Vector systems for prenatal gene therapy: principles of adeno-associated virus vector design and production.
Vectors based on adeno-associated virus (AAV) show great promise for safe, efficacious therapeutic gene transfer in extensive pre-clinical data and, recently, in clinical trials. Careful vector design and choice from a range of natural or synthetic pseudotypes allow targeted, efficient, and sustained expression of therapeutic genes. The efficiency of gene delivery can be further enhanced through the use of drug pre-treatment or co-infection with a suitable helper virus. This chapter describes current best practice for AAV production, including complete methods for: (1) efficient generation of vector without the use of helper viruses, simplifying the transition to GMP-grade production for clinical applications; (2) efficient and easily scalable purification of the virus by affinity chromatography, allowing rapid production of highly concentrated, high titre stocks; (3) reliable quantification and assaying of viral stocks, along with short- and long-term storage considerations.
Department of Haematology, UCL Cancer Institute, London, UK.
Christopher J
CJ
Binny
Amit C
AC
Nathwani
D002130:Calcium Phosphates / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D000229:Dependovirus / Q000235:genetics* / Q000302:isolation & purification; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000096:biosynthesis* / Q000235:genetics*; D016679:Genome, Viral / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D010957:Plasmids / Q000235:genetics; D011094:Polyethyleneimine / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011295:Prenatal Care / Q000379:methods*; D015203:Reproducibility of Results; D012394:Rosaniline Dyes; D013194:Staining and Labeling; D014162:Transfection
D002130:Calcium Phosphates; D012394:Rosaniline Dyes; C048139:Coomassie blue; D011094:Polyethyleneimine; C020243:calcium phosphate