Human immunodeficiency virus (HIV) is the primary cause of acquired immune deficiency syndrome (AIDS), which causes significant morbidity and mortality with a significant consequent decrease in the quality of patient’s lives. While combinatorial anti-retroviral therapy (cART) is very effective in controlling viral loads, people living with HIV now suffer from non-HIV conditions due to viral protein expression that cannot be controlled by cART.
Hence pathophysiological mechanisms with a focus on therapies that neutralize these HIV effects have gained increased attention. Gene therapy by engineering patient’s own blood cells to confer HIV resistance can potentially lead to a functional cure for AIDS. Additional RNA-based reagents such as RNA aptamers that are independent of base-pairing can further enhance combinatorial strategy for anti-HIV gene therapy.
Toward this goal, Pang et al. developed an anti-HIV lentivirus vector that deployed a combination of shRNA, ribozyme and RNA decoy overall generating a ‘chimeric’ expression construct with a very effective viral inhibition. To further improve this therapeutic vector against viral escape, multi-tagged RNA aptamers that target proteins with low solubility/stability such as the HIV integrase (IN) were developed using novel multi-tag SELEX. This method allowed the selection of aptamers against low solubility or unstable proteins while minimizing non-specific binding to the tags (Figure 1).
Figure 1. Schematic of multi-tag SELEX strategy. Typical change of aptamers binding during enrichment cycles monitored by the filter-binding assay. Odd number cycles were enriched by MBP-IN while even number cycles were enriched by His-IN. Alternate high and low binding at later cycles probably represent populations showing higher binding affinity to MBP-IN than HIS-IN.
Aptamers selected with a distinct secondary structure, namely S1R1, S3R1 and S3R3 were expressed in T cells or hematopoietic stem cells (HSC) for combinatorial gene therapy. By incorporating these aptamers into the terminal loop of anti-HIV Tat-Rev shRNA, long-term inhibition of HIV replication in a cell culture system was analysed by HIV-1 p24 ELISA kit assay (Figure 2). The shRNAs facilitated the export of the fusions to the cytoplasm where newly translated viral polyproteins might be more susceptible to aptamer binding.
Figure 2. shRNA–aptamer fusions confer long-term resistance to HIV replication in T cells. (A) Changes in virus concentration in CEM cells infected with pNL4-3 virus were monitored by p24 assays for 6 weeks. (B) p24 concentration at the end of week 6 post infection.
This novel strategy allowed efficient expression of the shRNA–aptamer fusions that targeted RNAs and proteins simultaneously and can be applicable for gene therapy against HIV and can potentially be adopted to treat other diseases. Moreover, by using multiplexed vectors, multiple shRNA–aptamer fusions can be expressed from a single transcript. This will allow inhibition of multiple targets at once and will be particularly useful to combat the ever-evolving HIV.
At Aptamer Group (AG) we offer the advantage of designing aptamers to be optimized for the conditions you want to use them in. This way they are engineered to bind to their target with high specificity and affinity. Moreover, AGL continuously aims to conduct further research in prevention, diagnosis and treatment of various such diseases. If you would like to know more about aptamers and their applications, please contact us using the form below.
Reference: Ka Ming Pang, Daniela Castanotto, Haitang Li, Lisa Scherer, John J Rossi, Incorporation of aptamers in the terminal loop of shRNAs yields an effective and novel combinatorial targeting strategy, Nucleic Acids Research, Volume 46, Issue 1, 9 January 2018, Page e6,