FAST BREAKING PAPERS - 2008

Why do you think your paper is highly cited?

Our study has reported novel findings and highlights ingenious ways to improve the delivery of small therapeutic molecules using cell-penetrating peptides.

Does it describe a new discovery, methodology or synthesis of knowledge?

We have systematically described our findings and the possible repercussions of the previous studies which rely heavily on pleiotropic effects of protein transduction domains (PTD) or Tat protein (the transactivator protein from lentiviruses, notably HIV) which may not necessarily be true. We have described here new ways which may help in the development of newly potent gene, protein, and drug-delivery vehicles for effective biodistribution.

Would you summarize the significance of your paper in laymen's terms?

We have elucidated several new properties of cell penetrating peptides (PTD) as a protein delivery and gene delivery vehicle which may have the potential for the development of novel therapeutics. We have described a stepwise strategy which will help in producing recombinant proteins for successful biodistribution.

In this manuscript, we have elucidated the possible mechanism of fusion protein entry into cells and resistance encountered by fusion proteins after entry. We have described ways to overcome the intracellular resistance and hence enable effective delivery and biodistribution. We have further described a way to predict the nature of fusion protein after fusion with PTD sequence and its ultimate fate (distribution) inside the cell.

We have highlighted shortcomings of this peptide (PTD) in general as a weak delivery vehicle, owing to its inefficient entry on fusion with different proteins. We infer that this peptide will be useful in delivering proteins, RNA, and DNA sequences; however, not in the native form. PTD's potential can profoundly be enhanced by fine-tuning of peptide sequence using available knowledge and cutting-edge technology.

How did you become involved in this research, and were there any problems along the way?

I started doing research on HIV-Tat protein and PTD in 1996 while working as a visiting scientist in the Departments of Neuroscience and Microbiology and Tumor Biology, at the Karolinska Institute in Sweden, and then I continued here in the United States at Johns Hopkins University in Baltimore. I am a virologist trained in RNA viruses and my research for the last 12 years has focused on HIV.

In the HIV life cycle, early Tat-protein has been described as pleiotropic in function apart from its primary transactivation function in HIV replication. I was working on HIV gene therapy to inhibit Tat protein and cripple the HIV life cycle. Tat protein is a mastermind of the HIV life cycle and its inhibition will curtail HIV-multiplication.

Tat protein has a unique property of entering other cells when added externally. This property of Tat is localized to a short stretch of sequence (PTD) in the middle of the protein. Since PTD is responsible for entry into the cell, I was specifically interested in delivering fusion-proteins into neurons. After complex designing and cumbersome experimentation with repeated failures, I finally succeeded in introducing fusion proteins into neurons and other cells. However, protein entry was not as impressive as was anticipated from earlier studies.

On further modifications in design and experiments, we came to the conclusion which was published in the Journal of Controlled Release in 2007. In science, when you are looking for something, you normally do not find precisely what you are looking for, instead you often discover something else. It is difficult to publish new concepts because people normally do not accept new ideas quickly. We encountered stiff resistance to publishing our findings; however, some people found our findings of interest and recommended that they be published.

Where do you see your research leading in the future?

We are headed towards the use of PTD or small cell-penetrating peptides and their permutations in delivering small therapeutic molecules such as siRNA and antisense. Small interfering RNA (siRNAs) are small synthetic inhibitors (gene silencers) designed to target specific viral or other genes, in our case HIV. We are at the final stage of concluding our investigations on the novel applicability of peptide-siRNA in HIV-inhibition. We believe that for safe drug and gene therapy in the future, non-viral delivery methods will be the ultimate choice and will be helpful in site-specific delivery of RNA and DNA sequences to correct disease states.

However, this kind of research has not yet picked up pace with other delivery systems. Indeed, we are fine-tuning these peptide sequences to give them target specificity and to have more potent and specific effects. Target-specific delivery will be our ultimate goal.

Do you foresee any social or political implications for your research?

Our aim is to look for an aggressive way to curtail HIV for the long term and for this we plan to impede the HIV life cycle at different steps, using our peptide in different forms. HIV is a very complex virus which affects millions of people around the globe. The disease is possible to treat, however, and difficult but not impossible to eradicate. Presently, the HIV life cycle is known somewhat, but not quite enough to control its activity completely.

Hence we are investigating the HIV life cycle in order to understand its weaknesses and to target those steps to give a death blow to the virus. Our research will have social and political implications since HIV, in particular, is a disease without borders. We also predict that our research will have implications in the rational designing of vaccines, potent therapeutic drugs, and in the novel development of gene delivery vehicles which will have implications for other chronic diseases such as neurodegeneration.

Ashok Chauhan, Ph.D.
Associate Professor
Department of Pathology, Microbiology, and Immunology
Adjunct Associate Professor
Department of Physiology, Pharmacology and Neuroscience
University of South Carolina
Columbia, SC, USA

Keywords: protein transduction domain (PTD), HIV Tat, protein delivery, cell penetrating peptides, HIV gene therapy, Small interfering RNA (siRNAs), peptide-siRNA, HIV-inhibition, HIV life cycle.