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Introduction to Gene Therapy
Maulik P. Suthar
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What is gene therapy? Why is it used?
Gene therapy = Introduction of normal genes into cellsthat contain defective genes to reconstitute a missingprotein product
GT is used to correct a deficient phenotype so that
sufficient amounts of a normal gene product aresynthesized to improve a genetic disorder
Gene therapy is a technique for correcting defectivegenes responsible for disease development.
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How is Gene Therapy Carried Out?
Modification of somatic cells by transferring desired gene
sequences into the genome.
Somatic cells necessary to ensure that inserted genes
are not carried over to the next generation.
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Approaches for correcting a
genetic defect
A normal gene may be inserted into a nonspecificlocation within the genome to replace a nonfunctionalgene. This approach is most common.
An abnormal gene could be swapped for a normal gene
through homologous recombination. The abnormal gene could be repaired through selective
reverse mutation, which returns the gene to its normalfunction.
The regulation (the degree to which a gene is turned onor off) of a particular gene could be altered.
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How does the normal gene
replace the abnormal gene?
A carrier molecule called a vectormust be used todeliver the therapeutic gene to the patient's target cells.
The most common vector is a virus that has beengenetically altered to carry normal human DNA.
Viruses have evolved a way of encapsulating anddelivering their genes to human cells in a pathogenicmanner.
Target cells such as the patient's liver or lung cells are
infected with the viral vector. The vector then unloads itsgenetic material containing the therapeutic human geneinto the target cell.
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Different Delivery Systems
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
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Cells removed from body
Transgene delivered
Cells cultured
Cells returned to the body
Ex Vivo In Vivo
Transgene delivered
directly into host
Strategies for Transgene Delivery
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Vectors
The way you insert the normal gene in the patients
cell is by vectors.
The most common vectors that are used in gene therapy
are virus vectors
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Why Viruses?
Viruses through the time of evolution have evolved toinfect the cells with great specificity
Viruses tend to be very efficient at transfecting their ownDNA into the host cell genome.
This allows them to produce new viral particles at theperiod of synthesis of the cell
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Naked DNANaked DNATargetTarget
CellCell
TherapeuticTherapeuticProteinProtein
AAVAAV
Retrovirus/LentivirusRetrovirus/Lentivirus
AdenovirusAdenovirus
NucleusNucleus
Gene Therapy Principles
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In vivo techniques
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 cells genome
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Types of Viral Vectors
Retroviruses - A class of viruses that can createdoublestranded DNA copies of their RNA genomes. Thesecopies of its genome can be integrated into the chromosomesof host cells. Human immunodeficiency virus (HIV) is aretrovirus.
Adenoviruses - A class of viruses with double-stranded DNAgenomes that cause respiratory, intestinal, and eye infections inhumans. The virus that causes the common cold is anadenovirus.
Adeno-associated viruses - A class of small, single-strandedDNA viruses that can insert their genetic material at a specific
site on chromosome 19. Herpes simplex viruses - A class of double-stranded DNA
viruses that infect a particular cell type, neurons. Herpessimplex virus type 1 is a common human pathogen that causescold sores.
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Adenovirus
36 kb Double Stranded DNA Genome
Entry through CAR receptor and integrin co-receptor
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E1A E3E1B
E2A E4E2B
L1 L2 L4L3 L5
Latest Generation Adenoviral VectorGutless; Helper-dependent; Minimal Ad
Therapeutic
TransgeneStuffer DNAStuffer DNA ITRITR
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Which Virus to Use?
Depends how well they transfer the genes to cells
which cells they can recognize and infect
and whether they alter the cells DNA permanentlyor temporarily
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Non viral methods
Direct introduction of therapeutic DNA into target cells (microinjection). Thisapproach is limited in its application because it can be used only with certaintissues and requires large amounts of DNA.
Artificial liposomes which carry the therapeutic DNA are capable of passing theDNA through the target cell's membrane. Can be non-specific to cell type.
Chemically linking the DNA to a molecule that will bind to special cell receptors.Once bound to these receptors, the therapeutic DNA constructs are engulfed bythe cell membrane and passed into the interior of the target cell. This deliverysystem tends to be less effective than other options.
A 47th artificial human chromosome. This chromosome would existautonomously alongside the standard 46 --not affecting their workings orcausing any mutations. It would be a large vector capable of carryingsubstantial amounts of DNA, and scientists anticipate that, because of itsconstruction and autonomy, the body's immune systems would not attack it.
A problem with this potential method is the difficulty in delivering such a large
molecule to the nucleus of a target cell.
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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.
