Remote regulation of glucose homeostasis in mice using genetically encoded nanoparticles

Abstract

Means for temporally regulating gene expression and cellular activity are invaluable for elucidating underlying physiological processes and would have therapeutic implications. Here we report the development of a genetically encoded system for remote regulation of gene expression by low-frequency radio waves (RFs) or a magnetic field. Iron oxide nanoparticles are synthesized intracellularly as a GFP-tagged ferritin heavy and light chain fusion. The ferritin nanoparticles associate with a camelid anti-GFP-transient receptor potential vanilloid 1 fusion protein, αGFP-TRPV1, and can transduce noninvasive RF or magnetic fields into channel activation, also showing that TRPV1 can transduce a mechanical stimulus. This, in turn, initiates calcium-dependent transgene expression. In mice with stem cell or viral expression of these genetically encoded components, remote stimulation of insulin transgene expression with RF or a magnet lowers blood glucose. This robust, repeatable method for remote regulation in vivo may ultimately have applications in basic science, technology and therapeutics.

Figure 1

In vitro optimization of gene expression and protein release with genetically encoded nanoparticles. (a) Schema of systems testing three alternate locations of genetically encoded ferritin to generate iron oxide nanoparticles to open the temperature-sensitive channel TRPV1 in response to RFs: cytoplasmic ferritin (left, TRPV1/ferritin); membrane-tethered ferritin, achieved by addition of an N-terminal myristoylation signal (middle, TRPV1/myrferritin); and channel-associated ferritin, achieved by adding a GFP-binding domain to the N terminus of TRPV1 and GFP to the N terminus of ferritin (right, αGFP-TRPV1/GFP-ferritin). P, phosphate; NFATc, cytoplasmic location of nuclear factor of activated T cells; NFATn, nuclear location of nuclear factor of activated T cells; SRE, serum response element; CRE, cyclic AMP response element; NFAT RE, nuclear factor of activated T cells response element. (b) Immunohistochemistry (IHC) for TRPV1 (red), GFP (green) and FLAG-tagged (blue) ferritin chimera in HEK 293T cells transfected with TRPV1/ferritin constructs confirmed membrane expression of TRPV1 and cytoplasmic expression of ferritin (top) in cells transfected with TRPV1/myrferritin; IHC confirmed membrane expression of both TRPV1 and ferritin (middle); in cells transfected with αGFP-TRPV1/GFP-ferritin, IHC confirmed membrane expression of TRPV1, GFP and ferritin (bottom). Scale bars, 50 μm. (c) Representative changes in Fluo-4 fluorescence after application of the TRP agonist 2APB to untransfected HEK cells or those transfected with αGFP-TRPV1/GFP-ferritin or left untreated. (d) Representative changes in Fluo-4 fluorescence after application of RF to HEK cells transfected with αGFP-TRPV1/GFP-ferritin or without RF treatment. (e) RF treatment increases insulin gene expression in HEK cells expressing TRPV1/myrferritin and αGFP-TRPV1/GFP-ferritin but not in those expressing TRPV1/ferritin. Each study was repeated on 2 (TRPV1/ferritin) or 3 occasions (TRPV1/myrferritin and GFP-TRPV1/GFP-ferritin) each with 4 replicates (see Online Methods). In all cases, columns represent mean and error bars show s.e.m. Data were analyzed by a two-tailed Mann-Whitney test. * and # indicate P < 0.05. (f) RF treatment increases proinsulin release from HEK cells expressing TRPV1/ferritin, TRPV1/myrferritin and αGFP-TRPV1/GFP-ferritin. Each study was repeated on 2 (TRPV1/ferritin) or 3 occasions (TRPV1/myrferritin and αGFP-TRPV1/GFP-ferritin) each with 4 replicates. In all cases, columns represent mean and error bars indicate s.e.m. Data were analyzed by a two-tailed Mann-Whitney test. *, # and & indicate P < 0.05.

Figure 2

RF-regulated gene expression in vivo using MSCs. (a) Schema for delivery and assessment of effects of RF treatment on blood glucose in mice with implanted MSCs expressing TRPV1/myrferritin or αGFP-TRPV1/GFP-ferritin and calcium-dependent human insulin. (b) IHC for TRPV1, EGFP and FLAG-tagged ferritin in sections of gelatin scaffold implants seeded with MSCs stably expressing TRPV1 and myrferritin (top) or αGFP-TRPV1 and GFP-ferritin fusion (bottom). TRPV1/myrferritin-expressing cells show TRPV1 and HA staining at the cell membrane, whereas αGFP-TRPV1/GFP-ferritin-expressing cells show TRPV1, FLAG and GFP staining at the cell membrane. Scale bars, 100 μm. (c) Effects of RF treatment on insulin gene expression in control (n = 7), TRPV1/myrferritin (n = 6) and αGFP-TRPV1/GFP-ferritin-expressing (n = 7) MSC implants. RF treatment substantially increases insulin gene expression in MSCs expressing TRPV1 and genetically encoded nanoparticles. *, # and & indicate P < 0.05. Data are shown as mean ± s.e.m. (d) Plasma insulin was substantially increased by RF treatment in mice implanted with MSCs expressing TRPV1/myrferritin (n = 6) or αGFP-TRPV1/GFP-ferritin (n = 6) but not in control mice (n = 5). * and # indicate P < 0.05 with analysis by paired t-test. Error bars indicate s.e.m. (e) RF treatment of mice implanted with MSCs expressing αGFP-TRPV1/GFP-ferritin (n = 7) or TRPV1/myrferritin (n = 6) reduces blood glucose compared with that seen in control mice (n = 7). *P < 0.05. Data are shown as mean ± s.e.m. (f) RF treatment reduced blood glucose over the course of the study in mice implanted with MSCs expressing αGFP-TRPV1/GFP-ferritin (n = 7) compared with RF treatment of mice with control MSC implants (n = 7). *P < 0.05. Data are shown as mean ± s.e.m.

