2.1 Designed and constructed the mini-dystrophin coding sequences

Previous studies demonstrated that a five-repeat micro-dystrophin gene could alleviate the DMD progression and restore muscle function in the severe DBA/2J-mdx model.³² In this study, the four modified human dystrophin gene sequences contained more genetic information for better therapeutic effects. The binding of the R1–R3 domains with the sarcolemma was added to modified sequences. The muscle-specific promoter Spc-5-12 was used to drive the expression of modified sequences.^{33, 34} Figure 1 depicts the diagrams of full-length (Figure 1A) and modified dystrophin (Figure 1B–E).

This study designed coding sequences of four mini-dystrophin proteins: Spc-5R, Spc-6R, Spc-9R, and Spc-11R. The translation product of the Spc-5R sequence (Figure 1B) was the same as that of the five-repeat micro-dystrophin sequence constructed by Hakim et al.,³² whose micro-dystrophin has achieved disease rescue in severe DMD model mice. As can be observed, the Spc-6R sequence (Figure 1C) contains the R1–R3 sequences, which are directed to bind with the sarcolemma.³⁵ From this, the translation product of Spc-6R might exhibit better sarcolemma localization properties, which could transform into better therapeutic effects. The Spc-9R sequence (Figure 1D) contained the R11–R15 domains, which directly interacted with F-actin and sarcolemma.³⁵ The Spc-11R sequence (Figure 1E) contains both the R1–R3 and R11–R15 domains, which might be better at preventing mechanical force damage. Codon optimization was performed in all of the nucleotide sequences for higher expression levels in mammals. For the more complete genetic information, the modified dystrophin sequence might be slightly larger. The larger packaging ability of a gene vector such as LVs could assist in this issue. Therefore, these sequences were constructed into lentiviral plasmids. For the long-term effect and less frequent dosing, LVs were appropriately used to deliver the modified genetic sequences into muscle cells. In summary, the modified dystrophin sequences and LVs might achieve better therapeutic effects in the gene therapy of DMD.

2.2 LV-mediated modified dystrophin expression in C2C12 myoblast cells

To explore whether the LVs could transfer the modified genetic sequences into muscle cells, the levels of modified dystrophin in C2C12 muscle cells transduced by LVs were examined. The results were divided into three parts, as follows. The level of green fluorescence protein (GFP) was observed by fluorescence microscopy in C2C12 myoblast cells (Figure 2A), which were transduced by LVs (Lv-Spc-5R-eGFP). The transduction efficiency was analyzed by flow cytometry (Figure 2B). The modified dystrophin expression level of transduced C2C12 cells was detected by western blot (Figure 2C). As illustrated in Figure 2A–C, green fluorescence is presented in transduced cells in vitro. According to Figure 2B, the transduction efficiency was acceptable at 83.99%. Figure 2C displays the western blot results. It could be observed that Lv-Spc-5R/6R/9R achieved distinct modified dystrophin expression. The band for full-length Spc-11R (233 kDa) was faint. This might be associated with larger coding sequence. In conclusion, LVs could be used for gene therapy for DMD.

2.3 Modified dystrophin expression following in vivo LV treatment

We intended to explore the therapeutic effects and feasibility of LVs in DMD. In this section, the modified dystrophin expression and distribution were detected by immunofluorescence at 4, 13, and 26 weeks after treatment. The immunofluorescence images are presented in Figure 3. As could be observed in the wild-type (WT) group, the dystrophin (green) expression in muscle tissue sections was rich and distributed on the cell membrane. In the mdx group, the case was the opposite. Consistent with a previous study,³⁶ the modified dystrophin of the LV-treated groups measurably increased and was distributed on the cell membrane, which was the same as that of the WT group. The modified dystrophin could also be stably expressed for up to 26 weeks, which might reduce the frequency of administration. And the percentage of dystrophin-positive fibers was restored after treatment (Figure S1). However, the expression of modified dystrophin gradually decreased. This might be attributed to the immunogenicity of modified protein, and future work should focus on decreasing the immune response. In conclusion, these immunofluorescence data indicated that the LVs could deliver relatively large modified dystrophin coding sequences into muscle tissues in vivo and express protein products for at least 26 weeks.

2.4 Reduction in pathology following treatment

As outlined in Section 1, we assessed the pathological alleviation of mdx mice after LV treatment. In this section, we analyzed the hematoxylin and eosin (H&E) staining images of tibialis anterior (TA) muscle tissues and measured the pull force of mice at 4, 13, and 26 weeks after treatment. The H&E images of TA and the results of the histological analysis are presented in Figures 4 and 5. The muscle tissues of the WT group presented little central nucleation and low infiltration of mononuclear cells. On the contrary, the mdx group displayed typical hallmarks of DMD muscle tissues, including a high percentage of central nucleation and high infiltration of mononuclear cells. The LV-treated groups (including Lv-Spc-5R, Lv-Spc-6R, Lv-Spc-9R, and Lv-Spc-11R) exhibited a measurably lower ratio between nuclei and muscle cells (Figure 5A). In addition, there was a lower percentage of centrally nucleated muscle cells (Figure 5B). These results demonstrated that LVs dramatically alleviated inflammation and pathological symptoms in vivo. To further examine the reduction in pathology, the pull force was measured. As presented in Figure 5C, the pull force of the LV-treated groups (including Lv-Spc-5R, Lv-Spc-6R, Lv-Spc-9R, and Lv-Spc-11R) was noticeably higher than that of the untreated mdx group but still lower than that of the WT group. However, the number of nuclei in treated groups gradually increased, which was always associated with the infiltration of mononuclear cells. In addition, the restoration of pull force slightly decreased. In the future, care should be taken to achieve more stable and longer therapeutic effects. It could be inferred that the LVs carrying modified dystrophin alleviated the inflammation, decreased the central nucleation cells, increased the pull force, and reduced the pathological signs in vivo.

