Gene delivery tool shows promise

K. S. Jayaraman

doi:10.1038/nindia.2010.106 Published online 6 August 2010

Scientists Prashant Mani (left) and Debi Sarkar.

Twelve years ago a team of biochemists led by Debi Sarkar at the Delhi University South Campus reported the potential use of 'Sendai virus' for gene therapy. They had then engineered the envelope of this harmless Japanese virus as a vehicle for targeted delivery of drugs or designer genes to the liver — a technique they hoped could some day be used for treating diseases such as hepatitis, jaundice, cancer and hemophilia.

That day does not seem far off. Now, along with Jayanta Roy-Chowdhury and his team at the Albert Einstein College of Medicine in New York (supported by a grant from the National Research Development Corporation, NRDC, New Delhi), they have successfully used this method to deliver the relevant gene to liver cells for treating jaundice in rats. Not only was the disease cured, the transgene got integrated into the genome of the liver cells.

"To our knowledge, this is the first report of long-term improvement of a liver metabolic disease through intravenous use of a plasmid entrapped in liposomes," the scientists have reported. Liposomes are tiny bubbles made out of the same material as a cell membrane and can be filled with drugs or relevant genes. Their application published by the US patents office describes the technique as "a process for producing modified reconstituted Sendai viral envelope specific for drug and/or gene delivery to liver cells." Some pharma companies are already showing interest in taking this development forward.

Sendai virus belongs to the family of paramyxoviruses. Its viral genome encodes two transmembrane glycoproteins — hemagglutinin-neuraminidase (HN) and fusion protein (F). While HN helps the virus to attach to its target cell membrane, F is essential for the fusion of the viral envelope with the host cell membrane at neutral pH, leading to internalization of the viral contents. However, this promising system lacks cell-type specificity because the HN protein binds to various cell types.

Sarkar's team overcame this problem by demonstrating that F protein (devoid of HN) can specifically bind and fuse with liver parenchymal cells. This enabled them to create an ideal vehicle for liver targeted delivery — aptly named FPL (F protein containing liposomes).

In the latest study the researchers used their delivery technique for treating Gunn rats suffering from an inherited liver disease marked by high levels of serum bilirubin due to lack of a gene called UGT1A1. The Indo-US team generated FPLs loaded with UGT1A1 gene and injected these 'biological missiles' into the rats hoping to correct gene deficiency in the rats.

Once the F-protein in the FPL binds to the liver cells, the contents of the liposome are deposited directly into the cytosol (the liquid inside cells), explained Prashant Mani who worked with Roy Chowdhury for nine months at the Einstein lab and played a crucial role in the experiment on Gunn rats. This method bypasses the classical endocytotic pathway where the delivered material gets largely degraded by enzymes present in lysosomes.

The researchers showed that the FPL carrying the therapeutic gene resulted in 'liver-specific delivery and expression of the transgene'. As proof they found that after the injection, the serum bilirubin levels in the rats declined by 30% in 2 weeks and remained at that level throughout the 7-month study duration. The scientists confirmed that the transgenes were delivered and expressed specifically in the liver, by performing both short-term and long-term studies.

Three days after FPL-mediated administration of a luciferase-expressing plasmid, the luciferase or light generating activity was detectable in the liver, but not in the other tissues tested. Similarly, in long-term experiments, 28 weeks after gene transfer, the transgene was detectable by polymerase chain reaction only in the liver. Therefore, according to the authors, "FPL is superior to existing methods of liver targeted gene delivery through injection."

Sarkar says success of the strategy hinged on two components of the gene transfer system: the FPL which permitted gene delivery exclusively to liver cells, and the plasmid construct, which was engineered in such a way that it promoted integration of the human UGT1A1 into the genome of the liver cells. "As inherited metabolic disorders are lifelong conditions, gene therapy for these conditions may be facilitated by integration of the transgene into the host genome, so that the gene would be transmitted to the progeny," the scientists reported.

Another advantage of FPL-based gene delivery is it obviates the use of recombinant viral vectors and does not result in an immune response and detectable toxic effect, Sarkar said.

"Thus our combined strategy of FPL-based hepatocytes targeted delivery of transposition competent DNA has the potential to be developed for gene therapy for inherited liver-based metabolic disorders and warrants further exploration toward clinical application," the authors said.


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