Human Gene Therapy

Genetic diseases

Ex vivo and in vivo gene therapy

Gene delivery systems

• Over 1000 human disorders are inherited

Most are rare, occurance is less than 1/100,000 persons

Ex. Severe combined immunodeficiency 1/1,000,000

Some are common, occurance is greater than 1/10,000 persons

Ex. Sickle-cell disease, 1/500 black africans

Cystic fibrosis, 1/2,500 caucasians

• Caused by one or more genes containing mutations
• Genetic basis of a disease can be established through:

Pedigree studies of families showing disease (occurence across generations)

Occurrence in twins and adoptive families

• A genetic disease resulting from a mutation in a single gene is easiest to identify (and treat by gene therapy)

Ex. Sickle cell disease

Single nucleotide mutation of b-hemoglobin gene changes the sixth amino acid from glutamic acid to valine

• Some cancers are caused by inherited mutations or by environmental factors that damage DNA

P53 tummor suppressor gene is mutated in ~ 50% of human cancers

Muscular dystrophy

Breast cancer

Colon cancer

Cystic fibrosis

Sickle cell disease

Alzheimer's disease

Parkinson's disease

Niemann-Pick disease

Sever combined immunodeficiency

Sclerosteosis (Bone density)

Hemochromatosis

Hemophilia A and B

Familial hypercholesterolemia

Huntington's disease

• Usaully aims to provide a functional copy of a defective gene (see note below)
• Confined to somatic cells so descendants will not be affected
• Germ line cell therapy considered to be unwise for now

-May be unintended consequences for future generations

• Applications still in research and testing phases

>4,000 patients treated in gene therapy clinical trials since 1990s

-Only a few clear successes

-At least one death that resulted in a systemic inflamatory immune response to the adenovirus vector

Patients lungs filled with fluid, causing respiratory failure

• Human gene therapy trials are highly regulated by the government
• Advisory committees to the National Institutes of Health must approve any human gene therapy experiments that are publically funded

Note: Rather than introducing a functional gene, other gene therapy approaches may involve inhibiting expression of a deffective gene or increasing expression of a normal gene.

Therapeutic agents undergo four levels of testing prior to final approval by the Food and Drug Administration

1. Preclinical phase using laboratory animals

2. Phase I trials using small number of healthy humans

To determine safety and maximum dosage levels

3. Phase II trials using larger numbers of diseased patients

To determine optimum dosage and if treatment is beneficial

4. Phase III trials using large numbers of patients

Further proof of safety and efficacy

• FDA gives final approval to market a therapy if all phases are successfully completed

Usually takes 7-9 years and ~$75 million

Several questions need to be resolved before gene therapy can be routinely used

1. How to access target cells with remedial gene?

2. How will gene be delivered to target cells?

3. What fraction of target cells must receive gene for therapeutic
effect?

4. What level of regulation of gene expression is needed?

5. Will overexpression cause other problems?

6. Will the cells maintain the gene for long periods of time?

7. Will treatments need to be repeated?

Delivery methods

I. Ex Vivo Gene Therapy

1. Collect cells from each individual patient --to avoid rejection by immune system

2. Grow cells in tissue culture

3. Transfect cells with cloned gene that corrects defective gene

4. Select and culture transfected cells

5. Transplant or transfuse cells back into patient

Advantages/disadvantages

• Can check transfected cells for problems before reintroduction into patient

Ex. Integration of therapeutic gene into tumor suppressor gene which might cause cancer

• Must be repeated unless pleuripotent stem cells can be isolated

Umbilical cord blood is good source of stem cells

Must be saved at birth and stored in liquid nitrogen

• Time consuming and costly

• Non-immunogenic universal donor cells could be used for all patients to avoid isolating and culturing each individual patient's cells

Currently do not exist

Cell Types Targeted for Ex Vivo Gene Therapy

1. Bone marrow stem cells

-Replicate and differentiate into B and T cells, macrophages, red blood cells and bone cells

-Transfected cells reintroduced into patient by bone marrow transplant

-Stem cells are difficult to isolate and culture

2.Cells from the liver and other organs

3. Blood vessel smooth muscle cells

-In contact with circulatory system

-Transfected cells transplanted to internal surface of an artery

Therapeutic gene product would be delivered to all tissues

4. Tumor-infiltrating lymphocytes for cancer treatment

-Transfection with gene for tumor necrosis factor

-Would invade tumors and kill cancer cells

II. In Vivo Gene Therapy

• Direct delivery of remedial gene to patient's cells without removal from body
• Eliminates need to isolate, culture and reintroduce cells for each patient

Target tissues
skin
muscle
lung
brain
colon
spleen
liver
pancreas
bone marrow
neural

• Specialized vectors are needed to target specific cell types

I. Viral vectors

• Retroviruses

-Insert their DNA randomly into host cell chromosomes

Gene stably maintained, treatment would not need to be repeated

Insertion into wrong site may cause cancer however

-Cells must be actively dividing

Might target rapidly dividing cancer cells but wouldn't work for neurons that do not divide very often

-Lack specificity for cell type

 

• Adenoviruses

-Fairly safe (cause only chest colds)

-Readily infect dividng and non-dividing cells

-Do not insert into chromosome

Eventually DNA is lost, therapy would need to be repeated

-Elicit strong immune response, transfected cells eliminated

Can't be administered repeatedly

-Lack specificity for cell type

• Adeno-associated viruses

-Nonpathogenic

-Amount of DNA they can contain is small

Some therapeutic genes would be too large

-Infect dividing and non-dividing cells

-Difficult to produce large quantities needed for therapy

• Herpes simplex virus

-Large DNA carrying capacity

-May be used to target therapeutic genes to nerve cells

• Gutless vectors

-Have problem genes deleted from viral genome

-Provides more room for larger genes and regulatory sequences

-Production of vector requires helper viruses or specialized cell lines that provide the functions of the deleted viral genes

Ex. replication and packaging

II. Nonviral gene delivery system

• Transfection efficiency is usually low and time of expression of remedial gene is too short for a therapeutic effects

Plasmids deleivered to cells:

-as naked DNA

-in liposomes

-by electroporation

-by microprojectile bombardmnent

• Human artificial chromosomes

-Provide long-term stability and expression of therapeutic gene

-Large carrying capacity for DNA

-Multiple and or large genes and their regulatory sequences can be included

• DNA conjugates

-Gene is complexed with poly-L-lysine and peptides that bond to specific cell receptors for entry into cell, protect DNA from degradation and direct DNA to the nucleus

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The End

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Last updated: December 5, 2007 /jdh

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