseases, immune system disorders

What is gene therapy?

History of gene therapy

1930’s “genetic engineering” - plant/animal breeding

60’s first ideas of using genes therapeutically

50’s-70’s gene transfer developed

70’s-80’s recombinant DNA technology

1990 first GT in humans (ADA deficiency)

2001 596 GT clinical trials (3464 patients)

Human Genome Project

• A genome is all the DNA in an organism, including its genes. Genes carry information for making all the proteins required by all organisms

• Variations in structure of person’s genes collectively helps define us as individuals

• Rationale for GT is based on knowledge of the human genetic code

• Began formally in 1990, set as a 13 year project.

HGP GOALS

• Identify aproximately 30,000 genes

• Determine 3 billion chemical base pairs that make up human DNA

• Store information in databases

• Improve tools for data analysis

• Address ethical, legal and social issues that might arise from this

HGP cont…

• Achieve these goals, researchers look at genetic makeup of other organisms

• This project is important because the government's dedication to the transfer of technology to the private sector

Three types of gene therapy:• Monogenic gene therapy

• Provides genes to encode for the production of a specific protein

• Cystic fibrosis, Muscular dystrophy, Sickle cell disease, Haemophilia, SCID

• Suicide gene therapy• Provide ‘suicide’ genes to target cancer cells for

destruction• Cancer

• Antisense gene therapy• Provides a single stranded gene in an’antisense’

(backward) orientation to block the production of harmful proteins

• AIDS/HIV

Different Delivery Systems are Available

• In vivo versus ex vivo– In vivo = delivery of genes takes place in the body– Ex vivo = delivery takes place out of the body, and

then cells are placed back into the body

Getting genes into cells

• In vivo versus ex vivo– In vivo = intravenous or intramuscular or non-

invasive (‘sniffable’)– Ex vivo = hepatocytes, skin fibroblasts,

haematopoietic cells (‘bioreactors’)

• Gene delivery approaches– Physical methods– Non-viral vectors– Viral vectors

• In vivo techniques usually utilize viral vectors– Virus = carrier of desired gene– Virus is usually “crippled” to disable its ability to cause disease– Viral methods have proved to be the most efficient to date– Many viral vectors can stable integrate the desired gene into

the target cell’s genome

In vivo techniques

– Problem: Replication defective viruses adversely affect the virus’ normal ability to spread genes in the body

• Reliant on diffusion and spread• Hampered by small intercellular spaces for transport• Restricted by viral-binding ligands on cell surface

therefore cannot advance far.

“ Viruses are highly evolved natural vectors

for the transfer of foreign genetic information into cells”

But to improve safety, they need to be replication defective

Viral vectors

Compared to nakedDNA, virus particlesprovide a relativelyefficient means oftransporting DNA into cells, for expression in the nucleus as recombinant genes(example = adenovirus).

Viral vectors

Viral vectors

• Retroviruses– eg Moloney murine leukaemia virus (Mo-MuLV)– Lentiviruses (eg HIV, SIV)

• Adenoviruses• Herpes simplex • Adeno-associated viruses (AAV)

YvectorVector uncoating

Therapeutic mRNAand protein

Episomal vectorIntegrated expression cassette

Target cell

Gene transfer

Delivery System of Choice = Viral Vectors

A. Rendering virus vector harmlessRemove harmful genes “cripple” the virus

Example – removal of env gene virus is not capable of producing a functional envelope

Vectors needed in very large numbers to achieve successful delivery of new genes into patient’s cells

Vectors must be propagated in large numbers in cell culture (109) with the aid of a helper virus

B. Integrating versus Non-Integrating Viruses

• Integrating viruses– Retrovirus (e.g. murine leukemia virus)– Adeno-associated virus (only 4kbp

accommodated)– Lentivirus

• Non-Integrating viruses– Adenovirus– Alphavirus– Herpes Simplex Virus– Vaccinia

Delivery System of Choice = Viral Vectors

Advantages• High transduction efficiency• Insert size up to 8kbHigh viral titer (1010-1013) • Infects both replicating and differentiated cellsDisadvantages• Expression is transient (viral DNA does not integrate)• Viral proteins can be expressed in host following vector

administration• In vivo delivery hampered by host immune response

Adenovirus

Advantages• Large insert size• Could provide long- term CNS gene expression• High titer

