A benefit of the voluminous wealth of research produced is that it allows us to stand on the shoulders of giants – we can take advantage of established facts, tools, and datasets. This may mean using a mutant library to find genes in your organism that are important for the process you study; accessing multiple microarrays to synthesize old datasets and uncover new effects; or screening molecules already approved by the FDA to find a therapy that can bypass the arduous task of becoming approved. This last method is particularly useful if you work in translational medicine, and have a specific molecular interaction on which you are working.
It may seem counterintuitive to screen FDA-approved small molecules for those that facilitate viral infection, but that is just what first author Sarah Nicolson and senior scientist R. Jude Samulski did in their recent Journal of Virology study. Their work centers on recombinant adeno-associated virus (rAAV)-mediated gene therapies, which are one of the best hopes to carry vital genes to the cells of patients born with genetic disorders. The more efficiently these vector-carrying viruses can get inside of host cells, the more host cells will have a repaired genetic system, and the more effective the targeted vector will be.
AAV has become a favored gene vector because there are no associated diseases known to be associated with AAV infection. AAV infection normally requires co-infection by adenovirus or herpes simplex virus, and use of AAV as a gene therapy vector requires additional factors to facilitate host cell entry, so several adenovirus helper factors have been engineered into rAAV vectors. The virus has a small genome (less than 5 kb) and has broad tropism, with different serotypes showing favorability toward different cell types, and therefore has potential to treat many different diseases (see schematic of rAAV used for gene therapy, left).
However, infected cells are still that: infected with a foreign microbe, which can signal the immune system for elimination through viral capsid MHC Class I antigen presentation, an idea first suggested based on clinical trials using an rAAV vector to treat hemophilia ($). The scientists involved in the current study hypothesized that an adjuvant, injected with the rAAV, might help increase efficiency of viral infection - also known as transduction, because the engineered virus carries a therapeutic gene. Increased efficiency would result in more infected (and thus treated) cells, along with a lower immune response that might eliminate these cells.
The researchers screened over 2000 FDA-approved small molecules for those that increased viral entry, and identified twelve potential candidates that fell into one of five functional categories. One group, the epipodophyllotoxins, induce DNA damage by inhibiting topoisomerase activity. The two small compounds from this group, teniposide and etoposide, increased rAAV transduction into a number of different cell types. Teniposide, approved for use in chemotherapy for some cancers, showed the highest increase in rAAV transfection.
Importantly, the results found in the in vitro studies were consistent when tested in vivo. Mice were injected with an rAAV-Luc construct, which contained a luciferase gene that allowed live-image visualization of the infection. Test animals were also administered one of five identified compounds, including teniposide, at doses comparable to their FDA-approved concentrations. Those treated with teniposide showed a nearly 100-fold increase in luciferase activity, indicating more of the animals’ cells expressed rAAV genes (see figure, right). Toxicity screens showed average levels for kidney and liver function assays that were comparable between drug- and vehicle-treated animals.
Despite this promising result, more questions must be answered before teniposide is coadministered with ongoing rAAV gene therapy trials. While the average increase in transduction efficiency was 100-fold, there was a lot of variability between individual subjects; understanding the cause of this variability is an important step toward generating a consistently beneficial treatment. Additionally, understanding how teniposide aids rAAV transduction, especially considering etoposide uses a similar mechanism but had different results, will also aid in fine-tuning treatment. The other identified compounds may also be worth following up, especially as the molecules that increased transduction efficiency fell into either DNA damage or proteasome inhibition. Combinations of these small molecules may yield yet different results, and the applicability to enhance particular tissue transduction is also an important area to explore.
Overall, the future of gene therapy looks bright (and not just due to the luciferase). The European Union approved the first rAAV-based therapy to confer a lipoprotein lipase transgene to some pancreatitis patients in 2012. Many clinical studies using rAAV vectors are ongoing, and while not yet approved in the United States, studies such as the one presented here may increase the claim of rAAV as a safe and, equally important, effective means to treat genetic disorders.
-- Julie Wolf