Enveloped viruses pseudotyped with mammalian myogenic cell fusogens target skeletal muscle for gene delivery

Abstract

Entry of enveloped viruses into cells is mediated by viral fusogenic proteins that drive membrane rearrangements needed for fusion between viral and target membranes. Skeletal muscle development also requires membrane fusion events between progenitor cells to form multinucleated myofibers. Myomaker and Myomerger are muscle-specific cell fusogens but do not structurally or functionally resemble classical viral fusogens. We asked whether the muscle fusogens could functionally substitute for viral fusogens, despite their structural distinctiveness, and fuse viruses to cells. We report that engineering of Myomaker and Myomerger on the membrane of enveloped viruses leads to specific transduction of skeletal muscle. We also demonstrate that locally and systemically injected virions pseudotyped with the muscle fusogens can deliver μDystrophin to skeletal muscle of a mouse model of Duchenne muscular dystrophy and alleviate pathology. Through harnessing the intrinsic properties of myogenic membranes, we establish a platform for delivery of therapeutic material to skeletal muscle.

Keywords: cell fusion; gene therapy; muscular dystrophy; myomaker; myomerger/myomixer; viral pseudotyping.

Figure 1. Engineering and characterization of lentiviruses pseudotyped with Myomaker and Myomerger.

(A) Schematic showing production of lentiviruses (LV) from Myomaker- and Myomerger-expressing HEK293T cells. (B) Western blot for Myomaker, Myomerger, and GAPDH on HEK293T cell lysates after various times of doxycycline treatment. (C) Western blot on concentrated viral particles for Myomaker, Myomaker, and VSV-G. (D) Western blot on Bald-LVs and Mymk+Mymg-LVs for viral components and the muscle fusogens. (E) Representative images from transmission electron microscopy on VSV-G-LVs and Mymk+Mymg-LVs. Scale bar, 100 nm. (F) Images from immunogold electron microscopy using the indicated antibodies on the pseudotyped lentiviruses. Myomaker and Myomerger antibodies were detected in the same sample using gold particles of different sizes. Scale bar, 200 nm. (G) Representative images of empty and Mymk+Mymg target cells after application of Bald GFP-encoding LVs or LVs pseudotyped with VSV-G or Myomaker and Myomerger. Nuclei were stained with Hoechst. Scale bar, 100 μm. (H) Quantification of titers for VSV-G-LVs and Mymk+Mymk-LVs prior to concentration of supernatants. Each sample is an average of 3–4 replicates from independent viral preparations. Titers were determined on Mymk+Mymg BHK21 cells. (I) Representative images from cultures of differentiated myotubes, proliferating myoblasts, and fibroblasts that were treated with Bald-LV-GFP or Mymk+Mymg-LV-GFP. Cells were stained with Phalloidin and Hoechst. Scale bar, 100 μm. GFP⁺ cells were quantified for each cell type and histograms are shown on the right. Data are presented as mean ± standard deviation. Statistical test used was an unpaired t-test with Welch’s correction; ***p<0.001, ****p<0.0001.

Figure 2. Mymk+Mymg-LVs transduce activated skeletal muscle in vivo.

