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Remote control of gene expression/magnetogenetics

Magnetogenetics

In collaboration with Jeffrey Friedman at the Rockefeller University and Sarah Stanley at the Icahn School of Medicine at Mount Sinai, we are combining nanotechnology and bioengineering to demonstrate that external and internally, genetically-encoded nanoparticles can be used in vivo to remotely regulate cellular activity. Calcium channels are of great therapeutic interest due to their numerous and varied functions throughout the body. One important channel, transient receptor potential vanilloid 1 (TRPV1), has gained great interest throughout the literature since its discovery. Understanding the channel’s role as a nociceptor has led to the development of treatments for a wide variety of diseases (e.g. pain caused by shingles). In particular, we have demonstrated that nanoparticles conjugated to TRPV1 can be used to remotely activate it and mediate cellular activity, such as neuron action potentials, gene transcription, and protein production.

We have shown that the channel can be manipulated remotely to regulate gene expression in mice1. This was achieved by decorating His6-tag modified TRPV1 channels (TRPV1His) with anti-His6 antibody-coated iron oxide nanoparticles (αHis6-IONPs) and subjecting them to a radiofrequency (RF) field (Figure MG-1). Upon exposure to the RF field, the IONPs activate the decorated channels and cause them to open, allowing calcium ion flux into the cytoplasm, which subsequently activates a gene under the control of a calcium-sensitive promoter. We tested the system in vitro in human embryonic kidney cells (HEK-293T) expressing TRPV1His and a bioengineered proinsulin gene under the control of a calcium promoter. Proinsulin levels were found to increase significantly when incubated with αHis6-IONPs and treated with RF (Figure MG-2). We also showed that exposure to RF stimulates insulin release from xenograft tumors and this lowers blood glucose in diabetic mice. These studies established the efficacy of a novel platform for using nanotechnology to remotely control cellular response.

 Figure41

Figure MG-1. TRPV1-IONP system. The IONP is covalently coated in anti-His6 antibodies allowing it to bind to the His6-tag on the channel. When subjected to RF waves, the nanoparticle activates the channel and causes it to open. This results in calcium ion flux into the cell and the subsequent expression of a calcium-promoted gene. [We would like to acknowledge UltraFlex Power Technologies for the custom RF induction system used to generate the RF waves in the in vitro and in vivo studies described herein.]

 Figure42

Figure MG-2. Efficacy of TRPV1-nanoparticle system in HEK-293T cells. Upon activation by RF waves, the particles cause the channel to open and the subsequent calcium flux promotes gene transcription. This effect is shut down by the channel antagonist Ruthenium red.

Temporally regulating gene expression and cellular activity are invaluable for elucidating underlying physiological processes and could have therapeutic implications. Building upon our TRPV1-IONP system, we developed a genetically encoded system for remote regulation of gene expression by either RF stimulation or exposure to a magnetic field2. Intracellularly, ferritin binds, converts, and stores excess iron ions as superparamagnetic iron oxide nanoparticles. We thus introduced and constitutively produced GFP-tagged ferritin light and heavy chain dimer fusion protein, which integrates with endogenous ferritin light and heavy chain monomers to form chimeric GFP-tagged ferritin 24-mers. The ferritin nanoparticles associate with a camelid anti-GFP nanobody TRPV1 fusion protein (αGFP-TRPV1), allowing for transduction of noninvasive RF or magnetic fields into channel activation. This, in turn, initiates calcium-dependent transgene expression (Figure MG- 3). In mice with viral expression of these genetically encoded components, remote stimulation of insulin transgene expression by RF or magnetic field exposure lowers blood glucose (Figure MG-4). This robust, repeatable method for remote regulation in vivo may ultimately have applications in basic science, technology, and therapeutics.

 Figure43

Figure MG-3. TRPV1-ferritin system. An anti-GFP camelid nanobody is expressed on the N-terminus of the TRPV1 and binds to a GFP-tag chimerically integrated into ferritin nanoparticle. When subjected to RF waves, the nanoparticle activates the channel and causes it to open. This results in calcium ion flux into the cell and the subsequent expression of a calcium-promoted gene.

 Figure44

Figure MG-4. Efficacy of TRPV1-ferritin system in mice. (a) RF treatment of mice implanted with mesenchymal stem cells (MSCs) expressing the TRPV1-ferritin system (Figure 3) and an analogous TRPV1-myrferritin system (not shown) were found to reduce blood glucose compared with control mice (* P < 0.05). (b) Total blood glucose reduced by TRPV1-ferritin and TRPV1-myrferritin systems over 60 min of RF treatment and 60 min following RF treatment as compared to the control (* P < 0.05). TRPV1-myrferritin system used myristoylated ferritin resulting in random integration of ferritin into the cell membrane, thus placing ferritin with varying proximity to the TRPV1 channels distributed throughout the membrane. In comparison to the αGFP-TRPV1 direct conjugation to the GFP-ferritin, data shows that close proximity has significant impact in improving response to RF stimulation. Data shown as Mean ± S.E.M.

