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Chapter: Pharmaceutical Biotechnology: Fundamentals and Applications - Gene Therapy

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General Anatomy and Production of a Gene Transfer Vector

A gene-based medicine typically consists of an expression cassette made of cDNA flanked by apromoter.

General Anatomy and Production of a Gene Transfer Vector

A gene-based medicine typically consists of an expression cassette made of cDNA flanked by apromoter on the 50 side and a transcription stop and polyadenylation site on the 30 side (Fig. 4A). This is incorporated into a DNA plasmid or a recombinant virus, based upon therapeutic requirements (Tables 4 and 5). The genes responsible for the pathogenicity of viral vectors are removed and replaced with the expression cassette in order to limit virus reproduc-tion and fulminant disease. In many vectors, all that remains of the original virus genome are long terminal repeats (LTRs), 5 and 3 terminal regions of the virus that control transcription for RNA viruses or inverted terminal repeats (ITRs), identical but oppo-sitely oriented sequences that drive DNA replication and stabilize the genome of DNA viruses. The packaging signal (y), responsible for virus assembly, is also kept intact. Genes for replication are supplied in trans by a producer/packaging cell line for large-scale production (Fig. 4B). Virus biology will dictate the manner in which the recombinant vector is produced. Careful thought must go into the design of a packaging cell line in order to minimize overlap between sequences in the virus genome and the cell that dictate replication and/or pathogenesis. If sub-stantial overlap exists, these sequences can inadver-tently be incorporated in the recombinant virus by homologous recombination. Replication competent (pathogenic) virus particles will then be produced with replication deficient particles. Preparations are often screened for replication competent virus (RCV) prior to clinical use. After production and harvest from a packaging cell line, recombinant vectors are purified, quantified and/or titered. Traditionally, purification strategies have relied upon density gradient ultracentrifugation to separate the vector from cellular proteins. This process is laborious, difficult to scale up and can reduce the effective titer of stock preparations by disrupting vector structure (Shamlou, 2003; Burova, 2005). Advances in column chromatography have mitigated these issues for many vectors, allowing them to be grown and purified to high concentrations needed for human use.




Given the diversity of diseases suitable for gene therapy, it is apparent that there can be no single vector that is appropriate for all gene transfer applications. Thus, selection of an appropriate vector for gene delivery requires careful consideration of a number of factors including: (a) size limitations for transgene cassettes, (b) transduction efficiency in therapeutic target (ability to infect dividing and/or non-dividing cells, appropriate receptors present on target cells), (c) duration of gene expression required for treatment (long-term vs. transient expression, integrating vs. non-integrating vectors), (d) necessity of temporal gene expression (inducible expression vs. constitutive expression), (e) maximum threshold of vector-induced immune response and toxicity acceptable for the host, (f) purity requirements for the vector and ease of large scale production, (g) route and ease of administration, (h) ability of the vector to protect the genetic material, and (i) robustness and physical stability of the vector system.






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