Challenges at the Crossroads: Myopathy Trials in 2020 Hindsight

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The Crossroads

Reports of clinical studies in gene therapy for two myopathies, Duchenne muscular dystrophy (DMD) and X-linked myotubular myopathy (XLMTM), have revealed tantalizing glimpses of efficacy, but the news has been partially offset by emerging safety concerns.^1,2 Although preclinical studies in disease models informed the design of these early-stage clinical trials, the emerging spectrum of safety issues urgently needs alternative models to enable mechanistic dissection and expedite development of improved gene therapies with much higher therapeutic indices. The etiologies of the vector-related serious adverse events (SAEs) are unclear, involve multiple organ systems, and are not necessarily identical across diseases and vectors. None were predicted by studies in existing pre-clinical myopathy models. Here we address a mix of common themes and disease-specific considerations, and attempt to identify opportunities for research to contribute evidence-based rationale to the choices at the crossroads.

Safety First

Similarities and distinctions between DMD and XLMTM, the vectors used, and the trial designs may be critical to the interpretation of the reported SAEs. For both diseases, respiratory insufficiency is a leading cause of death, but only in XLMTM is mechanical respiratory support often required at birth. DMD is typically asymptomatic in infancy, with an average age at diagnosis of 4 years, even later for the milder allelic variant of dystrophinopathy, Becker MD (BMD). DMD and BMD affect both skeletal and cardiac myocytes, while cardiomyopathy is not a recognized feature of XLMTM. Only XLMTM has an associated liver disease that, in severe cases, can autonomously progress to lethal “peliosis hepatis”. The phase 1 XLMTM ASPIRO trial showed impressive improvement in respiratory function as a primary endpoint in the initial 1.0 × 10¹⁴ adeno-associated virus (AAV) vg/kg patient group, partially de-risking the trial and incentivizing parents to consider enrollment of young XLMTM boys at the higher dose of 3.0 × 10¹⁴. However, three deaths occurred in the high-dose group months after vector administration, with a shared scenario of progressive liver dysfunction and intracellular cholestasis.² The available reports outline clinical evolution from liver insufficiency to lethal “sepsis,” raising concern for a delayed onset, treatment-related cytokine storm.

In the DMD trials, the SAEs included subacute renal failure in the setting of thrombocytopenia, hemolytic anemia, and complement activation resembling thrombotic microangiopathy (TMA), with or without subacute cardiopulmonary insufficiency. Thus, the pathomechanisms may be distinct, as are both the diseases and vectors used (XLMTM-AAV8, DMD-AAV9). However, both capsids are highly hepatotropic, and delayed but severe liver dysfunction has also been reported in post-approval studies of Zolgensma, the AAV9-based gene therapy for spinal muscular atrophy (SMA).³ In the search for plausible, unifying pathomechanisms to connect these disparate SAEs, we note a recent report of concurrent TMA and hemophagocytic lymphohistiocytosis (HLH) in a large series of patients with varying degrees of liver, renal, and respiratory failure associated with co-activation of interferon and complement pathways.⁴ Severe multi-organ failure was responsive to simultaneous inhibition of both pathways with Emapalumab and Eculizumab. In a potentially relevant mechanistic dissection, sequential activation of Toll-like receptor (TLR)9 and TLR4 has recently provided the first model of the fulminant hyperinflammatory syndrome of HLH.⁵ AAV vectors have the potential for activation of TLR9 by genomic CpG, while TLR4 activation could be driven by HMGB1 from hepatocytes sustaining dose-dependent direct toxicity during the intracellular trafficking of strongly hepatotropic vectors such as AAV8, AAV9, and AAV74.