Disadvantages• System currently under development• Current vectors provide transient expression• Low transduction efficiency

Herpes Simplex Virus

• Ex vivo manipulation techniques– Electroporation– Liposomes– Calcium phosphate– Gold bullets (fired within helium pressurized gun)– Retrotransposons (jumping genes – early days)– Human artificial chromosomes

Ex vivo

Electroporation

Ex vivo Electroporation

• In aqueous solution, polar phospholipids form ordered aggregates to minimize hydrophobic interactions

• Lipid shape and conditions of formation affect the final lipid organized structure

A phospholipid

Lipid Organization

Phopholipid Hierarchal Structures

Liposomes

Liposomes

• Liposomes are– not limited by size or number of genes– safe– easy to produce– short-term expression

DNAliposome

complexes

Liposomes

• Diverse manners of ‘lysing’ the liposome• Temperature sensitive• Target sensitive• pH sensitive• Electric field sensitive

Liposomes

Limitations of Gene Therapy

• Gene delivery– Limited tropism of viral vectors– Dependence on cell cycle by some viral vectors (i.e. mitosis

required)• Duration of gene activity

– Non-integrating delivery will be transient (transient expression)– Integrated delivery will be stable

• Patient safety– Immune hyperresponsiveness (hypersensitivity reactions

directed against viral vector components or against transgenes expressed in treated cells)

– Integration is not controlled oncogenes may be involved at insertion point cancer?

• Gene control/regulation– Most viral vectors are unable to accommodate full

length human genes containing all of their original regulatory sequences

– Human cDNA often used much regulatory information is lost (e.g. enhancers inside introns)

– Often promoters are substituted therefore gene expression pattern may be very different

– Random integration can adversely affect expression (insertion near highly methylated heterogeneous DNA may silence gene expression)

Limitations of Gene Therapy

• Expense– Costly because of cell

culturing needs involved in ex vivo techniques

– Virus cultures for in vivo delivery

– Usually the number of patients enrolled in any given trial is <20

– More than 5000 patients have been treated in last ~12 years worldwide

Limitations of Gene Therapy

3Other

196Cancer

21HIV

18Genetic disease

# Trials (total = 338)

Diagnosis

Gene Therapy Trials in U.S.

(Information from US NIH, Office of

Recombinant DNA Activities – 1999)

Applications of gene therapy

Diseases for applying gene therapy

Disease Defect Target cell

Severe combined Bone marrow cells or

immunodeficiency T-lymphocytes

Hemophilia Liver, muscle

Cystic fibrosis Lung Cells

Cancer Many cell types

Neurological diseases Parkinson’s/ Alzheimers Nerve Cells

Infectious diseases AIDS, hepatitis B White Blood Cells

Gene therapy could be very different for different diseases

• Gene transplantation (to patient with gene deletion)

• Gene correction (To revert specific mutation in the gene of interest)

• Gene augmentation (to enhance expression of gene of interest)

Cystic fibrosismost common lethal genetic disorder in Caucasian populations (1 in 2000 live births.) . Among African and Asian is really rare

a defect in the CFTR gene

Lungs create thick mucus secretion(prone to infections,

constant cough, leading cause of death)

Lungs in cystic fibrosisNormal lung CF lungs

dilated crypts filled with mucus and bacteria.

Normal alveolar appearance

CF lungs filled with mucus

lung did not collapse when it was removed postmortem

Example: Severe Combined Immunodeficiency Disease (SCID)

• Before GT, patients received a bone marrow transplant – David, the “Boy in the Bubble”, received BM from

his sister unfortunately he died from a a form of blood cancer

What is Severe Combined Immunodoficiency (SCID)?

> 8 new ear infections per year

> 2 serious sinus infections per year

> 2 month on antibiotics with little effect

> 2 pneumonias per year

-- failure to gain weight and grow

-- recurrent deep skin and organ abscesses

• SCID is caused by an Adenosine Deaminase Deficiency (ADA)

– Gene is located on chromosome #22 (32 Kbp, 12 exons)

– Deficiency results in failure to develop functional T and B lymphocytes

– ADA is involved in purine degradation– Accumulation of nucleotide metabolites = TOXIC to

developing T lymphocytes– B cells don’t mature because they require T cell help– Patients cannot withstand infection die if untreated

• September 14, 1990 @ NIH, French Anderson and R. Michael Blaese perform the first GT Trial

– Ashanti (4 year old girl)• Her lymphocytes were gene-altered (~109) ex

vivo used as a vehicle for gene introduction using a retrovirus vector to carry ADA gene (billions of retroviruses used)

– Cynthia (9 year old girl) treated in same year

• Problem: WBC are short-lived, therefore treatment must be repeated regularly

Parkinson's Disease Cont.