Figure 3

RF-regulated gene expression in vivo using adenoviral delivery of transgenes. (a) Schema for delivery and assessment of effects of RF treatment on blood glucose in C57BL/6 mice injected with replication-deficient adenovirus expressing LacZ, TRPV1/myrferritin or αGFP-TRPV1/GFP-ferritin and calcium-dependent human insulin. (b) IHC for LacZ or TRPV1, EGFP and FLAG-tagged ferritin in hepatic tissue infected with adenovirus expressing LacZ (top) or with adenovirus expressing αGFP-TRPV1 and GFP-ferritin fusion (bottom). Scale bars, 100 μm. (c) Effects of RF treatment on hepatic insulin gene expression in mice treated with adenovirus expressing LacZ, TRPV1/myrferritin or αGFP-TRPV1/GFP-ferritin and calcium-dependent human insulin. For hepatic insulin gene expression, control group: n = 3 no RF, 5 RF-treated; TRPV1/myrferritin: n = 3 no RF, 3 RF-treated; αGFP-TRPV1/GFP-ferritin: n = 3 no RF, 3 RF treated. RF treatment substantially increases insulin gene expression in hepatic tissue expressing αGFP-TRPV1/GFP-ferritin. *P < 0.05. Error bars indicate s.e.m. (d) Plasma insulin was increased by RF treatment in mice expressing TRPV1/myrferritin (n = 5) or αGFP-TRPV1/GFP-ferritin (n = 6) but not in control mice (n = 7). * and # indicate P < 0.05 with analysis by paired t-test. Data shown as mean ± s.e.m. (e) RF treatment of mice injected with adenovirus expressing αGFP-TRPV1/GFP-ferritin (n = 6) reduced blood glucose compared with that seen in control mice (n = 7). Asterisks indicated P < 0.05; data shown as mean ± s.e.m. (f) RF treatment substantially reduced blood glucose over the course of the study in mice expressing αGFP-TRPV1/GFP-ferritin (n = 6) compared with RF treatment of mice expressing LacZ (n = 7). *P < 0.05. Data are shown as mean ± s.e.m.

Figure 4

Repeated RF treatment to regulate protein delivery. (a) Effects of RF treatment at weeks 2–6 after virus injection on cumulative blood glucose in C57BL/6 mice injected with control (n = 7) or αGFP-TRPV1/GFP-ferritin (n = 6) expressing adenovirus. RF treatment reduced cumulative blood glucose in αGFP-TRPV1/GFP-ferritin-expressing mice at each assessment. *P < 0.05. Data are shown as mean ± s.e.m. (b) Plasma insulin was increased by RF treatment in mice expressing αGFP-TRPV1/GFP-ferritin (n = 6) but not in control mice (n = 7) at week 2. *P < 0.05. Error bars indicate s.e.m. (c) Plasma insulin was increased by RF treatment in mice expressing αGFP-TRPV1/GFP-ferritin (n = 6) but not in control mice (n = 6) at week 6. *P < 0.05 with analysis by paired t-test. Data are shown as mean ± s.e.m.

Figure 5

Remote activation of gene expression with a static magnetic field. (a) Effects of magnetic field on cumulative changes in Fluo-4 fluorescence in five representative HEK cells transfected with αGFP-TRPV1/GFP-ferritin or control cells. *P < 0.05. Data are shown as mean ± s.e.m. n = 6. (b) Magnetic field treatment increases proinsulin release from HEK cells expressing αGFP-TRPV1/GFP-ferritin and calcium-dependent human insulin. Data are shown as mean ± s.e.m. Each study was repeated on 3 occasions, each with 4 replicates. *P < 0.05 as analyzed by Mann-Whitney test. (c) Schema for delivery and assessment of effects of magnet treatment on blood glucose in C57BL/6 mice injected with replication-deficient adenovirus expressing αGFP-TRPV1/GFP-ferritin and calcium-dependent human insulin. (d) Plasma insulin is substantially increased in mice expressing αGFP-TRPV1/GFP-ferritin and calcium-dependent human insulin treated with an intermittent magnetic field compared with no magnet treatment. *P < 0.05 as analyzed by paired t-test. (e) Magnet treatment reduced blood glucose over the course of the study in mice expressing αGFP-TRPV1/GFP-ferritin compared with no magnet treatment. *P < 0.05. Data are shown as mean ± s.e.m. (f) Magnet treatment reduced blood glucose over the course of the study in mice expressing αGFP-TRPV1/GFP-ferritin compared with magnet treatment of mice expressing LacZ. *P < 0.05. Data are shown as mean ± s.e.m.