Long-term therapeutic levels of human alpha-1 antitrypsin in plasma after hydrodynamic injection of nonviral DNA

Abstract

he transfection efficacy of several vectors containing the full genomic hAAT gene with its natural promoter (pTG7101) and others containing the cDNA of hAAT gene driven by cytomegalovirus immediate-early promoter or the 0.5 kb upstream of hAAT gene sequence has been studied by hydrodynamic tail-vein injection (20 mg/mouse). pTG7101 (but not the other plasmids) results in therapeutic and stable concentration of hAAT in plasma. A dose-response study with this plasmid (0.3-320 mg/mouse) confirms that hAAT remains long-term stable in plasma, with therapeutic concentrations of hAAT (40.9 mg/ml). The parameters of the dose-response curve were: R: 0.98, E max 3449.07 279.7 mg/ml and EC 50 1.2 Â 10 12 plasmid-gene units. In addition, 4 months after transfection, the intrinsic efficacy of transgenic expression (amount of RNA/DNA) in mouse liver was 50-80% that normally expressed by the mouse gene. The important efficacy of nonviral genomic DNA opens a new avenue in the safety applications of human gene therapy.

Hydrodynamic liver gene transfer mechanism involves transient sinusoidal blood stasis and massive hepatocyte endocytic vesicles

“…The results obtained indicate that: (a) therapeutic plasma levels of hAAT can be achieved with good mouse tolerance employing cumulative dose; (b) hydrodynamic injection mediates the inversion of vein pressure between portal and central vein, supporting the possibility of retrodynamic blood flow; (c) the intravital observations show that hydrodynamic injection mediates transient intrahepatic flow inversion, with blood stasis lasting a few minutes, mainly in the sinusoids around the central veins; and (d) transmission electron microscopy shows hydrodynamic injection to promote massive fluid endocytosis in hepatocytes distributed around the central vein but not around the periportal vein. Recently, we have described 13 that increased plasma levels of hAAT can be observed in a dose-dependent response by hydrodynamic injection of single doses of pTG7101 plasmid, employing independent animal groups per dose. Since the procedure can mediate liver Figure 4 Intravital microscopy of liver blood flow.…”

“…9 However, hydrodynamic transfection has become widely used 10 because of its simplicity and high efficiency. By employing this procedure, high efficiencies of human alpha-1 antitrypsin gene transfer have been described, 11,12 and recently we have been able to demonstrate 13 that long-term therapeutic levels of human alpha-1-antitrypsin in mouse plasma can be achieved by this procedure.…”

Gene Therapy Progress and Prospects: Episomally maintained self-replicating systems

“…This plasmid mediated long-term (44 months) therapeutic levels of hAAT protein in mouse plasma, whereas cDNA constructs exhibited different biphasic kinetics, with the hAAT protein levels dropping approximately two orders of magnitude from day 1 to day 30. 17 The positive features of EBV components (episomal replication and retention) and of genomic sequences (tissue specificity, proper regulation and prevention of silencing of gene expression) were combined in fruitful constructs that provided therapeutic levels of proteins, such as AAT and factor IX (FIX), in mice. 16 Recently, by using a construct containing an hFIX minigene (comprised of apoE gene hepatic locus control region, the hepatocyte-specific hAAT promoter, part of the FIX intron A and the 3 0 -untranslated region) (Figure 1b).…”

Hydroporation as the mechanism of hydrodynamic delivery

“…1,2 This method, called the hydrodynamicsbased procedure, has been widely utilized by the gene therapy community for evaluating therapeutic activities of various genes (see, for reviews, Liu and Knapp 3 and Herweijer and Wolff 4 ). Other reported applications of this technique include studies to define the regulatory functions of DNA sequences, [5][6][7][8][9] investigations to evaluate gene suppression activity of siRNA, [10][11][12] and experiments to establish animal models for viral infection. 13,14 Despite many desirable features of the hydrodynamics-based procedure such as simplicity, convenience, and high efficiency, further improvement and new applications of this procedure require a full understanding of the mechanisms underlying this technique.…”