Studying Closed Hydrodynamic Models of "In Vivo" DNA Perfusion in Pig Liver for Gene Therapy Translation to Humans.
BACKGROUND
Expressing exogenous genes after naked DNA delivery into hepatocytes might achieve sustained and high expression of human proteins. Tail vein DNA injection is an efficient procedure for gene transfer in murine liver. Hydrodynamic procedures in large animals require organ targeting, and improve with liver vascular exclusion. In the present study, two closed liver hydrofection models employing the human alpha-1-antitrypsin (hAAT) gene are compared to reference standards in order to evaluate their potential clinical interest.


METHODS
A solution of naked DNA bearing the hAAT gene was retrogradely injected in 7 pig livers using two different closed perfusion procedures: an endovascular catheterization-mediated procedure (n = 3) with infrahepatic inferior vena cava and portal vein blockage; and a surgery-mediated procedure (n = 4) with completely sealed liver. Gene transfer was performed through the suprahepatic inferior cava vein in the endovascular procedure and through the infrahepatic inferior vena cava in the surgical procedure. The efficiency of the procedures was evaluated 14 days after hydrofection by quantifying the hAAT protein copies per cell in tissue and in plasma. For comparison, samples from mice (n = 7) successfully hydrofected with hAAT and healthy human liver segments (n = 4) were evaluated.


RESULTS
Gene decoding occurs efficiently using both procedures, with liver vascular arrest improving its efficiency. The surgically closed procedure (sealed organ) reached higher tissue protein levels (4x10^5- copies/cell) than the endovascular procedure, though the levels were lower than in human liver (5x10^6- copies/cell) and hydrofected mouse liver (10^6- copies/cell). However, protein levels in plasma were lower (p<0.001) than the reference standards in all cases.


CONCLUSIONS
Hydrofection of hAAT DNA to "in vivo" isolated pig liver mediates highly efficient gene delivery and protein expression in tissue. Both endovascular and surgically closed models mediate high tissue protein expression. Impairment of protein secretion to plasma is observed and might be species-related. This study reinforces the potential application of closed liver hydrofection for therapeutic purposes, provided protein secretion improves.
Departamento de Farmacología, Facultad de Medicina, Universidad de Valencia, Valencia, Spain.
Luis
L
Sendra
Departamento de Farmacología, Facultad de Medicina, Universidad de Valencia, Valencia, Spain.
Antonio
A
Miguel
Servicio de Radiología y Grupo de Investigación Biomédica en Imagen GIBI239, IIS La Fe y Hospital Universitario y Politécnico La Fe, Valencia, Spain.
Daniel
D
Pérez-Enguix
Departamento de Farmacología, Facultad de Medicina, Universidad de Valencia, Valencia, Spain.|Unidad de Farmacogenética, IIS La Fe y Área Clínica del Medicamento, Hospital Universitario y Politécnico La Fe, Valencia, Spain.
María José
http://orcid.org/0000-0002-6042-4185
MJ
Herrero
Unidad de Cirugía Hepatobiliopancreática y Trasplante, Hospital Universitario y Politécnico La Fe, Valencia, Spain.
Eva
E
Montalvá
CIBER e Instituto de Biomedicina de Valencia, CSIC, Valencia, Spain.
María Adelaida
MA
García-Gimeno
Servicio Central de Soporte a la Investigación Experimental (SCSIE), Universidad de Valencia, Valencia, Spain.
Inmaculada
I
Noguera
Servicio Central de Soporte a la Investigación Experimental (SCSIE), Universidad de Valencia, Valencia, Spain.
Ana
A
Díaz
Servicio de Anatomía Patológica, Hospital Universitario y Politécnico La Fe, Valencia, Spain.
Judith
J
Pérez
CIBER e Instituto de Biomedicina de Valencia, CSIC, Valencia, Spain.
Pascual
P
Sanz
Unidad de Cirugía Hepatobiliopancreática y Trasplante, Hospital Universitario y Politécnico La Fe, Valencia, Spain.
Rafael
R
López-Andújar
Servicio de Radiología y Grupo de Investigación Biomédica en Imagen GIBI239, IIS La Fe y Hospital Universitario y Politécnico La Fe, Valencia, Spain.
Luis
L
Martí-Bonmatí
Departamento de Farmacología, Facultad de Medicina, Universidad de Valencia, Valencia, Spain.|Unidad de Farmacogenética, IIS La Fe y Área Clínica del Medicamento, Hospital Universitario y Politécnico La Fe, Valencia, Spain.|Unidad de Farmacología Clínica, Área Clínica del Medicamento, Hospital Universitario y Politécnico La Fe, Valencia, Spain.
Salvador F
SF
Aliño
D000818:Animals; D002404:Catheterization; D004247:DNA / Q000008:administration & dosage*; D005260:Female; D015870:Gene Expression; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D006046:Gold; D006801:Humans; D057446:Hydrodynamics; D008099:Liver / Q000378:metabolism*; D008297:Male; D053768:Metal Nanoparticles; D051379:Mice; D009928:Organ Specificity; D010477:Perfusion / Q000379:methods*; D010957:Plasmids / Q000008:administration & dosage / Q000235:genetics; D013552:Swine; D019076:Transgenes; D057170:Translational Research, Biomedical / Q000379:methods; D000515:alpha 1-Antitrypsin / Q000097:blood / Q000235:genetics / Q000378:metabolism
D000515:alpha 1-Antitrypsin; D006046:Gold; D004247:DNA
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