19. Treatment of Genetic Diseases

Somatic Cell Gene Therapy

a). Treatment strategiesi). Metabolic manipulationii). Manipulation of the proteiniii). Modification of the genome

b). Strategies for gene transfer

Three categories of somatic cell gene therapy:1. Ex vivo – cells removed from body, incubated with

vector and gene-engineered cells returned to body.2. In situ – vector is placed directly into the affected

tissues.3. In vivo – vector injected directly into the blood stream.

Example of ex vivo somatic cell gene therapy

• Usually done with blood cells because they are easiest to remove and return.

• Sickle cell anemia

Examples of in situ somatic cell gene therapy

• Infusion of adenoviral vectors into the trachea and bronchi of cystic fibrosis patients.

• Injection of a tumor mass with a vector carrying the gene for a cytokine or toxin.

• Injection of a dystrophin gene directly into the muscle of muscular dystrophy patients.

Example of in vivo somatic cell gene therapy

• No clinical examples.

• In vivo injectable vectors must be developed.

Barriers to successful gene therapy:1. Vector development2. Corrective gene construct3. Proliferation and maintenance of target cells4. Efficient transfection and transport of DNA to nucleus for

integration into genome 5. Expansion of engineered cells and implantation into patient

Types of vectors• RNA viruses (Retroviruses)

1. Murine leukemia virus (MuLV)2. Human immunodeficiency viruses (HIV)3. Human T-cell lymphotropic viruses (HTLV)

• DNA viruses1. Adenoviruses2. Adeno-associated viruses (AAV)3. Herpes simplex virus (HSV)4. Pox viruses5. Foamy viruses

• Non-viral vectors1. Liposomes2. Naked DNA3. Liposome-polycation complexes4. Peptide delivery systems

Advantages:1. Randomly integrates into genome2. Wide host range3. Long term expression of transgeneDisadvantages:1. Capacity to carry therapeutic genes is small2. Infectivity limited to dividing cells3. Inactivated by complement cascade4. Safety

Adenovirus Advantages:

1. Efficiency of transduction is high

2. High level gene expression

3. Slightly increased capacity for exogenous DNA

Disadvantages:

1. Expression may be transient

2. Cell-specific targeting difficult to achieve

3. Virus uptake is ubiquitous

4. Safety

Other viral vectors

• Adeno-associated virus – infects wide range of cells (both dividing and non-dividing), able to integrate into host genome, not associated with any human disease, high efficiency of transduction.

• Herpes simplex virus, vaccinia virus, syndbis virus, foamy viruses – not yet widely studied

• Onyx virus – limited replicating adenovirus that replicates mainly in tumor cells.

1. Liposome 2. Cationic polymers

Non-viral vectors

May overcome limitations with viruses including small capacity for therapeutic DNA, difficulty in cell-type targeting and safety concerns.

4. Peptide-mediated gene delivery

3. Naked DNA

Synthesis of a retroviral gene therapy vector

Site of insertion of therapeutic gene

Selectable marker for transduced cells

Percent effort directed towards different gene therapy trials.

Examples of Gene Therapy Trials

• Adenosine deaminase gene transfer to treat Severe Combined Immuno-Deficiency (SCID)

• CFTR gene transfer to treat Cystic Fibrosis (CF)• Advanced Central Nervous System (CNS)

Malignancy• Mesothelioma• Ornithine Transcarbamylase Deficiency• Hemophilia• Sickle Cell DiseaseSickle Cell Disease

Stem Cell Transplantation

Harvest marrow

Radiation/Chemotherapy

Infuse normal donor cells

Donor

Patient

Stem Cell Gene Therapy

Harvest marrow

Introduce therapeutic gene

Radiation/Chemotherapy

Reinfuse corrected cells

Make genePut into vehiclefor delivery into cell

The Molecular Basis of Sickle Cell Anemia

2

1

s

LCR

s chains

chains

2 s2

Polymerization

Survives 15 - 25 daysSickled red cell

120 days

Sickle Cell

Normal

Preferential Survival of Normal Red Blood Cells

in Sickle Cell Anemia

20 days

Gene Therapy for Sickle Cell Anemia

2

1

s

LCR

s chains

chains

2 s

chains

Non-sickled red cellSurvives 120 days

No polymerization

LCR

Mixed Chimerism following BMT for Thalassemia and Sickle Cell

Disease• Occurs in a minority of patients (5 - 10%).• A minority of donor-origin progenitors (10 -

20%) is sufficient to ameliorate disease.

Thus, it may be possible to achieve therapeutic effects by gene transfer into only a fraction of stem cells.

Preferential Survival of Normal Red Blood Cells

20 / 120 = 1/6thnormal or corrected stem cells

= 50% corrected mature red cells

TURNOVERRATE

LOW

HIGH

Therapeutic effects from small numbers of normal

stem and progenitor cells in the marrow

BONE MARROW BLOOD

120 days

SSSSSS

N

20 days

Approaches to Improving the Efficiency of Gene Therapy Targeting

the Stem Cell

• Use selection to exponentially expand stem cells carrying the therapeutic gene.

• Use repeated treatments to additively expand stem cells carrying the therapeutic gene.

In Vivo Selection

Selectable gene =MDR1 (taxol, navelbine, vinblastine)DHFR (methotrexate)Other (MGMT, aldehyde dehydrogenase,

cytidine deaminase)

Therapeutic gene Selectable gene

In Vivo Selection of Genetically Modified Bone Marrow

Drug Treatment

Gene Therapy for Sickle Cell Disease

REPEAT In vivo selection

GCSF

MobilizeStem Cells

Introduce gene Re-infuse

One developing technology that may be utilized for gene therapy is nuclear transfer (“cloning”).

What’s in a Name? – Nuclear Transplantation vs. Therapeutic Cloning vs. Human

Reproductive Cloning.

Ethical Considerations

• Use of technology for non-disease conditions such as functional enhancement or “cosmetic” purposes – for example, treatment of baldness by gene transfer into follicle cells , larger size from growth hormone gene, increased muscle mass from dystrophin gene.

• In utero somatic gene therapy – only serious disease should be targeted and risk-benefit ratios for mother and fetus should be favorable.

• Potential for real abuse exists by combining human reproductive cloning and genetic engineering.