When it comes to RNA interference in human cells, delivery remains a top problem. Traditional transfection methods are effective, but it is difficult to achieve long-term silencing in vivo with them.
To solve this problem, some researchers are exploring viral vector-based RNAi techniques. Chang-Mei Liu and colleagues from the Chinese Academy of Medical Sciences and Peking Union Medical College, for example, published a report in the Jan. 16 issue of Biochemical and Biophysical Research Communications discussing the use of retrovirus vectors to knock down the tumor suppressor gene p53 in HepG2 cells.
In looking for an appropriate model to examine their ability to knock down the gene, the researchers wrote that they selected HepG2 because of its high levels of endogenous wild-type p53.
According to the researchers, they developed a retrovirus vector that permits delivery of stem-loop cassettes. To do so, they stated that they modified the retrovirus pLXSN by deleting the 260-bp NheI/XbaI fragment in the 3’LTR to generate pXSN. This was done in order to eliminate the 5’LTR counterpart after viral replication, thereby avoiding potential transcriptional interference from the 5’LTR promoter.
“The RNA polymerase III promoter of human U6+27 small nuclear RNA gene, used to drive high levels of expression of a small hairpin RNA, was inserted into the multiple cloning sites with reverse orientation to SV40 promoter-driven Neo gene transcription,” the authors wrote. “Subsequently, the synthesized inverted repeats with identical sequences to human p53 gene and EGFP gene were cloned to downstream of U6+27 promoter, using four thymidines as the terminal signal.”
Meanwhile, G418-resistant HepG2 cell pools were established after transduction with recombinant retroviruses. These were generated by co-transfecting GP-293 cells with retroviral vectors pXSNhU6+27sip53, pXSNhU6+27siegfp, and the envelope plasmid pVSVG, which confers the virus with pantropism, wrote the article’s authors.
In the end, “polymerase chain reaction demonstrated that aim fragments were integrated into cell pools,” the authors state. “Western blotting analysis demonstrated that the expression levels of p53 protein were reduced dramatically compared with those of beta-actin genes.”
Additionally, cell cycle analysis by FACS showed “decreases of G1 phase and increases of S phase in sip53-treated cell pools compared to control-treated cell pools,” the researchers stated. “Taken together, these results suggest that retrovirus-delivered RNAi can trigger down-regulation of p53 genes in an efficient and sequence-specific manner in HepG2 cells.”
The paper’s authors noted that generally, siRNAs used to knock down specific genes are delivered by “physical transfection methods, which limits the application of RNAi in long-term gene silencing in mammalian systems.”
By generating siRNA-expressing recombinant retroviruses that led to stable and sequence-specific knockdown of p53, the authors wrote that it appears that retroviruses may be an effective means for triggering long-term RNAi. “In addition,” they note, a “recombinant retrovirus pseudotyped with VSV-G can be concentrated by ultracentrifugation, and thus may be directly used in ex vivo and in vivo studies.”
The researchers add that retroviruses also appear to be exempt from RNAi degradation, despite the “presence of complementary sequences in the provirus.”
In conclusion, they wrote, “the development of appropriate siRNA delivery vectors may have important applications, especially in gene therapy and genomic research. Virus vectors, when combined with RNAi, may provide useful tools to elucidate gene function via analysis of loss-of-function phenotype.” They also added that lentiviral vector-mediated transgenic knockouts may make RNAi an alternative to homologous gene targeting for creating knockout mice.