The concept for target validation: Hitting the Achilles heel

In this week, we already introduced several high throughput approaches to identify disease-associated genes but the next question

is : “Are these genes real target for the disease?” This means whether the gene mutations or changed gene expression influence the disease progression.

You may hear about the Greek legend Trojans. The warrior Achilles was one of the great heroes of Greek mythology. He was extra-ordinary strong, courageous, loyal, and invulnerable everywhere because he was deep into the magic river. Wherever the magic river bathes the infant Achilles, the part becomes immune to human attacks. However, his heel was not covered by the water so he was killed by the fatal weakness. Therefore, target validation is trying to find Achilles heel for a disease.

Here we will introduce several common approaches for target validation. The first one is to introduce and overexpress the mutant genes into the normal cells or healthy animals. This method will interfere the normal gene function which called the dominant negative mutations. Using these methods, you can know the gene mutations is gain-of-functions or loss-of-functions or just passenger mutations. If you identify several gene mutations associated with the disease, you can clone the mutant gene from the patients and test whether it affects cell functions and disease progression. Second is RNA interference. This is a biotech that uses small piece of double-strand RNA suppress the gene expression.

If you find a gene abnormally up-regulated, in the disease, you can knockdown the gene expression by RNAi and examine the gene effect. The 3rd one is to use the antibody. The antibody can specifically block or neutralize the functions of membrane proteins or secreted proteins.

There are two levels in target validation, in vitro and in vivo. The in vitro means we can do the target validation in cell culture or test tubes. In vivo means we can test the function of genes in animals such as mice, rabbits, pigs, and so on. Usually, we will test our hypothesis in vitro and if we succeed we will continue the next level.

Lets look at how to perform the methods. The first is dominant-negative mutations.

You may find some gene mutations are associated with the disease. To determine whether the effect of mutant genes on the disease you can clone the mutant gene from the patients. You can isolate the mutant gene and insert it into a circular DNA called plasmids. Plasmids are small circular DNA that replicates separately from the bacterial chromosome and genetically engineered for gene cloning. After the mutant gene is inserted into the plasmid, the recombinant plasmid is then returned to the bacteria such as E.coli. And producing a recombinant bacteria cause high replication rate of E.coli we can get a clone or a population of E.coli and its plasmid DNA in a short time.

And then you can extract the mutant gene from the E.coli and send it to the normal cells to determine its functions. Or the proteins of the mutant genes can be purified to examine whether it have different structure or different function compared to the protein of normal gene.

The second is RNAi, RNA interference. RNAi is using a small piece of double-strand RNA usually 20 to 22 nucleotide that targets on pecific messenger RNA. These RNA we call it small interference RNA, siRNA. The siRNA can be sent into cells. After it going into cells, one strand will dissociate from the double strand RNA and will be recruited by a protein complex. The siRNA protein complex will reach, will search the target messenger RNA. If the siRNA sequence is completely matched with the messenger RNA, the RNA will degrade. But if the siRNA sequence is not completely matched with the messenger RNA, the complex will disrupt the gene translation.

The third one is to generate monoclonal antibody to neutralize or block the gene functions. Because the antibody-antigen interaction is a high affinity and specific binding.

Because the big size of antibody, it cannot penetrate cell membrane. So its main targets are membrane proteins or proteins in the extracellular space. You can design antibody target on membrane receptors which prevent the ligand binding or you can also design the antibody that inhibit the binding of ligand with the receptors. Both ways can inhibit the ligand effect on cell functions through prevention the receptor activation and the downstream signal transduction. The result of target validation provide you the

first information: go or no go for drug development. The information is very important for pharmaceutical company to decide whether the target deserves for the drug development. Because it cost billions of dollars to develop a drug. If you choose the wrong target all of the effort will be a waste of time and money.