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Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo.

Zuris JA, Thompson DB, Shu Y, Guilinger JP, Bessen JL, Hu JH, Maeder ML, Joung JK, Chen ZY, Liu DR - Nat. Biotechnol. (2014)

Bottom Line: Current methods of protein delivery commonly suffer from low tolerance for serum, poor endosomal escape and limited in vivo efficacy.This approach mediates the potent delivery of nM concentrations of Cre recombinase, TALE- and Cas9-based transcription activators, and Cas9:sgRNA nuclease complexes into cultured human cells in media containing 10% serum.Delivery of unmodified Cas9:sgRNA complexes resulted in up to 80% genome modification with substantially higher specificity compared to DNA transfection.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Chemistry &Chemical Biology, Harvard University, Cambridge, Massachusetts, USA. [2] Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA.

ABSTRACT
Efficient intracellular delivery of proteins is needed to fully realize the potential of protein therapeutics. Current methods of protein delivery commonly suffer from low tolerance for serum, poor endosomal escape and limited in vivo efficacy. Here we report that common cationic lipid nucleic acid transfection reagents can potently deliver proteins that are fused to negatively supercharged proteins, that contain natural anionic domains or that natively bind to anionic nucleic acids. This approach mediates the potent delivery of nM concentrations of Cre recombinase, TALE- and Cas9-based transcription activators, and Cas9:sgRNA nuclease complexes into cultured human cells in media containing 10% serum. Delivery of unmodified Cas9:sgRNA complexes resulted in up to 80% genome modification with substantially higher specificity compared to DNA transfection. This approach also mediated efficient delivery of Cre recombinase and Cas9:sgRNA complexes into the mouse inner ear in vivo, achieving 90% Cre-mediated recombination and 20% Cas9-mediated genome modification in hair cells.

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In vivo delivery of Cre recombinase and Cas9:sgRNA complexes to hair cells in the mouse inner ear. (a) The scala media (cochlear duct) of P0 floxP-tdTomato mice (n = 4) were injected with 0.3 µL of 23 µM (−30)GFP-Cre in 50% RNAiMAX or with RNAiMAX alone (control). After 5 days, tdTomato expression indicative of Cre-mediated recombination was visualized using immunohistology. Red = tdTomato; green = Myo7a; white = Sox2; blue = DAPI. Yellow brackets indicate the outer hair cell (OHC) region. (b) Ten days after (−30)GFP-Cre delivery, intact espin (Esp)-expressing stereocilia of tdTomato-positive outer hair cells were present (arrow), similar to stereocilia in control cochlea. Red = tdTomato; green = Esp; white = Sox2; blue = DAPI. (c) Identical to (a) except using Lipofectamine 2000 instead of RNAiMAX. (n = 4). The upper and lower panels are images of mice cochlea at low and high magnification, respectively, detailing the efficiency of delivery and the effect on cochlear architecture and hair cell loss. (d) The scala media of P2 Atoh1-GFP mice (n = 3) were injected with 0.3 µL of 33 µM Cas9, 16.5 µM EGFP sgRNA in 50% RNAiMAX or Lipofectamine 2000 commercial solutions. Cas9-mediated gene disruption results in the loss of GFP expression when visualized 10 days later. The upper panels show GFP signal only, while lower panels include additional immunohistological markers. Yellow boxes in the lower panels highlight hair cells that have lost GFP expression. No obvious OHC loss was observed in the Cas9 + RNAiMAX or Cas9 + Lipofectamine 2000 groups. Red = tdTomato; green = Myo7a; white/light blue = Sox2; blue = DAPI. All scale bars (white) are 10 µm.
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Figure 6: In vivo delivery of Cre recombinase and Cas9:sgRNA complexes to hair cells in the mouse inner ear. (a) The scala media (cochlear duct) of P0 floxP-tdTomato mice (n = 4) were injected with 0.3 µL of 23 µM (−30)GFP-Cre in 50% RNAiMAX or with RNAiMAX alone (control). After 5 days, tdTomato expression indicative of Cre-mediated recombination was visualized using immunohistology. Red = tdTomato; green = Myo7a; white = Sox2; blue = DAPI. Yellow brackets indicate the outer hair cell (OHC) region. (b) Ten days after (−30)GFP-Cre delivery, intact espin (Esp)-expressing stereocilia of tdTomato-positive outer hair cells were present (arrow), similar to stereocilia in control cochlea. Red = tdTomato; green = Esp; white = Sox2; blue = DAPI. (c) Identical to (a) except using Lipofectamine 2000 instead of RNAiMAX. (n = 4). The upper and lower panels are images of mice cochlea at low and high magnification, respectively, detailing the efficiency of delivery and the effect on cochlear architecture and hair cell loss. (d) The scala media of P2 Atoh1-GFP mice (n = 3) were injected with 0.3 µL of 33 µM Cas9, 16.5 µM EGFP sgRNA in 50% RNAiMAX or Lipofectamine 2000 commercial solutions. Cas9-mediated gene disruption results in the loss of GFP expression when visualized 10 days later. The upper panels show GFP signal only, while lower panels include additional immunohistological markers. Yellow boxes in the lower panels highlight hair cells that have lost GFP expression. No obvious OHC loss was observed in the Cas9 + RNAiMAX or Cas9 + Lipofectamine 2000 groups. Red = tdTomato; green = Myo7a; white/light blue = Sox2; blue = DAPI. All scale bars (white) are 10 µm.

