VIRAL VECTORS (I)

Eui-Cheol Shin, MD, PhD (ecshin@kaist.ac.kr; 4236; Bldg E7, Rm 4109) Graduate School of Medical Science & Engineering, KAIST

INTRODUCTION
1. Viral vectors: a tool to deliver genetic material into cells. 2. Performed inside a living organism (in vivo) or in cell culture (in vitro). 3. Viruses h 3 Vi have evolved specialized molecular mechanisms t l d i li d l l h i to efficiently transport their genomes inside the cells they infect. 4. Transduction: delivery of genes by a virus. (vs transfection, transformation etc)

KEY PROPERTIES
1. Safety y Deletion of a part of the viral genome critical for viral replication. Such a virus can efficiently infect cells but, once the infection has but taken place, requires a helper virus to provide the missing proteins for production of new virions virions. 2. Low t i it 2 L toxicity The viral vector should have a minimal effect on the physiology of the cell it infects.

KEY PROPERTIES
3. Stability y Some viruses are genetically unstable and can rapidly rearrange their genomes. This is detrimental to predictability and genomes reproducibility of the work. 4. Cell type specificity Most i l M t viral vectors are engineered to infect as wide a range of cell t i dt i f t id f ll types as possible. However, sometimes the opposite is preferred. The viral receptor can be modified to target the virus to a specific kind of cell.

APPLICATIONS
1. Basic research An alternative to transfection of naked DNA. High transfection rate (nearly 100% of cells). cells) Construction of a viral vector is a much more laborious process. 2. 2 Gene therapy Several gene therapy trials & a huge number of laboratory successes. Immune response to viruses. (a ( mortality case in 1999: an adenoviral vector) li i 1999 d i l ) Insert genomes on host chromosome: possibility of cancer formation. (two cases with leukemia in 2002: SCID retroviral gene therapy) AAV (adeno-associated virus)-based vectors are much safer: they always integrate at the same site in the human genome. 3. Vaccines Viruses expressing pathogen proteins. A kind of DNA vaccines which activate T cells cells. Adenoviruses are being actively developed as vaccines.

RETROVIRAL VECTOR 1. MMLV (moloney murine leukemia virus) y 2. Ability to integrate into the host genome in stable f hi t bl fashion ( (provirus) i ) 3. It has been used in a number of FDA-approved clinical trials (e.g. SCID-X1, insertion near LMO2). 4. 4 Target cells should be actively dividing for transduction (e.g. neuron?). 5. Concern for insertional mutagenesis (insertion at random position).

LENTIVIRAL VECTOR 1. A subclass of retroviruses. 2. Ability to integrate into the genome of non-dividing as well as di idi di idi ll dividing cells. ll 3. Concern for insertional mutagenesis (insertion at random position).

ADENOVIRAL VECTOR 1. Adenoviral DNA does not integrate into the g genome and is not replicated during cell division. 2. Used f 2 U d for gene therapy and vaccine. th d i 3. Naturally, adenoviruses cause respiratory, gastrointestinal and eye infections in human. 4. Pre-existing 4 Pre existing immunity in human ( failure in gene delivery or fatal side effect). 5. No possibility of insertional mutagenesis.

ADENOVIRUS-ASSOCIATED VIRAL (AAV) VECTOR 1. AAV is a defective virus and needs a helper virus (adenovirus) for its replication. 2. AAV is not known to cause disease. 3. AAV causes a very mild immune response. y p 4. AAV can infect both dividing & non-dividing cells. 5. 5 AAV incorporate its genome i t th h t genome. i t it into the host 6. Predictable insertion: insertion at a specific site (AAVS1) in the human 19th chromosome.

COMPARISON OF VIRAL VECTORS

PACKAGING OF VECTOR VIRUS