(A) Lentiviruses encoding for Cre recombinase and pseudotyped with Myomaker and Myomerger (Mymk+Mymg-LV-Cre) were produced and delivered to the tibialis anterior muscles of Rosa^tdTom mice through direct intramuscular injection. Some tibialis anterior muscles were injured with cardiotoxin prior to receiving lentivirus. (B) Representative images are shown displaying tdTomato⁺ myofibers after injury and regeneration. Nuclei were stained with DAPI. The percentage of tdTomato⁺ myofibers is shown (right panel). Scale bar, 100 μm. (C) Similar setup as in (A) except muscle overload, which causes hypertrophy, was performed and lentiviruses were injected into the plantaris. (D) Representative images are shown for sham and overloaded muscles treated intramuscularly with Bald-LV-Cre or Mymk+Mymg-LV-Cre. Nuclei were stained with DAPI. Scale bar, 50 μm. Quantification of the percentage of tdTomato⁺ myofibers (bottom panel). (E) Mdx^4cv; Rosa^tdTom mice were used as recipients for Bald-LV-Cre or Mymk+Mymg-LV-Cre. Tibialis anterior muscles receiving lentiviruses through intramuscular injection were uninjured or previously injured with cardiotoxin. (F) Representative images showing tdTomato⁺ myofibers with and without cardiotoxin injury. Nuclei were stained with DAPI. Scale bar, 100 μm. Quantification of the percentage of tdTomato⁺ myofibers (right panel). (G) Schematic for experimental plan to assess transduction of Myomaker and Myomerger-pseudotyped lentiviruses packaged with μDystrophin (μDys) after intramuscular injection. (H) Immunostaining for dystrophin from tibialis anterior muscles from wild-type or mdx^4cv mice that were injected with Bald- or Myomaker and Myomerger-lentiviruses encoding μDys. Nuclei were stained with DAPI. Scale bar, 100 μm. (I) Quantification of the percentage of μDys⁺ myofibers from (H). (J) Western blot for μDys from tibialis anterior muscle lysates after injection of Bald-LV-μDys or Mymk+Mymg-LV-μDys. Data are presented as mean ± standard deviation and combined from at least two independent lentiviral preparations. Statistical tests used were (B, D, F) Two-way ANOVA with Tukey’s correction for multiple comparisons; (I) One-way ANOVA with Tukey’s correction for multiple comparisons; **p< 0.01, ****p< 0.0001.

Figure 3. Myogenic progenitors in dystrophic mice are transduced by lentiviruses pseudotyped with Myomaker and Myomerger.

(A) Top panel displays experimental design for analysis of muscle progenitors. Middle panels show representative FACS plots and quantification for α7-Integrin⁺ myogenic cells (y-axis) and tdTomato⁺ cells (x-axis) from mdx^4cv; Rosa^tdTom muscle that received Bald-LV-Cre or Mymk+Mymg-LV-Cre. Bottom panels show representative FACS plots and quantification for tdTomato⁺ non-myogenic interstitial cells. (B) Top panel is the experimental design using lentivirus encoding for luciferase that was pseudotyped with Bald, VSV-G, or Myomaker and Myomerger. After cardiotoxin injury, lentiviruses were injected intramuscularly and bioluminesence measured through IVIS imaging multiple time points after viral delivery. Representative pseudocolored images show a progressive increase in bioluminescence in the muscles treated with Myomaker and Myomerger-pseudotyped virus. Bottom panel is quantification of bioluminescence for muscles transduced with lentivirus containing Myomaker and Myomerger. (C) Mdx^4cv; Rosa^tdTom mice received an intramuscular injection of Bald-LV-Cre or Mymk+Mymg-LV-Cre, then 2 weeks later some mice were injured with cardiotoxin. Representative images for tdTomato⁺ myofibers are shown to the right. Nuclei were stained with DAPI. Scale bar, 100 μm. Bottom, left panel shows quantification of the percentage of tdTomato⁺ myofibers. Data are presented as mean ± standard deviation; data in (A, B) combined from at least two independent lentiviral preparations; data in (C) is from one lentiviral preparation. Statistical tests used were (A) unpaired t-test with Welch’s correction; (B) Friedman test with Dunn’s correction for multiple comparisons; (C) Two-way ANOVA with Tukey’s correction for multiple comparisons; **p< 0.01, ****p< 0.0001.

Figure 4. Myomaker is needed on target cells for transduction by Myomaker+Myomerger lentiviruses.