2.5 A preliminary evaluation of the genetic safety of LV treatment in vivo

The property of integration into the host genome conferred the ability of long-term transgene expression to LVs. It is also accompanied by the risk of insertion mutations, which might result in cancer. In this part, we aimed to study the genetic safety of LVs. The genomes of mice were sequenced and analyzed by Oxford Nanopore Technologies. The results consisted of insertion sites and structural variation. The insertion site information is listed in Table 1. We compared these sites with candidate proto-oncogenes extracted from the Cancer Gene Census³⁷ and did not find the vectors integrated into the candidate genes. Integrative analysis of structural variation (Figure S2) also indicated that the vectors in this study had no detectable influence on proliferation or differentiation in vivo. These results preliminarily demonstrated the genetic safety of LVs.

4.1 Mice

Mdx (C57BL/10ScSn-Dmd^mdx/J) mice were obtained from the Jackson Laboratory. In addition, C57BL/6JNifdc mice were purchased from Charles River Laboratories. Because DMD is an X-linked recessive genetic disease, most patients were male. Hence, only male mice were used in this study. Mice were allocated at random to experimental groups. All mice were housed in specific pathogen-free conditions at Sichuan University. Animal experiments were approved by the ethics committee of Sichuan University.

4.2 Cell lines and reagents

The HEK293T cell line was obtained from American Type Culture Collection. The C2C12 cell line was purchased and authenticated by the National Collection of Authenticated Cell Cultures. The cells of both lines were cultured in Dulbecco's modified Eagle medium (Gibco, #11995065) with antibiotics (HyClone, #SV30010) and 10% fetal bovine serum (FBS, Gibco, #10099141C). HEK293T cells (5 × 10⁵) were plated in a 100-mm-diameter cell culture dish, and the cell cultures were split (1:5) every 2 days to ensure logarithmic growth. The C2C12 cells were seeded at 5 × 10³ cells/cm² and split (1:8) at low density to maintain an undifferentiated state.

4.3 Lentiviral transfer plasmids and viral production

This experiment involved the construction of four transfer plasmids, the preparation of four types of LVs, and the determination of viral vector titration. Codon optimization was performed in all of the transfer plasmids for higher expression and therapeutic effects. All the coding sequences were driven by the muscle- and cardiac-specific synthetic promoter Spc-5-12 to target expression. The LVs corresponding to each transfer plasmid were named Lv-Spc-5R, Lv-Spc-6R, Lv-Spc-9R, and Lv-Spc-11R. The LV envelope plasmid pMD2.G (Addgene, #12259) and the packaging plasmid psPAX2 (Addgene, #12260) were commercially available. The transfer plasmids were constructed by VectorBuilder. Self-inactivating LVs were generated by transient co-transfecting HEK293T cells. In brief, HEK293T cells were split to reach a confluency of 70%−90% at the time of co-transfection and cultured in Dulbecco's modified Eagle medium (Gibco, #11995065), 1% penicillin/streptomycin (HyClone, #SV30010), and 10% FBS (Gibco, #10099141C) at 37°C and 5% CO₂. For co-transfection in each dish, 8 μg of pMD2.G, 8 μg of psPAX2, and 8 μg of transfer plasmids were mixed in 700 μL of Opti-MEM, followed by the addition of 60 μg of polyethylenimine. The transfection reagents were dispersed in HEK293T cells after 20−30 min of incubation at room temperature, and the supernatant was replaced with fresh medium at 12−18 h post-transfection. The supernatant containing LVs was collected at 48 and 72 h, followed by filtering using 0.45-μm polyethersulfone membrane filters. Viral particles were concentrated by ultracentrifugation at 20,000 rpm for 120 min, aliquoted, and stored at −80°C. The titer of vector stocks was determined by measuring viral p24 antigen using the Lenti-X p24 Rapid Titer Kit (Clontech, #632200).

4.4 In vitro viral transduction

Transduction of LVs into C2C12 myoblast cells was performed using different multiplicities of infection. C2C12 cells were seeded to reach a confluency of 70%−90% at the time of transduction and cultured in a complete medium. For transduction, concentrated LVs were dispersed in complete media with 8 μg/mL of polybrene. At 16−18 h after transduction, the supernatant was changed with a fresh complete medium, and the expression peaked at 48−72 h after transduction.

