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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……