Efficient in vitro delivery of cationic nanoparticles loading siRNA targeting mutant huntingtin

Introduction: Clinical applica- tions of RNA-based therapeutics such as small interfering RNAs (siR- NAs) have been limited mainly due to low intracellular delivery efficien- cy in vitro and in vivo. To enhance gene delivery effect, various cationic complexes have been developed for delivering plasmid DNA, antisense, or siRNA into cells. However, the use of cationic vectors for clinical appli- cations has been severely limited by their high toxicity, low serum stabil- ity, nonspecific immune-stimulating effects, and poor biodegradability. In order to overcome these hurdles in gene therapy and improve gene delivery efficiency, we developed a copolymer composed of acetylated PEI (AcPEI) and poly(D,L-lactide- co-glycolide) (PLGA). The biode- gradable PLGA-AcPEI nanoparticles were tested by loading the siRNA of mutant huntingtin in fibroblasts from control and heterozygotic HD patients [1]. Materials and methods: The PLGA-based nanoparticles pre- sented in this study were synthesized by emulsion evaporation method, characterized by dynamic light scat- tering, and used as vector carrier for siRNA transfection in fibroblast cells. Quantitative PCR reactions were performed with primers to am- plify mutant huntingtin or β-actin mRNA using the Quantitect SYBR Green PCR kit. Data were analyzed using the 2-ΔΔCT method [S3] and β-actin mRNA for normalization. Results: PLGA-AcPEI nanoparticles exhibited a higher transfection ef- ficiency and lower cytotoxicity in fibroblasts from control and hetero- zygotic HD patients compared to the PEI/siRNA complex. Real-time PCR experiments showed decreased expression levels of mutant Htt and almost normal expression level of wild-type Htt. We achieved improved gene selectivity and allele selectivity of mutant HTT allele silencing. Con- clusions: Targeted reduction of mu- tant Huntingtin mRNA is considered an ideal strategy for treating HD. Our results demonstrate that PLGA- AcPEI nanoparticles are promising non-viral vectors for gene delivery in neurodegerative disease. Reference [1] Fiszer A, Mykowska A, Krzyzosiak WJ. Inhibition of mutant huntingtin expres- sion by RNA duplex targeting expanded CAG repeats. Nucleic Acids Res. 2011; 39: 5578-85.