Retrovirus in Health - Gene and Drug Delivery
Retroviruses are single stranded RNA viruses that replicates via a double stranded DNA intermediate produced by reverse transcriptase, the DNA then integrates into the host’s chromosome where it can be transcribed. This is the very reason scientists have started to use retroviruses as gene delivery vectors and drug delivery vectors, to help carry drugs or suppressor genes into an infected host cell.
Finding an Appropriate Viral Vector for a Disease
Ø Depends on the disease being treated
Ø Which types of therapy are intended
1) Which cell types or stages can be targeted
2) If and where a gene becomes integrated with the host genome
3) How long a nonintegrated gene actively makes product
Ø Size of gene for transmission packaging
Types of Viruses Used as Vectors
Ø Retroviruses- the most commonly uses is Moloney murine leukemia virus (MMLV)
Ø Adenoviruses-DNA-containing viruses typical of a common cold
Ø Adeno-associated viruses- small DNA viruses that do not cause know diseases in humans
Ø Viruses being studied-recombinant herpes simplex virus, lentiviruses (retrovirus)
Retroviruses
A potential problem with using retroviruses as retroviral vectors would be that retroviruses have high mutation rates. There are also benefits:
Ø Retroviruses are stable
Ø Strong promoters-that have effective expression of genes
Ø Retroviruses have a flexible genome, which is a full length RNA molecule
Ø Efficient gene transfer to different types of cells
Figure 1 The protocol for retroviral vectors. (4)
Problems in Developing Retroviral Vectors
Ø Obtaining efficient delivery
Ø Transduction of nondividing cells
Ø Sustaining long-term gene expression
Ø Cost effective development of the vectors
Ø
Quality control issues caused by mutations
Retroviral Vector Design
Ø Retroviral vectors need efficient gene transduction and integration; this depends on the inclusion of a number of cis-acting viral elements in the retroviral vector. (Figure 2)
Figure 2. Critical cis-acting elements in retroviral vectors and vector replication cycle. (U3) Unique 3
Viral elements include:
1) A promoter and polyadenylation signal in the viral genome
2) A viral packaging signal
3) Reverse transcription signal
4) Short partially inverted repeats located at the terminal end of the viral 5’ long terminal repeat for integration
Ø Introduce retroviral vectors into packaging cells:
· Direct transfection of DNA into the cells
· Infection with virus produced by DNA transfection of other packaging cells.
Figure 3. Strategies for packaging cell line construction. A generic helper virus modeled on Moloney murine leukemia virus (Mo-MLV) is shown at top. Strategies used to generate deleted viruses that provide all proteins required for viral replication but which cannot themselves replicate are shown. Deleted viruses of types A and B involve single DNA constructs, and those of type C involve two constructs. Nonviral transcription signals are shown as open boxes. The bottom construct in each set shows a generic retroviral vector. Potential mechanisms for recombination leading to helper virus production are indicated. Short arrows indicate promoters and direction of transcription.(5)
Moloney Murine Leukemia (MMLV)
Ø The most commonly used retroviral vector is based on MMLV
Ø MMLV can infect mouse cells and humans cells
Ø They only infect rapidly dividing cells, which can be a major impediment in using retroviruses as a vector
Ø A assortment of packaging systems have been developed to enfold the vector genome within the viral particles
Ø The vectors have all the viral genes removed
Lentivirus
Ø
Human Immune Deficiency Virus (HIV), most well-known member
of this type of virus
Ø Studies currently being done to use Lentivirus as a vector
Ø Benefits of using Lentivirus as a vector
1) Stable integration
2) Ability to change nondividing cells
Ø Public view of using a lentivirus such as HIV as vector could cause problems
References
1) M.T. Lotze and T.A. Kost, Viruses as gene delivery vectors: Application to gene function, target validation, and assay development, Cancer Gene Therapy 9 (2002), pp.692-699.
2) Su Su, Atsushi Watanabe, Motoko Yamamoto, Eiitsu Nakajima, Koichi Miyake, and Takashi Shimada, Mutations in p53 cDNA sequence introduced by retroviral vector, Biochemical and Biophysical Research Communications 2 (2002) (340), pp.567-572.
3) Lesney, Mark S., Genetic Transportation, Modern Drug Discovery 3 (Oct 2000) (8), pp.55-60.
4) Anderson, W. French, Human Gene Therapy, Nature Supplement (Apr 1998) (392), pp.25-30.
5) Coffin, John M., Hughes, Stephen H., Varmus, Harold E., Retroviruses, The National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/books/bv.fcgi?call=bv.View..ShowSection&rid=rv.chapter.4356