Duchenne Muscular Dystrophy Gene Therapy in 2023: Status,Perspective,and Beyond

In 1868, French physician Guillaume-Benjamin-Amand Duchenne reported 13 boys who lost the ability to walk due to a muscle-wasting disease. ¹ These boys showed progressive weakness in the lower limbs, slowly spreading to the upper limbs and respiratory muscles. Affected boys also had paradoxical hypertrophy of calf muscle, wide-based gait, and scoliosis in the advanced stage of the disease, and patients died before reaching their 20th birthday. Duchenne named the disease pseudohypertrophic muscular dystrophy. Today, the disease is known as Duchenne muscular dystrophy (DMD) (Fig. 1). ^2,3

OPEN IN VIEWER

Figure 1.

Development of dystrophin restoration gene therapy for Duchenne muscular dystrophy. (A) Affected boys are usually diagnosed between 2 and 5 years of age and lose ambulance in their early teens. The photographs show a patient at the early stage of the disease (left) and when wheelchair bound (right). Courtesy of Jen Portnoy at Hope for Javier. (B) Milestones in discovering the disease, the disease gene, and the genetic mechanism underlying the disease. (C) Milestones and major challenges in developing exon-skipping, microdystrophin, and CRISPR editing therapies.

In 1987, the Kunkel laboratory at Boston Children's Hospital discovered the DMD gene and its protein product dystrophin. ^4,5 In subsequent years, the Kunkel laboratory proposed the reading-frame theory. ^6,7 In-frame deletions of the DMD gene result in Becker muscular dystrophy, a mild form of myopathy with some patients ambulant till their 70s. Conversely, mutations that disrupt the open reading frame result in premature translation termination and clinically more severe DMD. These seminal findings raise the hope of treating DMD by dystrophin restoration. Unfortunately, early attempts with myoblast transfer and injection of the naked plasmid vector were unsuccessful. ^8,9

DMD is one of the most challenging diseases for gene therapy. Unlike diseases that affect a single organ (such as the retina), effective therapy for DMD requires systemic delivery to all the muscles (including the heart) throughout the body. Furthermore, gene therapy is complicated by muscle degeneration, inflammation, and fibrosis. Nevertheless, significant progress has been achieved in the past three decades. Of notice are exon skipping, microdystrophin, and CRISPR editing (Fig. 1).

Exon skipping aims to restore the reading frame by skipping out-of-frame exon(s) in the dystrophin transcript using antisense oligonucleotides (ASOs). Microdystrophins are highly abbreviated, partially functional, synthetic quasi-dystrophins. Microgenes carry about one-third of the dystrophin coding sequence, and they are delivered using adeno-associated viral (AAV) vectors. CRISPR editing restores the open reading frame by deleting or modifying frame-disrupting exon(s) in the genome using Cas endonuclease and guide RNA. CRISPR editing also has the potential to fill in the missing exon(s) to restore full-length dystrophin expression.

Exon skipping is the first to commercialize. Four exon-skipping ASOs (eteplirsen, casimersen, golodirsen, and viltolarsen) have been approved by regulatory agencies to treat patients who have mutations eligible for exon 45, 51, or 53 skippings. However, only minimal amount of dystrophin (≤5%) was detected in treated patients. The weekly dosing frequency is another nuisance. Furthermore, the current single exon-skipping strategy is highly mutation specific and unsuitable for a larger patient population. In this issue, Dr. Aartsma-Rus, a leader in exon skipping, discussed the future of exon-skipping therapy for DMD. ¹⁰ Novel strategies were elaborated for achieving persistent and more efficient dystrophin restoration in more patients (such as creative ASO modifications, viral vectorized ASOs, and multiexon skipping).

On January 4, 2018, Sarepta dosed the first patient for systemic AAV microdystrophin gene therapy (NCT03375164). On March 14 and March 22, 2018, Solid Biosciences (NCT03368742) and Pfizer (NCT03362502) dosed the first patient in its trial, respectively. Two more companies (Genethon and Regenxbio) recently initiated systemic AAV microdystrophin trials. Hundreds of patients have been dosed with AAV microdystrophin vectors at 1 × 10¹³ vg/kg or higher, with most ≥1 × 10¹⁴ vg/kg. Different AAV serotypes, different microdystrophin constructs, different muscle-specific promoters, and different AAV manufacturing methods were used in these trials. ¹¹

Despite the promising results, ^12,13 multiple clinical holds occurred at different stages in different trials, and one trial was suspended due to a serious adverse event. Reasons for the holds include complement activation-induced thrombotic microangiopathy causing renal dysfunction, AAV vector manufacture-related issues, and patient death. ^14,15 The cytotoxic T lymphocyte (CTL) immune response may play a crucial role in serious adverse events such as liver toxicity triggered by the AAV capsids-specific CTL responses; myositis and myocarditis caused by the CTL response to the new epitope in microdystrophin.

Besides innate and cellular responses, the humoral immune response represents another significant barrier to AAV-mediated DMD gene therapy. A large proportion of patients are excluded due to pre-existing antibodies. Antibodies induced by AAV administration prevent redosing. Several articles in this issue addressed the immune hurdle.