4.5 Western blot

Western blotting was used to determine modified dystrophin protein expression levels of viral vectors in C2C12 cells. This procedure involved protein extraction, bicinchoninic acid assay, and western blot. After transduction for 48 h, C2C12 cells were collected by scraping and centrifuged in microcentrifuge tubes. Total protein was extracted by radioimmunoprecipitation assay lysis buffer (Beyotime, #P0013B) for 15 min, and a phosphatase inhibitor cocktail (MedChemExpress, #HY-K0010) was added at a ratio of 1:100. The extracted protein was kept on ice during handling to inhibit protein degradation. The lysis buffer–containing protein sample was centrifuged at 10,000 × g for 5 min to pellet the cell debris. Then, the supernatant was transferred to a clean tube and stored. The protein concentrations were determined using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, #23225). The supernatant was mixed with 5× sample loading buffer (Biosharp Life Science, #BL502B) and heated at 99°C in a metal bath for 15 min in an attempt to denature proteins. Each sample was divided into 40-μL aliquots and stored at −80°C.

Equal amounts of protein lysates were loaded on a 7.5% sodium dodecyl sulfate–polyacrylamide gel and run at a constant voltage of 120 V for 1 h, followed by transfer onto a polyvinylidene fluoride membrane (Thermo Fisher Scientific, #88518) at 100 V for 2 h. Then, the membrane was blocked with 5% non-fat dry milk in Tris-buffered saline with Tween (TBST) for 3 h at room temperature. The membrane was incubated with dystrophin monoclonal antibody (Abcam, #Ab275391, 1:1000) or α-tubulin (Cell Signaling Technology, #2144, 1:1000) overnight at 4°C. The membranes were washed with TBST three times for 6 min and incubated with a secondary antibody (Bioss, #BJ08079044, 1:8000) for 1 h at room temperature. The membranes were washed with TBST three times for 20 min and incubated with horseradish peroxidase substrate (Cofitt, #LBP7518-50), and the proteins were detected using an eBlot Touch Imager.

4.6 Intramuscular injection of TA muscles

LVs were injected intramuscularly into TA muscles. Briefly, male mdx mice aged 2 days were randomly allocated into five groups (each group contained 5−15 mice) and anesthetized by hypothermia. Then, a total of 5-μL viral preparations with a titer of 4.0–13.0 × 10⁸ IFU/mL were injected into the TA muscles of 2-day-old mdx mice, and phosphate-buffered saline (PBS) was used in the mdx mice as the control group. C57BL/6JNifdc mice of the same age and sex were used as WT groups. The living conditions of the mice were monitored daily. Then, muscle tissues were harvested and recorded at 4, 13, and 26 weeks after transduction.

4.7 Immunofluorescence staining of muscle sections

All TA muscles were harvested from mdx or WT mice, embedded in O.C.T. compound (Sakura, #4583), and snap-frozen. The embedded muscle samples were sectioned at a 5-μm thickness and stored at −80°C for subsequent staining. For immunofluorescence staining, frozen sections were fixed with 4% formaldehyde (Biosharp, #BL539A) for 15 min and washed with PBS three times. The frozen sections were blocked for 1 h with 10% donkey serum (Solarbio Life Science, #SL050). Then, the sections were stained with dystrophin monoclonal antibody (Abcam, #275391, 1:200) overnight at 4°C, followed by three washes with PBS. The muscle sections were incubated with secondary antibodies (Abcam, #Ab150073, 1:100) for 1 h at room temperature, followed by three washes with PBS. The sections were then stained with 10% DAPI (4‘,6-diamidino-2-phenylindole) dihydrochloride (Beyotime, #C1005) for 15 min at room temperature. Finally, the sections were observed immediately using a fluorescence microscope (Leica CM1950).

4.8 Hematoxylin and eosin staining

H&E staining was performed according to a routine protocol. In brief, tissues were paraffin-embedded and sectioned. The sections were dewaxed and rehydrated, followed by staining with hematoxylin solution for 5 min. The sections were differentiated with 1% acid ethanol (1% HCl in 75% ethanol) for several seconds and rinsed in water. Then, the sections were incubated in eosin solution for several seconds and rinsed in water. Finally, the tissue sections were dehydrated in a graded series of alcohol, embedded in resin according to standard procedures, and observed.

4.9 Grip strength

Muscle strength was determined by a grip strength meter (Columbus Instruments, #180199). In brief, the tail of the mice was lifted at the same height as the grip strength meter grid. Then, the mice were moved horizontally and pulled away from the grid until the grasp was released. The grip strength meter automatically recorded the values of the force.

4.10 Statistical analysis

GraphPad Prism 8.4.0 software (GraphPad) was used for all data analyses. The D'Agostino–Pearson normality test was used to assess the normality of the data (n > 8). The distribution followed the normal distribution and passed the D'Agostino–Pearson normality test (α = 0.05). All values are expressed as the mean ± standard deviation. Significance was determined using an unpaired t test and one-way analysis of variance. A value of p < 0.05 was considered statistically significant. In addition, the researchers were blinded to the treatment of each sample at the time assays were performed.