Yonsei Med J.  2010 Jan;51(1):52-57. 10.3349/ymj.2010.51.1.52.

Delivery of Factor VIII Gene into Skeletal Muscle Cells Using Lentiviral Vector

• Jeon HJ
• Oh TK
• Kim OH
• Kim ST

• KMID: 1779606
• DOI: http://doi.org/10.3349/ymj.2010.51.1.52

Abstract

PURPOSE
This study was designed to investigate whether transduction of lentiviral vectors (LV) carrying human coagulation factor VIII (hFVIII) cDNA into skeletal muscle could increase circulating hFVIII concentrations. MATERIALS AND METHODS: A LV containing bacterial LacZ gene as a control or human FVIII gene was intramuscularly administered into the thigh muscle of 5 weeks old Sparague-Dawley rats. The plasma human FVIII concentration and neutralizing anti-FVIII antibodies were measured for up to 12 weeks in these experimental animals. RESULTS: The plasma human FVIII levels in the rats injected with LV carrying FVIII cDNA peaked at post-injection 1st week (5.19 +/- 0.14 ng/mL vs. 0.21 +/- 0.05 ng/mL in control rats , p < 0.05). Elevated hFVIII concentrations were maintained for 4 weeks (2.52 +/- 0.83 ng/mL vs. 0.17 +/- 0.08 ng/mL in control rats, p < 0.05) after a single intramuscular injection. In the Bethesda assay, neutralizing antibodies for FVIII protein were detected only in FVIII-LV injected rats by the 10th week, but not in control rats. CONCLUSION: This study suggested that a single administration of an advanced generation LV carrying the human FVIII cDNA resulted in elevation of FVIII level in immune competent rats, and that this gene transfer approach to the skeletal muscle could be an effective tool in treatment of hemophilia A.

Keyword

Gene therapy; human coagulation factor VIII; hemophilia A; lentiviral vector

MeSH Terms

Animals
Antibodies/blood/immunology
Factor VIII/genetics/immunology/*metabolism
Gene Therapy
Genetic Vectors/genetics
Hela Cells
Hemophilia A/therapy
Humans
Lentivirus/genetics
Male
Muscle, Skeletal/*metabolism
Rats
Rats, Sprague-Dawley
Transduction, Genetic
beta-Galactosidase

Figure

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  Fig. 1 In vitro production of human coagulation factor VIII in HeLa cells transduced with lentiviral vectors (LV). Hela cells (5×105 cells) were transduced with increasing dose of lentiviral vector (ng p24 Gag antigen, which is an index of titer) carrying the human B-domain depleted coagulation factor VIII cDNA. The media were changed 24 hours after initial transduction, and the cells were incubated for an additional 48 hours prior to harvesting the media for the assessment of factor VIII concentration by enzyme-linked immunosorbent assay (ELISA) (n = 2, independent experiments).

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  Fig. 2 X-gal staining of tissue harvested from lentiviral vector-injected rats. Four weeks after intramuscular injection of lentivirus containing lacZ gene (1.5×107 infectious units), the following organs were harvested; the lentiviral vector-injected skeletal muscle (A), liver (B), lung (C) and spleen (D). The tissues were sectioned (10 µm) and stained with X-gal to examine the expression of lacZ in vivo, following lentiviral vector transduction. (A) Shows definitive X-gal stained myocytes (×400), (B-D) but no other X-gal-positive cells are detected in the other three organs (×400).

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  Fig. 3 Temporal expression of plasma human factor VIII levels following lentiviral vector administration. Plasma factor VIII levels isolated from rats which were injected with lentiviral vector expressing human factor VIII (●, n = 4) or bacterial lacZ (control) rats (□, n = 4) over the course of the experiment. Data are expressed as mean±SD. *p< 0.01.

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  Fig. 4 Bethesda assay for anti-human factor VIII antibody. Analysis for antibody against human factor VIII showed that neutralizing antibodies developed in rats injected with pLV-factor VIII after 10 weeks, but not in control rats.

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