Here we present a series of experiments designed to detect transfer of RNAi molecules from MSC to neighboring cells of neuronal or glial lineage. RNAi therapies are difficult to translate, mainly due to the difficulties in delivering RNAi molecules to the affected cells, and their transient effects. MSC are currently being considered for the treatment of neurodegenerative diseases due to their innate homing abilities, established safety profile, anti-inflammatory effects, and the broad array of cytokines that they secrete into the microenvironment (Joyce et al., 2010
). For these reasons, we believe that MSC could make an excellent delivery vehicle for RNAi molecules to treat disorders such as Huntington’s disease, combining their innate reparative abilities with the disease silencing power of targeted siRNA.
Several groups have reported the use of RNAi targeted to single nucleotide polymorphisms (SNPs) to specifically silence a single HTT allele, used to knockdown HD HTT and spare normal HTT genes (Boudreau et al., 2009
; Davidson and Paulson, 2004
; Lombardi et al., 2009
; Pfister et al., 2009
; Rodriguez-Lebron et al., 2009
; van Bilsen et al., 2008
; Zhang et al., 2009
). Additionally, other researchers have shown that while HTT knockouts are embryonic lethal in mice, conditional knockouts in adult mice have little effect (Boudreau et al., 2009
; DiFiglia et al., 2007
; Drouet et al., 2009
; Harper et al., 2005
). For this report, we chose to use a shRNA sequence that would knock down all HTT as an initial proof of concept study. The shRNA sequence could be easily switched to a sequence specific to a common familial HTT allele.
Lentiviral vectors were constructed to create both shRNA donor and target cells in a co-culture system. shRNA vector expression did not result in any significant effect on MSC viability, growth or differentiation potential, as assayed by growth curve analysis and differentiation into adipogenic and osteogenic lineages. Genetically transduced and expanded MSC retained a normal karyotype without any detected chromosomal aberrations. As a potential human therapy it was imperative for the shRNA to be well tolerated by MSC, and we demonstrated that the MSC were essentially unaffected by the RNAi production.
The direct activity of the shRNA vectors was established by transducing U87 cells previously transduced with a vector that carried both the mutant HTT fragment and EGFP (U87HTTgfp). shGFP significantly reduced the expression of GFP as measured by flow cytometry. Likewise, shHTT was effective in reducing mutant HTT levels as assayed using densitometry after gel electrophoresis and Western blot. No changes in protein levels were detected with the scrambled control (shSCRAM), demonstrating that the reduction in protein was due to specific shRNA activity and not a byproduct of either lentiviral integration or shRNA expression.
Specific protein reduction in a targeted cell population was detected in co-culture systems. MSC expressing either a shRNA targeting GFP or HTT as a control were co-cultured with SH-SY5Y cells expressing a short-lived GFP variant and GFP expression per cell was quantified using flow cytometry on day 4 and day 10. The shGFP and shHTT cultures went from indiscernible at day 4 to very different on day 10, with GFP expression sharply reduced in the shGFP culture. A separate co-culture featured MSC expressing either shGFP as a control or shHTT mixed with U87 cells expressing a mutant HTT fragment and an eGFP marker that had been pretreated with mitomycin C to decrease proliferation. HTT levels were assayed over time by Western blots quantified using densitometry, revealing a decrease in HTT in both cultures when normalized to actin, but a larger decrease from the shHTT containing co-culture. We found that normalizing to actin, a common control protein, is insufficient for our conditions due to the presence of otherwise undetected MSC in the culture. To address this, we constructed new co-cultures using shSCRAM as a control instead of shGFP in order to normalize HTT expression to the GFP used as a transduction marker and co-expressed with HTT. In co-cultures conducted simultaneously in either reduced serum or serum-free conditions, we found a reduction of mutant HTT through day 5 of cultures, as compared to GFP expression.
The sporadic successes of our co-cultures are due, in part to the technical challenges of keeping two very different cell populations alive and healthy for prolonged periods of time while maintaining a cell density high enough to encourage substantial exchange of RNAi by either secreted microvesicles or direct cell to cell transfer. Approximately 5 days after being seeded at the moderate density of 1000 cells/cm2 most cultures were completely confluent. As the cultures overgrew, the medium would become toxic to one or both cell populations. The more tolerant cell population would then dominate the culture and cause spurious measurements late in the time-courses. In order to address these obstacles, we examined a number of culture conditions and treatments; changing cell types, assays, sera concentration, and using both mitomycin C and gamma-irradiation to induce growth arrest. While U87 and MSC co-cultures exhausted themselves quickly, MSC and SH-SY5Y often persisted a few additional days before becoming errant. Mitomycin C treatment of MSC was largely ineffective at preventing overgrowth, but did manage to reduce U87 growth in the cultures. We conducted kill-curves to optimize gamma radiation doses for the individual cell types to cause growth arrest without apoptosis/necrosis, but found that irradiation did not produce reliable cultures or positive outcomes. These data might indicate that the mechanisms that MSC can use to transfer macromolecules to neighboring cells are adversely affected by radiation.
A number of different basal media (both catering to MSC and U87/ SH-SY5Y) were used with varying amounts of serum to abrogate overgrowth of the cultures. We found that as little as 1% FBS induced sufficient growth to overwhelm the culture, probably due to the large number of autocrine and paracrine growth factors produced by MSC. The varied conditions with which we experienced successful outcomes speak to the transient and delicate nature of the transaction. A large number of culture variables were created in an effort to control cell growth with irradiation and chemical treatments. These treatments could have inhibited RNAi transfer either directly, through their effect on the cells or, indirectly through something as small as pH changes in the medium. A better understanding of the nature of intercellular RNAi transfer from MSC to target cell at the molecular level will allow our group and others to design better platforms for RNAi delivery in the future. Knowledge of the molecular processes involved in the RNAi transfer, whether by gap junction, nanotubes, exosomes, virtosomes, or some other mechanism, could allow screening of MSC batches to find those most likely to be robust RNAi donors. Additionally, we are currently optimizing mouse models in which we can assay RNAi transfer from human MSC in vivo. In an animal model there is no need for extensive cell manipulations to arrest cell overgrowth, and there is greater cell-cell contact as MSC are surrounded by tissue, and the physiological conditions may encourage more native interactions.
We believe that the data presented here is compelling evidence that RNAi molecules can be transferred from one cell population to another. The results presented in this report demonstrate that specific protein reduction in a recipient cell population can be achieved through intercellular transfer of RNAi from MSC to target cell. These findings are novel, are encouraging and warrant further study to describe and optimize mechanisms of RNAi exchange. The ability to use MSC as a vector for intercellular delivery of RNAi molecules holds promise for a wide variety of cellular therapies. A number of genetic diseases that are currently incurable and often untreatable could benefit greatly should MSC, or another cell type, be able to deliver efficacious amounts of RNAi directly to affected cells over a prolonged period of time. The potential benefits of cellular therapies and RNAi therapies combined may be sufficient to delay or even halt disease progression, which could dramatically improve quality of life for patients suffering from degenerative diseases like HD.