• The gene transfer procedure utilized the AAV (adeno-associated virus) vector, a virus that has been used safely in a variety of clinical gene therapy trials, and the vehicle that will be used in all of the company's first generation products, including epilepsy and Huntington's disease. In its Parkinson's disease trial, Neurologix used its gene transfer technology.

Ornithine transcarbamylase (OTC) deficiency

• September 17, 1999

– Ornithine transcarbamylase (OTC) deficiency• Urea cycle disorder (1/10,000 births)• Encoded on X chromosome

– Females usually carriers, sons have disease– Urea cycle = series of 5 liver enzymes that rid the

body of ammonia (toxic breakdown product of protein)• If enzymes are missing or deficient, ammonia

accumulates in the blood and travels to the brain (coma, brain damage or death)

• Severe OTC deficiency– Newborns coma within 72 hours

• Most suffer severe brain damage• ½ die in first month• ½ of survivors die by age 5

– Early treatment• Low-protein formula called “keto-acid”

– Modern day treatment• Sodium benzoate and another sodium derivative• Bind ammonia helps eliminate it from the body

Ornithine transcarbamylase (OTC) deficiency

• Case study: Jesse Gelsinger

– GT began Sept. 13, 1999, Coma on Sept. 14, Brain dead and life support terminated on Sept. 17, 1999

– Cause of death: Respiratory Disease Syndrome– Adenovirus (a weakened cold virus) was the vector of

choice (DNA genome and an icosahedral capsid)– Chain reaction occurred that previous testing had not

predicted following introduction of “maximum tolerated dose”

• Jaundice, kidney failure, lung failure and brain death• Adenovirus triggered an overwhelming inflammatory

reaction massive production of monokine IL-6 multiple organ failure

Ornithine transcarbamylase (OTC) deficiency

• AIDS– HIV patients T lymphocytes treated ex vivo

with rev and env defective mutant strains of HIV– Large numbers of cells obtained

• Injected back into patient• Stimulated good CD8+ cytotoxic T cell

responses (Tcyt)

• Familial Hypercholesterolemia– Defective cholesterol receptors on liver cells

• Fail to filter cholesterol from blood properly• Cholesterol levels are elevated, increasing risk of

heart attacks and strokes– 1993 First attempt

• Retroviral vector used to infect 3.2 x 109 liver cells (~15% of patients liver) ex vivo

– Infused back into patient– Improvement seen

– Has been used in many trials since then

• Lesch-Nyhan Disease – Candidate– Early days confined to animal models and in vitro

tests– Defect in producing HGPRT enzyme (hypoxanthine-

guanine phosphoribosyl transferase)• Defective metabolism of hypoxanthine and guanine

Uric acid accumulates– Gout, Kidney disease, cerebral palsy, mental

retardation, head banging, profanity, spitting, mutilation of fingers

• Gaucher’s disease– Glucocerebrosidase gene defect– RAC approved clinical tests – 1993– Affects CNS, enlarged spleen and liver, long

bone erosion and discoloration of skin

• Liposomes coated in polymer PEG – can cross the blood-brain barrier (viral vectors are too big) (January 2003)

• Case Western Uni. & Copernicus Therapeutics able to create tiny liposomes 25nm across to carry therapeutic DNA through pores in nuclear membrane

• New gene approach repairs errors in mRNA • Thalassaemia • Cystic fibrosis• Some cancers

Recent Developments in Gene Therapy

• 2003 – temporary hold on all gene therapy trials including retroviral vectors in blood stem cells

• Too early to tell

• Desperately need improved DELIVERY …could liposomes be the answer?

Future?

• 2003 – temporary hold on all gene therapy trials including retroviral vectors in blood stem cells

• Too early to tell

• Desperately need improved DELIVERY …could liposomes be the answer?

Future?