Immunology and Infection
Published: September 5th, 2016
Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.
Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.
A limitation of current HIV-1 treatments is that they must be chronically administered to prevent disease progression. Transplant of HIV-1 resistant T lymphocyte, or hematopoietic stem cells, has the potential to provide long term control of HIV-1 replication in the absence of drug therapy1,2 and may also be an effective approach to attain an HIV-1 cure3. One way to render cells resistant to HIV-1 replication is to insert one or more genes coding for anti-HIV-1 RNAs or peptides into an infected individual's cells during an autologous transplant4. Several candidate anti-HIV-1 genes have been designed with some entering clinical tria....
1. Cells and Transfections
A general schematic of the procedures is shown in Figure 2 with an example transfection plan for three test RNAs and a control RNA provided in Figure 2B. For viral production and cell viability assays, the read-out for each test construct is normalized to a negative control. Replicates are transfected in sets, so that each test RNA is normalized to its adjacent negative control. This is done to avoid inaccurate data related to the time between complexing .......
The HIV-1 production assay described was performed using HEK293T cells (Figure 2) and is similar to assays used to screen HIV-1 RNA for effective ribozyme13, shRNA10,29, siRNA30, and U1i RNA11,31 target sites. Using different methods to quantify HIV-1 production, most studies have measured viral production 48 hr after co-transfection of an HIV-1 expression plasmid with candidate RNAs. Following the production of HIV-1, immature virions undergo proteolytic cleav.......
|Cell culture plates, 96 well, 24 well, 6 well.
|353075, 353047, 353043
|Micro tubes Axygen
|Low molecular weight Poly I:C
|transfection reagent for synthetic RNAs
|transfection reagent for RNA expression plasmids
|Nonidet P40 (NP-40)
|poly(A) RNA template
|oligo(dT)12-18 DNA primer
|DEAE filtermat paper
|Microplate scintillation counter
|Lysis buffer tablets
|protease and phosphatase inhibitors
|Gel running chamber
|Semi-dry transfer cell
|Protein ladder EZ-Run
|Antibody stripping solution
|ECL - Pierce
|from Dr. B.L. Bass
|from Dr. A. Hovanessian
|Peroxidase-labeled goat anti-rabbit
|Peroxidase-labeled goat anti-mouse
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