Preclinical Development Strategies for Novel Gene Therapeutic Products

Systemic circulation of poly(l-lysine)/DNA vectors is influenced by polycation molecular weight and type of DNA: differential circulation in mice and rats and the implications for human gene therapy

“…Preparation of plasmid DNA and 32 P-, 35 S-labeled DNA A 6.4-kb plasmid encoding lac Z under transcriptional regulation of the cytomegalovirus immediate early promoter was used throughout these studies. Plasmid DNA was prepared in bulk by growth in Escherichia coli and purified using Qiagen columns (Qiagen, Crawley, United Kingdom).…”

“…Purity of the DNA was checked by ethidium bromide visualization of agarose gels. 32 P-or 35 S-labeled expression vector was prepared as follows: linearized plasmid was radiolabeled by template extension with 32 P dCTP or 35 S dCTP (Amersham Pharmacia, Little Chalfont, United Kingdom) using the Ready-to-Go Oligolabeling kit (Amersham Pharmacia). Unincorporated nucleotides were removed using microspin columns (Amersham Pharmacia), and the purity of the labeled DNA was checked after agarose gel electrophoresis and quantitative analysis with a PhosphorImager (Molecular Dynamics, Chesham, United Kingdom).…”

Latest Developments in Gene Transfer Technology: Achievements, Perspectives, and Controversies over Therapeutic Applications

“…The vectors that have already been applied in clinical trials are based on retroviruses [68][69][70][71][72], adenovirus [73][74][75][76][77][78], AAV [79][80][81][82][83][84][85], vaccinia virus [86,87], canarypox virus [87], herpes simplex virus (HSV) [88], cationic liposomes [89][90][91][92], polylysine-DNA complexes [93,94], and injection of naked DNA [22,26,27,30]. As anticipated, the pathological conditions with which gene therapy has dealt so far comprise: cancer [2], inherited or acquired monogenic disorders [3,4], AIDS [3], and cardiovascular diseases [19][20][21]. In addition, vectors based on vaccinia virus, canarypox virus, injection of naked DNA and other nonviral vectors have been used in the AIDS vaccination programs in the USA [22][23][24].…”

“…The transfection procedure usually takes between 48 to 72 h to produce the retroviral vector stocks [5]. So far, the retroviral vectors used in clinical trials derive from conventional packaging cell lines, which were previously approved for clinical applications by the U.S. Food and Drug Administration [3,157]. Studies are currently addressing the issue of generating clinical grade retroviral vector stocks by transient transfection systems [158].…”

“…Indeed, gene therapy is one of the fastest growing areas in experimental medicine. As of June 1998, in the USA there were 244 gene therapy clinical trials that were either active or in the evaluation phase by the Recombinant DNA Advisory Committee (RAC) of the National Institutes of Health (NIH) [3]. Most of these trials were either phase I or phase II.…”