Development Of An In Vivo Gene Repair Kit: Towards A Cure For Cystinosis

Patrick T. Harrison
Department of Physiology, University College Cork, Ireland

Cystinosis is caused by a defect in a protein called cystinosin. To understand the role of this protein, imagine the acid-rich organelles inside every cell in the human body known as lysosomes. These organelles, the so-called stomachs of the cell, are responsible for ingesting old proteins and breaking them down into their building blocks, known as amino acids. These amino acids can then be recycled to make fresh proteins, but first the amino acids must be transported back into the main part of the cell called the cytoplasm.

There are many different transporters which move the 20 or so different amino acids back to the cytoplasm. One of them, cystinosin, allows the recycling of the amino acid cystine. In patients with cystinosis, this cystine transporter is either absent of malfunctional. Thus, cystine has no escape route and accumulates as crystals within the lysosome. This reduces the life span of cells and ultimately leads to the kidney and visual problems associated with this disease.

The current therapeutic approach to this disease is to use a drug called Cystagon, which can help breakdown the crystals, and slow onset of symptoms. However, this drug does not cure the disease, and patients receiving Cystagon still require additional medication.

So, are there any alternatives? Given that cystinosis is caused by the lack or malfunction of cystinosin, an obvious approach would be to explore techniques that can restore the function of this transporter protein.

The technology to restore a missing protein, such as cystinosin, is called gene therapy. Genes make proteins using a messenger molecule called RNA. Patients with cystinosis have defects in both copies of the cystinosin gene which means either no messenger RNA is made, or that it contains errors which result in the production of a malfunctional transporter.

For more than 25 years, scientists have been developing methods to deliver a stable version of the messenger RNA molecules, so-called complementary DNA or cDNA, to cells. These cDNA molecules are literally a DNA copy of the messenger RNA, and are, in essence, a shortened version of the gene. The aim of this approach to date has been to add the cDNA to cells in the hope that it will compensate for the gene defect and allow a functional version of the missing or malfunctional protein to be made. If normal protein function is restored in a sufficient number of cells, the hope is that disease symptoms can be slowed, or even reversed.

This approach has significantly increased the understanding of the basis of many genetic disorders, although clinical success has lagged behind, with a positive outcome in just a handful of cases. However, when successful, the outcome has been quite dramatic ranging from successfully treating cancer, partially restoring vision, or repairing defects to the immune system.

Some of the challenges associated with gene therapy using the cDNA addition strategy include difficulties in delivery of the cDNA to cells, the lack of long-term correction, and, in some cases, differences in function; remember the cDNA is only a copy of the messenger RNA molecule, not the whole gene.

What then are the alternatives? In many genetic diseases, including cystinosis, it is unusual for the whole gene to be absent, rather, mutations or small deletions within a gene are more common. Thus, is it possible to repair these defects?

The answer is a qualified “yes”. Gene repair is possible by exploiting a natural phenomenon known as homologous recombination. If a DNA molecule, the so-called donor sequence which contains the correct genetic sequence, is added to cells, it can serve as a template for repair of the related, or homologous, defective gene by a process known as recombination. This phenomenon forms the basis of the 2007 Nobel Prize-winning gene-targeting technology, which has revolutionised our understanding of disease processes. However, this gene-targeting approach works in only approximately one in every million treated cells rendering it of little therapeutic value.

For years, scientists wrestled with ideas to improve this efficiency, but the breakthrough came in 2005 by using the donor sequence in conjunction with a group of enzymes called Zinc Finger Nucleases, or ZFNs. These enzymes were developed a few years earlier to be able to cut DNA at just a single gene with the cell.

It was postulated that these enzymes might increase the efficiency of homologous recombination by breaking the gene sequence at or near the genetic defect; this proved to be the case. Moreover, ZFN-mediated homologous recombination can trigger gene repair in as many as one in five cells, a 200,000-fold improvement in efficiency! Since its initial description, this technology has been shown to be able to correct mutations and even medium-sized deletions in at least four different genes.

So, can this technology be applied to the treatment of cystinosis? My research group in University College Cork began using this technology in 2005 and two of my research students, Ciaran Lee and Rowan Flynn, have created ZFNs that can cut defined gene sequences in an efficient and specific manner.

We now have funding through Cystinosis Ireland and the Health Research Board to develop ZFNs which can target the cystinosin gene. Katrin Kaschig has recently joined the lab to work on this project, and has designed and is now synthesising suitable ZFNs. These will then be used in conjunction with candidate donor DNA sequences which should have the potential to repair not only mutations in the cystinosin gene, but also the 57 kilobase pair deletion which occurs in many patients.

The second part of the project will be to determine if the gene repair system can be delivered to cells using a virus vector. This work, conducted in collaboration with Martina Scallan from the Department of Microbiology, University College Cork, is a crucial step if this technology is to be evaluated in vivo, and ultimately in a clinical situation.

We have a long road ahead – may the road rise up to meet us!

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