Kumar et al. reviewed immune responses observed in muscle-directed AAV gene therapy trials and discussed strategies for managing immune toxicity. ¹⁵ Verma et al. and Anthony et al. reported findings on the seroprevalence of AAV-binding/neutralizing antibodies and the pre-existing dystrophin-specific T cell response, respectively, in DMD patients. ^16,17 Neutralizing antibodies were detected in ∼30–50% of patients. ¹⁶ Binding antibodies were found in nearly all patients. ¹⁶ Pre-existing dystrophin-specific T cell responses were observed in ∼10–30% of patients. ^17,18

Owing to the AAV packaging capacity limitation, only selected elements of dystrophin are included in microdystrophin. It is yet unclear whether the function of the current microdystrophins has reached the limit. Recent studies, including the one by Wasala et al. (this issue), have dived deep into the microdystrophin structure in the hope of developing more potent next-generation constructs to enhance muscle and heart protection and minimize dystrophin-specific T cell responses. ^{19 –21}

Five years have passed since the start of systemic AAV microdystrophin trials. Long-term data suggest durable motor function benefits. ^12,13 Consistently, Wasala et al. (this issue) showed lifelong motor and cardiac function improvements after single intravenous dosing in young mdx mice, a mouse model for DMD. ²² However, clinical disease is mild in mdx mice. ²³ Furthermore, mice have a much lower body weight (body size) and a much shorter life span than humans. It is yet to be determined how long will the therapeutic effect last in DMD patients. ²⁴

The U.S. Food and Drug Administration is reviewing a microdystrophin drug developed by Sarepta for accelerated approval. The advisory committee met on May 12, 2023, and the committee voted 8-6 in favor of approving the treatment. Traditionally, the approval of a new drug requires a demonstration of clinical benefits in a phase III trial. The accelerated approval pathway was established by the U.S. Food and Drug Administration in 2012 to fill unmet medical needs for serious diseases. This pathway allows the use of a surrogate endpoint. In this issue, Chamberlain et al. provided a strong argument supporting microdystrophin expression as the surrogate endpoint. ¹¹

AAV is currently the only vector that can efficiently deliver a therapeutic gene to all muscles in the body. ²⁵ There are hundreds of naturally existing and laboratory-engineered AAV variants. ²⁶ AAV8, AAV9, and AAVrh74 are used in systemic gene therapy trials to treat DMD and other neuromuscular diseases. A major drawback of these AAV vectors is the dependence on the high dose (≥1 × 10¹⁴ vg/kg). Infusion of trillions of AAV particles has caused fatalities and other severe adverse events in patients with DMD, myotubular myopathy, and spinal muscular dystrophy. ^15,27,28

There is an urgent need for myotropic super AAV variants. The Grimm laboratory has a long-standing interest in AAV capsid engineering. ²⁹ In this issue, Dr. Grimm and colleagues provided a comprehensive and critical review of the ever-expanding and improving portfolio of techniques for developing next-generation muscle gene therapy AAV vectors. ³⁰ In preclinical studies, the newly developed AAVMYO and MyoAAV variants showed superior muscle performance and liver detargeting over the existing AAV capsids in rodents and nonhuman primates. ^{31 –33}

The combined use of top–down methods (such as structure-guided rational design) and bottom–up approaches (such as machine learning) is expected to yield more potent and less immunogenic AAV capsids. Nevertheless, cross-species translation to diseased humans remains a major challenge. ³⁰

An exciting development in the past decade is the CRISPR editing technology. Numerous laboratories have applied this revolutionary strategy to treat DMD and other neuromuscular diseases (such as spinal muscular atrophy, myotonic dystrophy, congenital muscular dystrophy, and facioscapulohumeral muscular dystrophy). In this issue, two excellent review articles by Chemello et al. and Fatehi et al. discussed the latest developments and future directions in this ever-evolving field. ^34,35 Many innovative approaches have been investigated using Cas9, dead Cas9, base-editor, or prime editor to correct mutations (deletion, duplication, point mutation, and insertion) to restore the expression of the therapeutic (even full-length) proteins; or to modulate transcription of therapy-relevant proteins without correcting the mutations.

Success has been achieved in mouse and large animal models and patient cells. The first-in-human CRISPR therapy for neuromuscular diseases was conducted on a late-stage DMD patient in 2022, though the patient died after therapy. ³⁶ Delivery and safety are two major hurdles limiting the translation of CRISPR editing to clinical benefits. AAV is the most used delivery platform in animal studies. Other vehicles (such as lipid nanoparticles, extracellular vesicles, and virus-like particles) are actively pursued, but their delivery efficiency still needs to improve for human translation.

Immunotoxicity of the AAV vector is discussed by Kumar et al. in this issue. ¹⁵ Pre-existing and induced immune responses to Cas9 are another major obstacle. ^37,38 The risk of off-target editing and undesirable on-target genome modifications remains a safety concern. Muscle stem cell editing has been demonstrated in mouse models. ^39,40 However, the muscle-specific promoters are less active in muscle stem cells. ^40,41 The development of novel satellite cell-targeting muscle-specific promoters should address this issue.

The articles collected in this issue have focused on exon skipping, microdystrophin, and CRISPR editing. However, dystrophin-independent gene therapies are also being actively pursued. Of notice are strategies aimed at overexpressing compensatory proteins (such as microutrophin) or targeting disease mechanisms (such as cytosolic calcium overload). ^{42 –45} These emerging approaches may have immunological advantages since they target proteins already in DMD patients. Transgene product-specific immune responses will be less of a concern.

Optogenetics is a powerful technique to control cellular activity with light. It is widely used in neuroscience as a basic research tool and a potential therapy for diseases such as retinitis pigmentosa. In this issue, Cohen et al. discussed muscle optogenetics, a relatively new field. ⁴⁶ AAV has been used to deliver light-sensitive proteins (such as channelrhodopsin-2) to cardiac, skeletal, and smooth muscles. Animal studies have revealed the massive potential of using muscle optogenetics to regulate cardiac rhythm or to trigger skeletal muscle and smooth muscle contraction.

There are >1,173 inherited neuromuscular diseases in humans. ⁴⁷ At least 658 genes have been linked to these disorders. Progress in DMD gene therapy will pave the way for treating these diseases with gene therapy. The newly developed muscle optogenetics will further expand the portfolio of muscle gene therapy.