Using a similar approach, we further targeted and temporally regulated neural modulation to determine the physiological roles of specific neural populations or circuits. We induced neuronal activation remotely using either RF or magnetic fields via Cre-dependent expression of the aforementioned GFP-tagged ferritin fusion protein tethered to the αGFP-TRPV1 fusion protein3. Neuronal inhibition via the same stimuli is achieved by mutating the TRPV1 pore, rendering the channel chloride-permeable. These constructs were targeted to glucose-sensing neurons in the ventromedial hypothalamus in glucokinase–Cre (GK-Cre) mice, which express Cre in glucose-sensing neurons. Acute activation, via Ca2+ flux, of glucose-sensing neurons in this region was found to increase plasma glucose and glucagon levels, lower insulin levels, and stimulate feeding (Figure MG-5a). Conversely, inhibition via Cl- flux through the mutant TRPV1 resulted in reduced blood glucose levels, increased insulin levels, and the suppression of feeding (Figure MG-5b). These results suggest that pancreatic hormones function as an effector mechanism of central nervous system circuits controlling blood glucose and behavior. Our method obviates the need for permanent implants and could potentially be applied to study other neural processes or used to regulate other, even dispersed, cell types.

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Figure MG-5. Activation and inhibition of glucose-sensing neurons in the ventromedial hypothalamus of mice. (a) RF treatment of GK-Cre (Gck) and wild-type (WT) mice expressing the TRPV1-ferritin system (Figure 3) showed significant reduction in insulin levels, increase in glucagon levels, and upregulation in glucose-6-phosphatase expression (* P < 0.05, *** P < 0.005). (b) RF treatment of GK-Cre (Gck) and wild-type (WT) mice expressing the mutant TRPV1-ferritin system showed significant increase in insulin levels and downregulation in glucose-6-phosphatase expression (* P < 0.05). Data shown as Mean ±S.E.M.

These approaches provide a platform for using nanotechnology to remotely control cellular response through cell signaling, gene transcription, and protein expression. We are now broadening the capabilities of this platform, exploring the range of potential nanoparticle interactions available to remotely regulate cellular activity.

Near-Infrared Activation of Gene Expression

In addition to RF stimulation, we have demonstrated that photostimulation of gold nanorods (AuNRs) using a tunable near-infrared (NIR) laser at specific longitudinal surface plasmon resonance wavelengths can induce the selective and temporal internalization of calcium in HEK-293T cells via TRPV1 activation leading to gene expression4. Biotin-PEG-Au nanorods coated with streptavidin Alexa Fluor-633 and biotinylated anti-His antibodies (Figure MG-6a) were used to decorate cells genetically modified with His6-tagged TRPV1 temperature-sensitive ion channel (Figure MG-6b) and AuNRs conjugated to biotinylated RGD peptide were used to decorate integrins in unmodified cells (Figure MG6c). Plasmonic activation can be stimulated at weak laser power (0.7-4.0 W·cm-2) without causing cell damage. Selective activation of TRPV1 channels could be controlled by laser power between 1.0-1.5 W·cm-2. Integrin targeting robustly stimulated calcium signaling due to a dense cellular distribution of nanoparticles (Figure MG-7). Such an approach represents a functional tool for combinatorial activation of cell signaling in heterogeneous cell populations. Our results suggest that it is possible to induce cell activation via NIR-induced AuNR heating through the selective targeting of membrane proteins in unmodified cells to produce calcium signaling and downstream expression of specific genes with significant relevance for both in vitro and therapeutic applications.

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Figure MG-6. TRPV1-AuNR and integrins-AuNR systems. (a) Antibody functionalization of gold nanorods using biotin-PEG, streptavidin, and biotinylated anti-His6 antibody. For RGD functionalized AuNR, biotinylated RGD peptide was used in place of biotinylated antibody. (b) The anti-His6 antibody functionalized AuNR is conjugated to the His6-tag on the TRPV1 channel. When subjected to NIR light, the nanoparticle activates the channel and causes it to open. This results in calcium ion flux into the cell and the subsequent fluorescence of Fluo-4 AM. (c) The RGD peptide functionalized AuNR is conjugated to integrins on the cell surface. When subjected to NIR light, the nanoparticle likely polarizes the cell membrane, resulting in calcium ion flux into the cell and the subsequent expression of a calcium-promoted gene.

 Figure47

Figure MG-7. Response to NIR light stimulation of functionalized AuNRs on HEK-293T cells. Comparison of the Fluo-4 AM signal after NIR treatment of NR720 functionalized with either anti-His6 or RGD peptide at 0.8, 1.5, and 4.0 W·cm-2. Anti-His6 antibody AuNRs produced fluorescence in HEK-293T cells above a threshold of ~ 1.0 W·cm-2 while RGD peptide AuNRs showed substantial fluorescence even at 0.8 W·cm-2.

References

  1. S.A. Stanley, J.E. Gagner, S. Damanpour, M. Yoshida, J.S. Dordick, and J.M. Friedman (2012), “Radio-wave heating of iron oxide nanoparticles can regulate plasma glucose in mice,” Science 336, 604-608.
  2. S.A. Stanley, J. Sauer, R.S. Kane, J.S. Dordick, and J.M. Friedman (2015), “Remote regulation of glucose homeostasis in mice using genetically encoded nanoparticles,” Nat. Medicine 21, 92-98.
  3. S.A. Stanley, L. Kelly, K.N. Latcha, S.F. Schmidt, X. Yu, A.R. Nectow, J. Sauer, J.P. Dyke, J.S. Dordick, and J.M. Friedman (2016), “Bidirectional electromagnetic control of the hypothalamus regulates feeding and metabolism,” Nature 531, 647-650.
  4. S.P. Sanchez-Rodriguez, J.P. Sauer, S.A. Stanley, X. Qian, A. Gottesdiener, J.M. Friedman, and J.S. Dordick (2016), “Plasmonic activation of gold nanorods for remote stimulation of calcium signaling and protein expression in HEK 293T cells,” Biotechnol. Bioeng. 113, 2228-2240.