Direct myocardial transfection in two animal models. Evaluation of parameters affecting gene expression and percutaneous gene delivery.
BACKGROUND
Gene therapy represents a novel approach to the treatment of a variety of disease states. Direct injection of pure untreated DNA into skeletal and cardiac muscle is sufficient to perform gene transfer in vivo. Little information is available, however, regarding the extent to which individual parameters of direct gene delivery affect the efficiency of myocardial transfection. Moreover, the fact that all previously reported studies of successful myocardial transfection were performed using open chest surgery to visualize the heart for direct injection of DNA constitutes a potential limitation to clinical applications of myocardial gene transfer. The objectives of the present study were: (a) to determine the extent to which gene expression is altered by varying the amount of DNA and the volume of the delivery vehicle; (b) to study whether protracted expression of DNA persists after direct myocardial transfection; and (c) to test the feasibility of percutaneous, transthoracic myocardial injection of DNA.


METHODS
New Zealand white rabbits (N = 30) were transfected with the firefly (Photinus pyralis) luciferase reporter gene. Twenty-three rabbits were segregated into 5 groups according to the amount of DNA and volume of delivery vehicle injected. These rabbits were sacrificed at 5 days posttransfection to measure the level of luciferase gene expression. Three more rabbits were designated exclusively for in situ hybridization. Two rabbits were sacrificed at 5 and 6 months, respectively to evaluate long-term expression. Two additional rabbits were used as controls. Yucatan microswine (N = 4) were used to evaluate the feasibility of percutaneous, transthoracic myocardial transfection with beta-galactosidase or luciferase reporter genes. Microswine were sacrificed immediately post-sham transfection (N = 2), or 5 days postmyocardial transfection (N = 2).


RESULTS
Among 11 rabbits, transfection with 10 micrograms of DNA (N = 4), 25 micrograms of DNA (N = 3), and 50 micrograms of DNA (N = 4) in 100 microL of injectate yielded a step-wise but statistically insignificant increase in luciferase activity. Among 16 rabbits, transfection with 50 micrograms of DNA in injectate volumes of 50 microliters (N = 4), 100 microliters (N = 4), 150 microliters (N = 4), and 300 microliters (N = 4) yielded a statistically significant (p < 0.05) increase in luciferase activity in those hearts transfected with 300 microliters compared with those hearts transfected with either 50 or 100 microliters. Luciferase activity at 5 and 6 months postmyocardial transfection was 7.14 and 68.8 Turner light units, respectively. In situ hybridization confirmed that myocytes represented the site of luciferase expression. Percutaneous myocardial transfection was successfully accomplished with both reporter genes.


CONCLUSIONS
The results of the present study demonstrate that increasing the amount of DNA employed for direct myocardial transfection fails to produce a statistically significant increase in the level of gene expression. In contrast, increasing the volume of injectate used to directly transfect a constant amount of DNA (50 micrograms) produced significantly augmented expression of the reporter luciferase gene. Luciferase expression was detected 6 months posttransfection. Successful transfection after fluoroscopic-guided direct percutaneous delivery suggests that it may be feasible to deliver genetic material into the myocardium of patients in a similar fashion.
Gal
D
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Department of Medicine (Cardiology), St. Elizabeth's Hospital, Tufts University School of Medicine, Boston, Massachusetts.
Weir
L
L
Leclerc
G
G
Pickering
J G
JG
Hogan
J
J
Isner
J M
JM
D000818:Animals; D004247:DNA / Q000378:metabolism; D015870:Gene Expression; D017403:In Situ Hybridization; D007267:Injections; D009206:Myocardium / Q000378:metabolism / Q000473:pathology; D011817:Rabbits; D013552:Swine; D013556:Swine, Miniature; D013997:Time Factors; D014162:Transfection / Q000379:methods*
D004247:DNA