23402863 Lecture1 Introduction to Gene Therapy

8/2/2019 23402863 Lecture1 Introduction to Gene Therapy

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2009, Maulik P. Suthar

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.

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23402863 Lecture1 Introduction to Gene Therapy