Viral Vectors Part I

Viral Vectors 416521 Medical DNA Technology Graduate Course, First Semester, 2009

Assist. Prof. Somchai Saengamnatdej, Ph.D. Thursday 25th June 2009

Outline (4 periods) 1.

2.

3. 4.

Introduction and Concepts 

Definition & related terms

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Non-viral/ Viral vectors

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Systemic/ Local gene deliveries

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Gene delivery barriers

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Appropriate vectors: Factors to be considered

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The potential use of viral vectors

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Rationale for some experiments

Types of viral vectors 

Viruses : Review of features & their replication cycle

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Viral vectors ; structure, experiments, advantages, and limitations

Summary Perspectives

CONCEPTS

Vector Definition  Vehicle

for delivery of genetic material to host cells Replication-Deficient DNA

Viruses

Plasmids.

Other

Non-viral vectors Virosome

Gene Delivery 1.        

Systemic gene delivery. Via bloodstream. Distribute throughout the body. For target tissue that cannot be reached directly. Often low specificity of gene expression. Require large concentration. May influence & damage the function of normal healthy tissues. High titre could carry immunologic or toxicity concerns. Inappropriate for many musculoskeletal system injuries due to lack of blood supply to various tissues (such as cartilage or meniscus).

Gene delivery 2. Local gene delivery.  In vivo: direct injection of vector/ DNA complex into the host tissue. • Ex vivo: Growing cells in culture delivery gene (alone or with growth factors, differentiation signals) into the cells or with other chemical or physical alterations to the culture environment Transplant into patients.

EXAMPLE: Ex vivo TKD/IL-2 activated, patient-derived autologous NK cells. (Note: Hsp70-peptide TKD, a 14-mer immunogenic peptide derived from the C-terminal domain of Hsp70 protein, in combination with IL-2 or IL-15.)

(http://www.onkologie-regensburg.de/Forschung/Englisch/Multhoff/Immunotherapy_neu.htm 13 Nov2006)

Terms In vivo Ex vivo Autologous Xenogeneic Allogeneic Syngeneic

Gene delivery barriers 1. systemic (pre-existing immunity) 2. cellular (binding to cell surface)

Factors: to select appropriate vectors.  2.

Duration of expression required (transient or long term.) Nature of target cells   

   

ease of transduction receptor expression dividing (cancer) or non-dividing (neuron, islets, muscle cell).

Route (Ex vivo or In vivo). Maximum threshold of vector-induced immune response acceptable for the host. Level of expression. Desired temporal regulation of transgene expression 

Inducible or Constitutive.

[One vector unlikely appropriate for all]

Non-viral vectors Delivery:  Direct injection: simple

& safe (dividing & non-dividing cells).

 Electroporation.  Encapsulated

within liposomes.  Gene-activated matrices.  Natural/ Synthetic polymers.  Attached to gold microparticles for gene gun.

Non-viral vectors  Limitation: Low transfection

efficiency. Transient expression. Non-selective cell targeting.  Advantages: Easy

to manufacture. Able to incorporate large sequences of DNA. Cost-effective. Display low immunogenicity in vivo.

Viral Vectors  Engineering the virus to express the gene of interest. Advantages:  Prolonged transgene expression.  Markedly higher transduction efficiency (more efficient than physical methods).

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Treatment

Inherited congenital disorders :     

Cystic fibrosis Phenylketonuria Adenosine deaminase deficiency Haemophilia B. Inherited retinal degenerations 

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Acquired diseases: 

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Mutations in genes expressed in photoreceptors or the retinal pigment epithelium

AIDs, Cancer, Cardiopathy.

Tissue engineering: 

Trauma, burns, degenerative diseases, and diseases that produce tissue or organ failure.

Rationale for Some Experiments (1) 

To deliver growth factors that control the formation of new vasculature to ischemic tissues in order to restore the blood supply that has been pathologically disrupted or reduced [therapeutic angiogenesis].

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Bone fractures: [ bone healing].  

non-healing or delayed union defects caused by    

age-related bone deterioration traumatic injury tumour resection, or osteolytic disease

 ~10%

Require some forms of orthopaedic graft

Rationale for Some Experiments (2) 3. Use of tumour-selective replicating viral vector may enable the targeting of the gene-modified viruses to malignant tissue without toxic effect (happened with standard approaches) [anti-cancer]. 4. To introduce genes designed to specifically block or inhibit the gene expression or function of gene products [intracellular anti-microbial]. 5. To inhibit the spreading by expression of a secreted inhibitory protein or stimulation of a specific immune response [extracellular antimicrobial]