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Ex vivo manipulation
Ex vivo manipulation techniques
Electroporation
Liposomes
Calcium phosphate
Gold bullets (fired within helium pressurized gun) Retrotransposons (jumping genes early days)
Human artificial chromosomes
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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
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Patient safety
Patient safety
Immune hyperresponsiveness (hypersensitivityreactions directed against viral vector components oragainst transgenes expressed in treated cells)
Integration is not controlled oncogenes may beinvolved at insertion point cancer?
More than 5000 patients have been treated in last
~12 years worldwide
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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)
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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)
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Expense
Costly because of cell culturing needs involved in ex
vivo techniques
Virus cultures for
in vivodelivery
Usually the number of patients enrolled in any given
trial is
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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
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
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ADA is involved in purine degradation
Accumulation of nucleotide metabolites = TOXIC to
developing T lymphocytes
B cells dont mature because they require T cell help
Patients cannot withstand infection die if untreated
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Severe OTC deficiency
Newborns coma within 72 hours
Most suffer severe brain damage
die in first month 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
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Other Examples of Gene Therapy: Single Gene Defects
= Most Attractive Candidates
Cystic fibrosis
Crippled adenovirus selected (non-integrating,
replication defective, respiratory virus)
Gene therapy trials 3 Research teams, 10patients/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 adenovirusdoes not integrate into genome like retroviruses
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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)
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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
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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, mentalretardation, head banging, profanity, spitting,
mutilation of fingers
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Gauchers disease
Glucocerebrosidase gene defect
RAC approved clinical tests 1993
Affects CNS, enlarged spleen and liver, long boneerosion and discoloration of skin
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Gene Therapy of Cancer
Cancer results from multiple mutations
Limited success with p53--growth arrest versusapoptosis
Target the Rb pathway
INK/ARF locus--two potential targets inhibit cyclin PTEN expression alters metastatic potential and reduces
vascularization
New tumor suppressors such as mda-7 (melanoma) and
OPCML(ovarian cancer)
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Methods for gene therapy of cancer
Viruses
Naked DNA (vector-free)
Liposomes
Protein-DNA complexes
Gene gun
Calcium phosphate precipitation
Electroporation
Intracellular microinjection
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Reasons for lack of clinical success
Low transduction frequency
Insufficient expression in vivo
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Strategies
#1: Strengthening of the immune response against a tumor B7 expression on tumors may provide necessary second signal (co-
stimulation) required forTcyt cell activation
Improve antigen presenting properties of tumor cells
Antibodies target tumors for destruction by immune system
Monoclonal antibody binding to tumor antigens can stimulate:
NK cell killing via antibody-dependent cell-mediated cytotoxicity Phagocytosis via FcR-binding on macrophages
Complement activation opsonization
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Attract cells of the immune system to the tumor
Example: Transfect tumor cells with cytokine genes GM-CSF
Recruits dendritic cells to site of tumor
Dendritic cells pick up tumor antigen, process it, and travel to draining
lymph nodes to present antigen to T cells
Load professional Antigen Presenting Cells of patient withtumor antigen in vitro and then administer them back to patient
T cells of patient can now recognize tumor cells and will destroy them
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#2: Repair of cell cycle defects caused by
loss of tumor suppressor gene
e.g. p53 = DNA repair enzyme Guardian of the
genome Faulty p53 allows cells carrying damaged DNA to survive
when they would normally die
Mutations passed to progeny
Can accumulate additional mutations lethal tumor
In most human cancers, the p53 gene appears defective
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#3: Repair of cell cycle defects caused
by inappropriate activation of oncogenes
Oncogenes = mutant versions of normal
genes (proto-oncogenes) that drive cellgrowth
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Example: ras gene (20-30% of all human
cancers have an abnormal ras gene)
Normally = relay switch within the signal pathway
that tells the cell to divide In absence of external stimulation ras is off
Mutant ras = switch stuck in the on position
misinforming cells mitosis
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Problems to overcome
Short-lived nature of gene therapy
Immune response
Problems with viral vectors
Multigene disorders
Current Gene Therapy
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Current Gene Therapy
progress
Into the brain using liposomes coated in a polymer callpolyethylene glycol (PEG).
RNA interference or gene silencing may be a new way totreat Huntington's.
New gene therapy approach repairs errors in messengerRNA derived from defective genes. Technique haspotential to treat the blood disorder thalassaemia, cysticfibrosis, and some cancers.
Gene therapy for treating children with X-SCID (severcombined immunodeficiency) or the "bubble boy"
disease is stopped in France when the treatment causesleukemia in one of the patients.
Sickle cell is successfully treated in mice.