Mentions: We previously showed that (+36)GFP-Cre could be delivered to mouse retina,15 although the protein resulted in only modest levels of recombinant conversion suggestive of inefficient in vivo delivery. For our initial inner ear delivery trials, we complexed (−30)GFP-Cre with RNAiMAX and injected the complex into the cochlea of postnatal day 1 (P1) reporter mice with a genomically integrated floxed-STOP tdTomato reporter. As with our in vitro Cre reporter cell line, functional delivery of Cre to the inner ear cells, followed by endosomal escape, nuclear localization, and Cre-mediated recombination results in expression of tdTomato. After injection, the cochleas were harvested for immunolabeling with inner ear cell markers for co-localization with tdTomato. RNAiMAX injection alone was used as control. Five days following injection of (−30)GFP-Cre and RNAiMAX, cochlear outer hair cells, a type of auditory sensory cells that detect sound, showed strong tdTomato signal that co-localized with the hair cell marker myosin VIIa (Myo7a), demonstrating functional Cre delivery to hair cells (Figs. 6a–b). No tdTomato expression was detected in control cochleas (Fig. 6a). The tdTomato signal was concentrated in the region of the injection site at the basal turn of the cochlea. On average 33±3% of outer hair cells were tdTomato positive at the base of the cochlea (P < 0.001; mean ± SEM, n = 4) and intact sterocilia were observed indicative of healthy hair cells (Fig. 6b). We also tested delivery using Lipofectamine 2000 due to its higher potency in vitro (Supplementary Fig. 7a) and observed dramatically higher recombination efficiency: 91 ± 5% outer hair cells in cochleas treated with (−30)GFP-Cre + Lipofectamine 2000 were tdTomato positive (Fig. 6c). In comparison to control samples, some outer hair cell loss was observed (Fig. 6c), consistent with our previous observation of higher cell toxicity of Lipofectamine 2000, although overall cochlear architecture was preserved.


Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo.

Zuris JA, Thompson DB, Shu Y, Guilinger JP, Bessen JL, Hu JH, Maeder ML, Joung JK, Chen ZY, Liu DR - Nat. Biotechnol. (2014)

