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Chapter: Pharmaceutical Biotechnology: Fundamentals and Applications - Gene Therapy

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General Considerations - Vectors For Gene Transfer

Preparations for gene delivery are colloidal suspen-sions consisting of complex molecules with average hydrodynamic radii ranging from 40 to 1,000 nm.

VECTORS FOR GENE TRANSFER

General Considerations

 

Preparations for gene delivery are colloidal suspen-sions consisting of complex molecules with average hydrodynamic radii ranging from 40 to 1,000 nm. Particles of this size are readily taken up by organs of the reticuloendothelial system (RES) such as the liver, spleen, bone marrow and adrenal glands and effi-ciently cleared from the circulation. They are also highly susceptible to opsonization, where they are coated with serum proteins such as complement and taken up by macrophages in the liver and spleen within minutes after intravenous administration (Dash, 1999; Read, 2005). Conjugation of vectors with biodegradable polymers has been shown to success-fully prevent interaction with the RES and serum components (Oupicky, 2002; Demeneix, 2004; Kommareddy, 2005), however, this effect is often avoided by instillation of the vector directly into the target organ or tissue.

Even though a vector is administered by direct injection, it still must overcome certain epithelial barriers designed to restrict entry of macromolecules into the target tissue/organ. Cells lining the organ surface are often covered with a thick mucous layer that physically prevents contact with cellular targets and contains enzymes capable of rapidly degrading the vector. Cells under this layer are often ciliated and form tight junctions, which restrict entry. Many vectors carry a negative charge on their surface, making interaction with the cell, also negatively charged, difficult. Use of cationic compounds and mucolytic reagents to facilitate cellular interactions and hypotonic formulations to disrupt tight junctions and enhance cellular penetration has successfully enhanced gene transfer (Weiss, 2002; Tiera, 2006).

Once a vector enters a cell, it must next escape the endosomal/lysosomal environment. Some vectors have been administered with compounds that swell within the acidic endosomal environment and protect the genetic material from exposure to significant changes in pH and enzymatic digestion and promote vector release (Alexander, 2006). Others have used inactivated virus as well as specific components from bacteria, viruses and toxins that promote endosomal escape (Wagner, 2005). Once in the cytoplasm, the vector must successfully reach the nucleus where it can use the cells endogenous transcriptional machinery to express the therapeutic transgene. To accomplish this, non-viral and viral gene transfer vectors are used.

 

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