Risk-Benefit and Equipoise in Dose Escalation Trials

Efficacy must offset risk in individual pediatric research subjects, at least as reasonably extrapolated from preclinical studies. In life-long diseases such as DMD, durability is a requirement of effective gene therapy, at least until the safety of re-administration has been established. The longevity of systemic AAV gene therapy for myopathies therefore depends on “anti-dilution” strategies. Efficient transduction of myonuclei is required to enable adequate transgene expression to accommodate the expanding nuclear domains of transduced cardiac and skeletal myonuclei throughout growth to skeletal maturity. Transgene products must be sufficiently myoprotective by both design and level of expression to minimize necrotic turnover of transduced post-mitotic myonuclei. Capsids derived from the natural serotypes AAV8, AAV9, and AAVrh74 have been shown to support systemic gene transfer for this remarkable task, but only at the extraordinarily high doses required for the vectors to cross the continuous endothelium of muscle. These AAVs were initially recovered from liver, a tissue perfused by fenestrated endothelia. While the inherent hepatotropism of these AAVs prompted their initial commercial development, it now probably contributes strongly to their potential dose-limiting toxicity. Although the cell surface receptors mediating endocytosis of these capsids are not fully defined, none identified to date are muscle specific. Rationally designed or unbiased modifications in capsid structure may enhance myotropism, minimize off-target transduction, or both. This might shift the balance in favor of benefit over risk in future trials with the caveat that off-target effects are to be expected if modifications increase affinity for receptors also expressed in non-muscle tissues. Nonetheless, capsid engineering represents a key opportunity for improvement in the therapeutic index by selectively augmenting muscle transduction, lowering the total dose requirement, and minimizing dose-limiting off-target toxicity.

Strong muscle-specific promoters

Strong muscle-specific promoters have the potential to further minimize the systemic vector dose required to achieve a given level of transgene expression in myopathies while downregulating transgene expression within specialized antigen presenting cells (APCs). Because muscle naturally harbors some of the most abundant proteins in the body (myosin, actin, etc.), the transcriptional networks that drive the robust expression of these genes may be co-opted to induce transgene expression with similar strength and muscle specificity. Use of such promoters to limit robust transgene expression to myonuclei might minimize toxic expression in off-target cell types collaterally transduced by AAV. While muscle-specific promoters may limit transgene expression in APCs, and thereby reduce the immunogenicity of the therapeutic transgene product, muscle-resident APCs remain capable of presenting any exogenous muscle antigens that are released from necrotic muscle fibers. Therefore, if the therapeutic efficacy of a muscle-specific gene therapy is insufficient to prevent ongoing myonecrosis, the exogenous transgene product will still be abundantly presented on muscle-resident APCs to drive cellular immunity against therapeutic proteins not protected by central tolerance.

Ameliorating Dystrophinopathy

The molecular pathogenesis of BMD is relevant to any discussion of efficacy and safety of DMD gene therapy. The majority of mutations that cause DMD and BMD are large genomic deletions or duplications in the dystrophin gene, but the nuance is the integrity of the reading frame—preserved in BMD but destroyed in DMD such that dystrophin epitopes may never be presented in the thymus. The spectrum of BMD includes cases almost as severe as DMD, even when associated with relatively small deletions in the repetitive rod domain, raising questions about the function of the spectrin-like triple helices that constitute the majority of the cytoskeletal protein. The challenge for DMD gene therapy is the design of transgenes encoding maximally functional surrogate proteins in which ∼80% of the triple helices are deleted, much smaller than dystrophins in even severe BMD patients. Recent insights into the molecular evolution of dystrophin and its paralog dystrophin-related protein or utrophin suggest alternative designs that might simultaneously optimize function and safety. This includes compelling evidence from the study of adaptive immunity in a deletional canine model for DMD in which recombinant utrophin was protected by central immunological tolerance while myocytes expressing recombinant dystrophin were targeted as non-self and destroyed by effector T cells.⁶ Future studies should help resolve gaps in our understanding of the biomechanics of dystrophin and utrophin to enable the design of improved transgenes to confer more durable functional rescue in DMD. Analogous approaches may facilitate progress in XLMTM, where many mutations undermine the development of central tolerance for myotubularin but do not affect the paralogous products of the MTMR2–14 genes.

Synergy

For patients, the most direct stakeholders affected by this therapeutic development campaign, the central quandary is whether to seek immediate benefit with the first-generation vectors, despite the acknowledged risk of SAEs, or wait for further improvements at the risk of irreversible muscle loss in the interim. We can anticipate future improvements in vector capsid, transcriptional control, and transgene design, with potentially vast and synergistic enhancements in biodistribution and functional rescue to widen the safety margin. Admittedly, the historical development of therapies for many severe diseases justified narrow safety margins early on (e.g. King Claudius’ paraphrase of an already 2,000-year-old Hippocratic proclamation: “diseases desperate grown, by desperate appliance are relieved, or not at all” Hamlet, Act 4, Scene 3, Shakespeare). In 2020 AD, there are many more layers of regulation in place to minimize the risk to vulnerable research subjects, especially if mitigating technologies are close at hand.