In vivo delivery of Cre recombinase and Cas9:sgRNA complexes to hair cells in the mouse inner ear. (a) The scala media (cochlear duct) of P0 floxP-tdTomato mice (n = 4) were injected with 0.3 µL of 23 µM (−30)GFP-Cre in 50% RNAiMAX or with RNAiMAX alone (control). After 5 days, tdTomato expression indicative of Cre-mediated recombination was visualized using immunohistology. Red = tdTomato; green = Myo7a; white = Sox2; blue = DAPI. Yellow brackets indicate the outer hair cell (OHC) region. (b) Ten days after (−30)GFP-Cre delivery, intact espin (Esp)-expressing stereocilia of tdTomato-positive outer hair cells were present (arrow), similar to stereocilia in control cochlea. Red = tdTomato; green = Esp; white = Sox2; blue = DAPI. (c) Identical to (a) except using Lipofectamine 2000 instead of RNAiMAX. (n = 4). The upper and lower panels are images of mice cochlea at low and high magnification, respectively, detailing the efficiency of delivery and the effect on cochlear architecture and hair cell loss. (d) The scala media of P2 Atoh1-GFP mice (n = 3) were injected with 0.3 µL of 33 µM Cas9, 16.5 µM EGFP sgRNA in 50% RNAiMAX or Lipofectamine 2000 commercial solutions. Cas9-mediated gene disruption results in the loss of GFP expression when visualized 10 days later. The upper panels show GFP signal only, while lower panels include additional immunohistological markers. Yellow boxes in the lower panels highlight hair cells that have lost GFP expression. No obvious OHC loss was observed in the Cas9 + RNAiMAX or Cas9 + Lipofectamine 2000 groups. Red = tdTomato; green = Myo7a; white/light blue = Sox2; blue = DAPI. All scale bars (white) are 10 µm.
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Figure 6: In vivo delivery of Cre recombinase and Cas9:sgRNA complexes to hair cells in the mouse inner ear. (a) The scala media (cochlear duct) of P0 floxP-tdTomato mice (n = 4) were injected with 0.3 µL of 23 µM (−30)GFP-Cre in 50% RNAiMAX or with RNAiMAX alone (control). After 5 days, tdTomato expression indicative of Cre-mediated recombination was visualized using immunohistology. Red = tdTomato; green = Myo7a; white = Sox2; blue = DAPI. Yellow brackets indicate the outer hair cell (OHC) region. (b) Ten days after (−30)GFP-Cre delivery, intact espin (Esp)-expressing stereocilia of tdTomato-positive outer hair cells were present (arrow), similar to stereocilia in control cochlea. Red = tdTomato; green = Esp; white = Sox2; blue = DAPI. (c) Identical to (a) except using Lipofectamine 2000 instead of RNAiMAX. (n = 4). The upper and lower panels are images of mice cochlea at low and high magnification, respectively, detailing the efficiency of delivery and the effect on cochlear architecture and hair cell loss. (d) The scala media of P2 Atoh1-GFP mice (n = 3) were injected with 0.3 µL of 33 µM Cas9, 16.5 µM EGFP sgRNA in 50% RNAiMAX or Lipofectamine 2000 commercial solutions. Cas9-mediated gene disruption results in the loss of GFP expression when visualized 10 days later. The upper panels show GFP signal only, while lower panels include additional immunohistological markers. Yellow boxes in the lower panels highlight hair cells that have lost GFP expression. No obvious OHC loss was observed in the Cas9 + RNAiMAX or Cas9 + Lipofectamine 2000 groups. Red = tdTomato; green = Myo7a; white/light blue = Sox2; blue = DAPI. All scale bars (white) are 10 µm.
Mentions: We previously showed that (+36)GFP-Cre could be delivered to mouse retina,15 although the protein resulted in only modest levels of recombinant conversion suggestive of inefficient in vivo delivery. For our initial inner ear delivery trials, we complexed (−30)GFP-Cre with RNAiMAX and injected the complex into the cochlea of postnatal day 1 (P1) reporter mice with a genomically integrated floxed-STOP tdTomato reporter. As with our in vitro Cre reporter cell line, functional delivery of Cre to the inner ear cells, followed by endosomal escape, nuclear localization, and Cre-mediated recombination results in expression of tdTomato. After injection, the cochleas were harvested for immunolabeling with inner ear cell markers for co-localization with tdTomato. RNAiMAX injection alone was used as control. Five days following injection of (−30)GFP-Cre and RNAiMAX, cochlear outer hair cells, a type of auditory sensory cells that detect sound, showed strong tdTomato signal that co-localized with the hair cell marker myosin VIIa (Myo7a), demonstrating functional Cre delivery to hair cells (Figs. 6a–b). No tdTomato expression was detected in control cochleas (Fig. 6a). The tdTomato signal was concentrated in the region of the injection site at the basal turn of the cochlea. On average 33±3% of outer hair cells were tdTomato positive at the base of the cochlea (P < 0.001; mean ± SEM, n = 4) and intact sterocilia were observed indicative of healthy hair cells (Fig. 6b). We also tested delivery using Lipofectamine 2000 due to its higher potency in vitro (Supplementary Fig. 7a) and observed dramatically higher recombination efficiency: 91 ± 5% outer hair cells in cochleas treated with (−30)GFP-Cre + Lipofectamine 2000 were tdTomato positive (Fig. 6c). In comparison to control samples, some outer hair cell loss was observed (Fig. 6c), consistent with our previous observation of higher cell toxicity of Lipofectamine 2000, although overall cochlear architecture was preserved.

Bottom Line: Current methods of protein delivery commonly suffer from low tolerance for serum, poor endosomal escape and limited in vivo efficacy.This approach mediates the potent delivery of nM concentrations of Cre recombinase, TALE- and Cas9-based transcription activators, and Cas9:sgRNA nuclease complexes into cultured human cells in media containing 10% serum.Delivery of unmodified Cas9:sgRNA complexes resulted in up to 80% genome modification with substantially higher specificity compared to DNA transfection.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Chemistry &Chemical Biology, Harvard University, Cambridge, Massachusetts, USA. [2] Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA.

ABSTRACT
Efficient intracellular delivery of proteins is needed to fully realize the potential of protein therapeutics. Current methods of protein delivery commonly suffer from low tolerance for serum, poor endosomal escape and limited in vivo efficacy. Here we report that common cationic lipid nucleic acid transfection reagents can potently deliver proteins that are fused to negatively supercharged proteins, that contain natural anionic domains or that natively bind to anionic nucleic acids. This approach mediates the potent delivery of nM concentrations of Cre recombinase, TALE- and Cas9-based transcription activators, and Cas9:sgRNA nuclease complexes into cultured human cells in media containing 10% serum. Delivery of unmodified Cas9:sgRNA complexes resulted in up to 80% genome modification with substantially higher specificity compared to DNA transfection. This approach also mediated efficient delivery of Cre recombinase and Cas9:sgRNA complexes into the mouse inner ear in vivo, achieving 90% Cre-mediated recombination and 20% Cas9-mediated genome modification in hair cells.

Show MeSH