Dr. Nilanjana Basu,Vice Principal, Asst. Professor,Department of Biotechnology,

T.John College,Bangalore

CANCER is a complex of diseases which occurs when normal cells mutate into abnormal cells ,that take over normal tissue , eventually harming and destroying the host.

Uncontrolled growth and spread of abnormal cells

Proliferation (rapid reproduction by cell vision)

Metastasis (spread or transfer of cancerous cells from one organ or part to another not directly connected)

PHYSICAL AGENTS: Radiation( Ionizing : radon gas), Irritants, Exposure to sunlight(melanoma).

CHEMICAL AGENTS: Smoking (nitrosamines, and aromatic hydrocarbons) dietary ingredients, Drugs, alcohol, asbestos fibers (mesothelioma).

GENETICS AND FAMILY HISTORY(breast and ovarian cancer)

DIETARY HABITS: Low fiber, High Fat, Processed food, grilled food ( benzopyene carcinogen present in foods cooked at high temperature).

VIRUSES AND BACTERIA: DNA viruses, Hepa B, Herpes, EBV, Human Papilloma Virus; RNA Viruses: HIV ( viral oncogenes, insertion near protooncogene in host); Bacteria- H. pylori ( gastric cancer)

All the factors lead to series of mutations in a cell

Epigenetics: Non mutational changes in DNA alters gene expression. Loss of DNA methylation( keeps oncogene s silent) and acetylation of histones .

Genes controlling cell growth and differentiation may get altered

Two categories of genes get affected: Oncogenes( gain of function), and Tumor suppressor genes ( loss of function)

May produce hormones which encourage cell division by passing the message to cell nucleus on binding to receptors

Part of the signal transduction pathway

May produce mitogens

May be involved in trancription of DNA and Protein synthesis

Mutations in protooncogenes may produce oncogenes

In dominant gain of function mutation , change in one copy of protooncogene enough to produce cancer.

Example –ras- oncogene (has come into humans from Rous sarcoma Virus)

CODE FOR ANTIPROLIFERATION SIGNALS , SUPRESS MITOSIS AND CELL GROWTH

DNA DAMAGE TRIGGERS ACTIVATION OF TUMOR SUPRESSOR GENES

ARRESTS CELL CYCLE TO CARRY ON REPAIR

EXAMPLE: P53 ACTIVATED DURING HYPOXIA AND UV DAMAGE

MUTATION MAY ALTER TSG: DAMAGED DNA ACCUMULATES LEADING TO CNACER.

MUTATIONS OF TSG IN GERMLINE IS CARIED ON TO OFFSPRING

IS RECESSIVE LOSS OF FUNCTION MUTATION TWO COPIES OF THE GENE NEED TO BE ABSENT FOR CANCER DEVELOPMENT

MAY BE INHERITED : IN LI-FRAUMENI SYNDROME ( PANCREATIC CANCER), RETINOBLASTOMA (Rb)

Surgery: removal of tumors: preventive, diagnostic or curative.

Chemotherapy: use of antineoplastic cytotoic drugsdugs to promote cell death by interfering with cellular functions and reproduction. Alkylating agents: create defect in DNA, antimetabolites

Radiotherapy: high energy ionizing radiations directed to destroy malignant tumors without harming surrounding

Immunotherapy: Use of chemicals r microbial agents to induce mobilization of immune defense. Modifiers to improve the immunologic relationship between tumor and host in beneficial way.

Angiogenesis inhibitors: prevent growth of blood vessels that tumors need to live

Hormone blockers: for testosterone and estrogen in prostrate and breast cancer.

Gene therapy: Replacement of mutated genes :in clinical trials.