(A) Lentiviruses pseudotyped with Myomaker and Myomerger were placed on BHK21 target cells expressing one or both fusogens. Representative images for GFP⁺ cells are shown. Scale bar, 100 μm. Quantification of GFP⁺ cells, as a metric for viral transduction, is shown to the right. (B) Mdx^4cv; Mymk^loxP/loxP; Pax7^CreER mice were used to delete Myomaker in myogenic progenitors. Top panel shows schematic for Myomaker deletion and intramuscular delivery of Bald-LV-μDys or Mymk+Mymg-LV-μDys. Bottom left panel shows representative images of sections immunostained with dystrophin antibodies. Nuclei were stained with DAPI. Scale bar, 100 μm. Bottom right panel shows quantification of μDys⁺ myofibers. Data are presented as mean ± standard deviation. Statistical tests used were (A) One-way ANOVA (Kruskal-Wallis test) with Dunn’s correction for multiple comparisons; (B) Two-way ANOVA with Tukey’s correction for multiple comparisons; **p<0.01, ****p<0.0001.

Figure 5. Tropism of Myomaker+Myomerger lentiviruses is specific for skeletal muscle after systemic delivery.

(A) Schematic of experimental plan to assess transduction of lentiviruses delivered to mdx^4cv; Rosa^tdTom mice through three retro-orbital injections two weeks apart. (B) Representative images showing tdTomato⁺ myofibers from various skeletal muscle tissues after retro-orbital delivery of Cre-encoding lentiviruses pseudotyped with Bald or Myomaker and Myomerger. Nuclei were stained with DAPI. Scale bar, 100 μm. (C) Quantification of the percentage of tdTomato⁺ myofibers. (D) Representative images analyzing tdTomato⁺ cells from non-skeletal muscle tissues. No positive cells were detected. Quantification is shown on the right. Nuclei were stained with DAPI. Scale bar, 100 μm. Data are presented as mean ± standard deviation and combined from at least two independent lentiviral preparations. Statistical tests used were (C) Unpaired t-tests with Welch’s correction; *p<0.05, ****p<0.0001.

Figure 6. Lentiviruses pseudotyped with the muscle fusogens deliver therapeutic material to skeletal muscle.

(A) Design to assess systemic delivery of Mymk+Mymg-LV-μDys to mdx^4cv mice. (B) Immunostaining for dystrophin in mdx^4cv diaphragms treated with Bald-LV-μDys or Mymk+Mymg-LV-μDys. Nuclei were stained with DAPI. Scale bar, 50 μm. (C) Quantification of μDys⁺ myofibers in diaphragms. (D) Quantification of centrally nucleated myofibers in the diaphragms from wild-type mice and mice treated with Bald-LV-μDys or Mymk+Mymg-LV-μDys. (E) Masson’s trichrome staining of diaphragms treated systemically with the indicated lentiviruses. Scale bar, 100 μm. (F) Quantification of blue trichrome area from (E). (G) Schematic for long-term assessment of dystrophic muscle after treatment with Mymk+Mymg-LV-μDys. (H) Representative images from diaphragms showing Evan’s blue dye (EBD) myofibers. Scale bar, 100 μm. Quantification is shown on the right. (I) Specific force from wild-type gastrocnemius muscles, or mdx^4cv gastrocnemius muscles after systemic treatment with Bald-LV-μDys or Mymk+Mymg-LV-μDys. (J) Loss of force from gastrocnemius muscles from the indicated mice after eccentric contractions. (K) Analysis of EBD in gastrocnemius muscles at baseline or after eccentric contractions in mdx^4cv mice treated with Bald-LV-μDys or Mymk+Mymg-LV-μDys. Scale bar, 200 μm. Quantification is shown on the right. Data are presented as mean ± standard deviation and combined from at least two lentiviral preparations. Statistical tests used were (C,D,F, I) One-way ANOVA with Tukey’s correction for multiple comparisons; (H) Mann-Whitney test; (J) Repeated measures two-way ANOVA with Geisser-Greenhouse correction and Holm-Sidak’s corrections for multiple comparisons; (K) Two-way ANOVA with Tukey’s correction for multiple comparisons; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.