Hepatic Gene Therapy Technique Developed

Researchers have developed a technique for selective expansion of genetically modified hepatocytes, resulting in expression of high levels of transgenes in livers of mice.

The tyrosine catabolic pathway. Genetic deficiency of Fah causes hereditary tyrosinemia type 1 due to accumulation of FAA in hepatocytes. The disease can be treated pharmacologically or by shRNA knockdown of the genes required for making FAA. CEHPOBA (4-[(2-carboxyethyl)-hydroxyphosphinyl]-3-oxobutyrate) inhibits FAH and causes accumulation of FAA. TAT, tyrosine aminotransferase; MAI, maleylacetoacetate isomerase.

The tyrosine catabolic pathway. Genetic deficiency of Fah causes hereditary tyrosinemia type 1 due to accumulation of FAA in hepatocytes. The disease can be treated pharmacologically or by shRNA knockdown of the genes required for making FAA. CEHPOBA inhibits FAH and causes accumulation of FAA. TAT, tyrosine aminotransferase; MAI, maleylacetoacetate isomerase.

Sean Nygaard et al (Oregon Health and Science University) describe their system for in vivo selection and expansion of genetically modified hepatocytes in the June 8 issue of Science Translational Medicine.

Science Daily wrote that the new technique could help overcome one of the largest hurdles in gene therapy—the ability to generate a large pool of gene-corrected cells that repair injury and repopulate in vivo.

Nygaard et al created an adeno-associated viral vector that integrates at the albumin gene locus and contains a transgene encoding human coagulation factor IX. Coagulation factor IX is one of the serine proteases of the coagulation system (it belongs to peptidase family S1). Deficiency of this protein causes hemophilia B.

The vector also contained a short hairpin RNA (shRNA) that allows hepatocytes to become resistant to the selective, toxic agent (see figure). This shRNA targets the tyrosine catabolic enzyme 4-OH-phenylpyruvate dioxygenase and protects the hepatocytes from 4-[(2-carboxyethyl)-hydroxyphosphinyl]-3-oxobutyrate (CEHPOBA)—a small-molecule inhibitor of fumarylacetoacetate hydrolase (FAH).

Previous studies have shown that transplanted hepatocytes can be selected with CEHPOBA in mice if they carry a genetic mutation in a tyrosine catabolic enzyme.

After selection of the modified hepatocytes, transgene expression increased 10- to 1000-fold in livers of mice (to 50,000 ng/ml). The authors state that this CEHPOBA-resistance system can be used to express transgenes at therapeutically relevant levels in hepatocytes, in any setting. They say that the approach could also be used in bone marrow, intestine, skin, or kidney tissues.

The Scientist explained that scientists have attempted to use recombinant adeno-associated viral vectors to treat liver disorders such as hemophilia with some success. However, low doses of viral vectors do not usually lead to efficient transduction, and high doses can produce an immune response and possibly activate oncogenes.

Previous studies have shown that gene-corrected hepatocytes have a selective advantage in rodent models of Wilson’s disease and progressive familial intrahepatic cholestasis.

The Scientist wrote that genetic and acquired liver disorders are amenable to gene and/or cell therapy. However, the efficiencies of cell engraftment and stable genetic modification are low and often sub-therapeutic. In particular, targeted gene modifications from homologous recombination are rare events. These obstacles can be overcome if hepatocytes that have undergone genetic modification are selectively amplified or expanded, as in the system of Nygaard et al.

“The new method paves the way to therapies for many metabolic diseases,” said study coauthor Adi Barzel (Stanford University) in a press briefing.

“The risk from this approach is acute side effects of using a drug to impair the ability of nonmodified cells to divide,” admitted the study’s senior author, Markus Grompe (Oregon Health and Science University) in the press briefing. “However, we know that because the liver is so regenerative, any such intervention is likely to be completely reversible upon withdrawal of the agent”.

To date, efforts to modify and deliver genetically modified cells to treat various disorders have required the delivery of thousands of cells, many of which don’t survive. This has limited the application of gene therapy, along with other concerns in the field, like the safety of the delivery vector.

This universal method of selection could be used to support cell-based treatments for neonatal metabolic liver diseases and genetic disorders, such as hemophilia B, say the authors. The technique could also be used to expand a therapeutic population of gene-corrected cells in many tissues that proliferate after injury, such as cells found in bone marrow, skin, or the intestine.

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