It is a technique for correcting defective genes that are responsible for disease development

There are four approaches:1. A normal gene inserted to compensate for a

nonfunctional gene ( gene supplementation).2. An abnormal gene traded for a normal gene

(gene replacement)3. Change the regulation of gene pairs (targeted

inhibition of gene expression)4. Targeted killing of specific cells

Naked DNANaked DNATarget Target

CellCell

Therapeutic Therapeutic ProteinProtein

AAVAAV

Retrovirus/LentivirusRetrovirus/Lentivirus

AdenovirusAdenovirus

NucleusNucleus

Gene Therapy Principles

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

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

The first gene therapy was performed on September 14th, 1990 Ashanti DeSilva was treated for SCID Defective ADA gene which 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 Doctors removed her white blood cells, inserted the missing

gene into the WBC, and then put them back into her blood stream.

This strengthened her immune system Only worked for a few months

Gene Therapy is used to correct a deficient phenotype so that sufficient amounts of a normal gene product are synthesized to improve a genetic disorder by Introduction of normal genes into cells (that contain defective genes) to reconstitute a missing protein product

Modification of somatic cells by transferring desired gene sequences into the genome.

Somatic cells and not germ line cells are necessary to ensure that inserted genes are not carried over to the next generation.

Cells removed from body

Transgene deliveredCells cultured

Cells returned to the body

Ex Vivo In Vivo

Transgene delivereddirectly into host

Strategies for Transgene Delivery

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 stablely integrate the

desired gene into the target cell’s genome

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.

Ex vivo manipulation techniques Electroporation: pores by High voltage shock. Liposomes: DNA to be transferred is packaged with

cationic :outside, or anionic:inside. Calcium phosphate: DNA ppts. Phagocytosed by cells Particle bombardment :Gold bullets (fired with helium

pressurized gun) Receptor mediated endocytosis : transfer to lysosomes to

be prevented Human Artificial Chromosomes

Viruses through the time of evolution have evolved to infect the cells with great specificity

Viruses tend to be very efficient at transfecting their own DNA into the host cell genome.

New Viral particles produced which can infect neighboring cells

Retrovirus Adenovirus Lentiviruses Poxviruses Herpes Viruses

Depends▪ how well they transfer the genes to cells▪ which cells they can recognize and infect ▪ and whether they alter the cell’s DNA permanently or

temporarily

Adenovirus

36 kb Double Stranded DNA Genome

Entry through CAR receptor and integrin co-receptor

Are double stranded DNA genome that cause respiratory, intestinal, and eye infections in humans

The inserted DNA is not incorporate into genome remains non integrated as an episome in the nucleus

Do not replicate at the same rate as cells Has to be reinserted when more cells divide

Ex. Common cold

35kb of therapeutic gene can be inserted

Adenovirus Cell Entry

E1A E3 E1B

E2A E4E2B

L1 L2 L4L3 L5

Latest Generation Adenoviral Vector

“Gutless”; Helper-dependent; Minimal Ad

Therapeutic Transgene

Stuffer DNAStuffer DNAψ ITRITR

RNA Viruses, possess reverse transcriptase, synthesizes c-DNA copy of genome

Random Integration into host genome

gag ,pol ,env removed provided by packag ing cell line or helper phages

Nuclear membrane has to dissolve

Only rapidly dividing cells

Limits potential target cells like mature neurons

Good for actively dividing cancer cells in non dividing brain tissues

Cloning capacity 8kb

Single stranded DNA genome

Need adenovirus to replicate

Site specific Integration into chromosomal DNA

Long term expression without risk of insertional mutation

rep gene needed, provided by packaging cell line

4.5 kb inserts

To infect central nervous system

Double stranded DNA genome

Life long latent infection in sensory ganglia as non integrated extrachromosomal element

Exploited for long term treatment

Delivery f genes to neurons for treatment of Parkinson's disease and CNS tumors.

cloning capacity 30kb

Gene delivery Limited tropism of viral vectors Dependence on cell cycle by some viral vectors

(i.e. only actively dividing cells)

Duration of gene activity Non-integrating delivery will be transient

(transient expression) Integrated delivery will be stable.(Integration may

alter gene expression of the gene where attachment takes place)

