April 22, 2003

Bio. 551 Rec. DNA

Dr. Michelle Mardahl

Correcting human disorders in vitro:

Human CF- cells were infected with retroviral vector w/ CFTR- cells expressed protein and had restored channel function

Animal Models- Genetic Defects have been corrected in Transgenic Mice

Shiverer- develop trembles and die at youn aged. Offspring are deficient in myelin in the CNS due to a mutated meylin basic protein gene (MBP). Transgenic mice w/ 1 copy- had 8% of normal; Mice with 2 copies of nomral MBP- 26% and were phenotypically normal

A new age of medicine began in 1990 when gene therapy was used for the first time to treat a young girl with an inherited severe combined immune deficiency (SCID).

Laura Cay Boren- from her birth in July 1982- she fought infection after infection. In 1983 & 194, she received bone marrow transplants from her father- only temp. relieve. By 1985, she was gravely ill. She was injected with ADA-PEG (polyethylene glycol). Within hours- her ADA increase 20 fold. By summer 1988 she could play with other kids.

In 1990, a 4 yr. old girl with ADA-deficient SCID, became the first person treated with gene therapy.
Her own T-cells were collected and transformed with a gene for ADA.

Hemophilia- In 1999 Don Miller was the first patient to receive a disable virus that delivered a functional gene for clotting factor VIII to his bloodstream. He came in 3 infusions one a day for 3 days. As of 2000- there were no side effects. 14 weeks after the treatment he stopped having spontaneous bleeding. 2 nosebleeds stopped within minutes.

Protein-based therapies replace gene products and treat the phenotype.

Gene therapies replace malfunctioning or absent genes.

Germline gene therapy targets gametes or fertilized ova and is heritable.

Somatic gene therapy targets various types of somatic tissue as well as cancer cells and is not heritable.

Ex vivo gene therapy- removes cells from a patient, manipulates them, and returns them.

In situ gene therapy delivers a gene directly to an accessible body part.

In vivo gene therapy delivers the vector into the body.

Chimeraplasty - use small RNA-DNA oligonucleotides to stimulate DNA repair and restore a normal gene sequence.

1. Stem cells from bone marrow and other tissues are being pursued as sites since under certain conditions they can specialize into a variety of cell types

2. Endothelium implanted in blood vessels secretes needed proteins into the bloodstream.

3. Skin cells genetically altered in culture, expanded, then grafted onto animals, where they secrete foreign gene products.

4. Muscle - accessible, near a blood supply, and abundant.

5. Liver is the largest organ, has many functions and can regenerate. Liver cells from an individual can be removed, genetically altered, and grafted back. Liver cells infected with engineered viruses (Hepatitis) introduced into an artery leading to the liver.

6. Lung - use disabled cold viruses to deliver genes

7. Nerve cells- Altered herpes viruses, or treated by genetically engineering fibroblasts or glia around them.

8. Cancer cells are targets for two different approaches of gene therapy.

Suicide gene therapy delivers genes that cause cancer cells to self-destruct.

Cancer vaccines mark tumor cells for destruction by the immune system.

1. Liposomes: Mixture of neutral & cationic lipids in vesicle form. Since DNA delivered via liposomes is usually a plasmid, the DNA remains extra chromosomal.

Advantages:

1. The size of DNA is not limited.

2. The vector can not replicate or recombine to form an infectious agent

Disadvantages:

1. Mode of delivery is inefficient & requires thousands of plasmid copies to be presented to the cell to achieve gene transfer

2. Liposomes may induce a limited inflammatory response.

3. DNA is short-lived w/in the cell and requires repeated doses for continued therapy

4. The effects of repeated exposure are unknown

2. Conjugated DNA- a particle coated w/ DNA delivered directly to target tissue or via blood stream

Particle:

Receptor specific ligands

Example- Peanut allergies cause anaphylactic shock killing approx. 100 people/year.

Animal model- delivered DNA to epithelial gut lining to expose animal to peanuts building immunity. Particle of Arah2 (Principle allergen in peanuts & chitosan (biodgradable component of crustacean shells). The particle protects the DNA delivers it to epithelial cells where gene is expressed. Exposure to small amounts of the protein allowed the animal to develop tolerance to doses of peanuts

3. Viral Vectors- As of 1998, retrovirus, adenovirus, herpesvirus and adeno-associated virus (AAV) vectors have been developed for use in human gene therapy and have reached phase 1 clinical trials. Transgenes may be incorporated into viral vectors by addition or replacement of viral genes. Viral vectors differ in the type of cell they can enter, and in how large a gene they can carry.

Replication competent or helper independent- if transgene replaces viral genes that are non-essential for replication

Replication Dependent- or Helper —dependent — if transgene replaces an essential viral gene. Gut-less vectors contain just the cis-acting elements required for packaging and genome replication. The advantage is

1) High capacity for foreign DNA

2) No viral gene products are made so has no cytotoxic effects

3) Care to prevent the helper virus to complete its own infection cycle so that only the recombinant vectoris pakacaged.

Retrovirus- RNA virus w/ DNA intermediate. Integrates into the chromosome as a single copy resulting in a permanent modification. Recombinant Retroviruses accomodates ~ 8kb DNA insert

Characteristics-

1. Stable but imprecise integration.

2. Long term expression.

6. Production problems- recombine, rearrange and low titers (10^5-8 pfu/ml)

Applications:

1. Gaucher disease

2. HIV infection

3. Several cancers

4. ADA deficiency

Adenovirus- infects most human cells. Viral DNA integrates at low frequency. Simple life cycle. Infects both replicating & non-replicating cells. High titer- 10^13 pfu/ml

Accomodates up to 7 kb, needs a helper virus to package.

Characteristics:

APPLICATIONS

1. Cystic fibrosis

2. Hereditary emphysema (lacking alpha1-antitrypsin

Characteristics:

1. Integrates into specific chromosomal sites

2. Long term expression

3. Non toxic

4. Infects dividing and nondividing cells

5. Carries small gene

APPLICATIONS:

1. Cystic fibrosis

2. Sickle cell disease

3. Thalassemias

Charcteristics:

1. Long-term expression

2. Infects nerve cells

Applications

Brain tumors

Viral particles that make it to target cells face a second obstacle: a tough membrane.

Retroviruses insert the therapeutic gene at a random position in the cell's DNA-might interrupt an important sequence, actually harming the cell or new genes often end up in dormant stretches of DNA where they do not get switched on frequently enough to make much of a difference to the patient.

4. Transposable Elements

Hyperammonemia- genetically engineer bacteria

Chimoplasty for mutations associated w/ mental retardation

Ex- vivo Lentiviral Stem Cell For MPS

Genetically engineered bone marrow

Hydroxyurea to reactivate fetal hemoglobin genes are being applied to sickle cell disease.

1. Which cells should be treated?

2. What proportion of the targeted cell population must be corrected to alleviate or halt progression of symptoms

3. Is overexpression of therapeutic gene dangerous

4. Is it dangerous if the engineered gene"escapes" and infiltrates other tissues?

5. How long will the affected cells function?

6. Will the immune system attack the introduced cells?

1. Does the participant in a gene therapy trial truly understand the risks?

2. If a gene therapy is effective, how will recipients be selected, assuming it is expensive at first?

3. Should rare or common disorders be the focus of gene therapy research and clinical trials?

4. What effect should deaths among volunteers have on research efforts?

5. Altering human germline

6. Potential for creating new infectious agents