Patient safety Immune hyper responsiveness (hypersensitivity

reactions) Immune response may make the virus refractory :

prevent spread of infection. 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)

Expense Costly because of cell culturing needs involved in

ex vivo techniques Virus cultures needed for in vivo delivery Usually the number of patients enrolled in any

given trial is <20

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

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

“Every realm of medicine has its defining moment, often with a human face attached. Polio had Jonas Salk. In vitro fertilization had Louise Brown, the first test-tube baby. Transplant surgery had Barney Clark, the Seattle dentist with the artificial heart. AIDs had Magic Johnson. Now gene therapy has Jesse Gelsinger.”

Sheryl Gay Stolberg, The New York Times.

Cystic fibrosis “Crippled” adenovirus selected (non-integrating,

replication defective, respiratory virus)

Gene therapy trials – 3 Research teams, 10 patients/team▪ 2 teams administered virus via aerosol delivery into nasal passages

ad lungs▪ 1 team administered virus via nasal passages only▪ Only transient expression observed because adenovirus does not

integrate into genome like retroviruses

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

Cancer results from multiple mutations

Viruses Naked DNA (vector-free) Liposomes Protein-DNA complexes Gene gun Calcium phosphate precipitation Electroporation Intracellular microinjection

Low transduction frequency Insufficient expression in vivo

Use of antisense molecules

Synthetic oligo deoxy nucleotides(ODN’s)

Hybridize to mRNA

ds RNA’s not transcribed destroyed in the cell

ODN’s modified to increase stability :non bridging oxygen atom in inter nucleotide phosphate linkage replaced with sulphur: phosphorothioate ODN’s .

antisense nucleotides may also be expressed from plasmid transfected into the cell .ODN’s against oncogenes k-ras,c-myc,bcr-abl and bcl-2 in clinical trials Phase I-II. Found to down regulate target BCL-2 protein in metastatic cancer anti tumor response in 6/14 patients.

Genetically alter immune cells to increase their functions

Therapeutic cells introduced into tumor cells or effector cells like T-lymphocyte

Introducing cytokine ( help in chemotaxis, and communication between different WBC’s)

Recruitment of inflammatory cells by such tumor cells ,inhibit tumor growth.

Tumor antigen priming of the host immune cells. Enhanced tumor immunogenicity resulting in tumor regression.

Lymphokine activated killer cells LAK can be grown invitro in presence of interleukin (IL2) and adoptively transferred to cancer cells.

Tumor infiltrating lymphocytes isolated from patients tumors may be grown in cultures in presence of IL2 and re-transplanted back to the patient. TIL may me modified with cytokine genes TNFalpha helplng in tumor regression .

Cytokine genes are introduced into APC’s and dendritic cell which are able to prime T-lymphoytes ,increasing antigen specific immune response.

Potential tumor vaccines IL2,IL4,IL7 etc.

For efficient activation of T cells in addition to antigen specific signal received by T-cell receptor /CD3 complex non specific signal are needed by CD28 and their ligands B7 family.

B7 molecules expressed on APC’s – produces cytokines-proliferation activation maturation of T cells . Tumor cells produce Tumor specific antigens but in absence of co-stimulatory molecules helper T-cells needed .

B7 molecules may be expressed in cells exvivo , transferred into tumor cells lead to stimulation of T-cells for tumor killin

good response seen in squamous cell carcinoma when HLA-B7 was injected intra tumorally into 9 patients

New strategy for immunization

Genes coding tumor specific antigens intramuscularly injected

Synthesize protein

Stimulates antibody –mediated response as well as cytotoxic T-cell response

DNA into biodegradable polymer –long term release-no booster dose needed

Major problem of solid tumors towards conventional treatment is resistant to apoptosis

Defective p-53 responsible as it is the gate keeper in inducing apoptosis

Adenoviral p53 transfer in squamous cell carcinoma cell lines has been found to be effective

Wildtype p53 may be used alone or in combination with other apoptosis inducing signals along with radiotherapy

Apoptosis inducing key enzyme Caspase 8 may be expressed in some cells

Preclinical trials of caspase 8 expression able to induce cell death in glioma’s and sensitize radio refractory cells to radiotherapy

p53-dependent apoptosis still occurs when either nuclear translocation is blocked with WGA, or protein synthesis is blocked by cycloheximide. Transcription-independent apoptosis pathways may include direct p53 effects on Bax, or p53-dependent release of BID from Bcl-xL sequestration.

Used for solid tumors

Therapeutic gens to target cells, very cell expressing it gets destroyed

Suicide genes produce enzymes which can activate prodrugs into toxic compounds

Example HSV-TK ( herpes simplex virus thymidine kinase) or yeast enzyme CD (cytosine deaminase )

HSV-TK phosphorylates Gancyclovir to its toxic form and CD changes 5-FC( 5 flurocytosine )to 5FU ( flurouraacill)

Used in preclinical trials of breast, head and neck cancer

Suicide gene placed under tumor specific promoters

Intrroduced via adenovirus or retrovirus intratumorally

Followed by GCV injections

Expression of gene only in tumor cells and thus killing of those cells selectively.

Transfection efficiency low thus depends on bystander effect for its success

Tumors require oxygen, nutrition hormones for growth provided by newly formed blood vessels

Inhibiting angiogenesis can regress experimental tumors

Inhibitors of angiogenesis are angiostatin and endostatin

Genes coding for angigenesis inhbitor can be introduced directly or exvivo by manipulating generic cells to over express the protein.

Interesting results seen in brain tumors

Bone marrow cells are highly susceptible to killing by chemotherapeutic drugs

To protect bone marrow MDR –I gene introduced in bone marrow

MDR-I produces 170 kDa glycoprotein which is an energy dependent cellular pump which actively effluxes toxic drugs like paclitaxel and anthracycline.

Helps to protect bone marrow in patients with metastatic breast cancer

RAC (recombinant DNA advisry committee of NIH)established in October 7, 1974

Oversees federally funded research involving recombinant DNA

Provides advice concerning advances in: Recombinant technology New organisms under investigation Public attitudes associated with research in molecular

biology

Examines clinical trials that involve the transfer of recombinant DNA to humans

All trials funded by NIH are registered with the RAC Advisory to the Director – NIH

Before any human gene transfer trials can take place, the RAC reviews proposals and comments on the feasibility, risks, etc.

Multilayered review system: Institutional review boards FDA (drugs involved) RAC of the NIH Human gene therapy subcommittee of RAC

Safety concerns Toxicity Contaminants Safe vectors? Side effects

Gene therapy only a decade old Has shown promising results in treatment of

monogenic disorders like SCID, Hemophilia etc. At present in phase II of trials for cancer treatment May be best for early stage of disease and those with

minimal residual disease. Success depends on ability to target every cancer cell,

and high levels of expression Minimize toxicity by delivering genes to specific cells Main key to success will be the ability to have a

simple injectable form of the treatment.

FDA hasn’t approved any human gene therapy product for sale

Reasons: 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

Short Lived Hard to rapidly integrate therapeutic DNA into genome and rapidly dividing

nature of cells prevent gene therapy from long time Would have to have multiple rounds of therapy

Immune Response new things introduced leads to immune response increased response when a repeat offender enters

Viral Vectors patient could have toxic, immune, inflammatory response also may cause disease once inside

Multigene Disorders Heart disease, high blood pressure, Alzheimer’s, arthritis and diabetes are

hard to treat because you need to introduce more than one gene

May induce a tumor if integrated in a tumor suppressor gene because insertional mutagenesis

SiBiono Genetech Co. Shenzhen Guandong Province is commercially producing Gendicine : adenovirus carrying P53 for tumor shrinkage in later stages of conventional treatments.

Oncorine : oncolytic recombinant adenovirus injection H103 – producing anti tumor response ( In combination with chemotherapy)

Thank you……