ASOs, DMD and CPPs

A bit of background research on antisense oligonucleotides, Duchenne muscular dystrophy and where cell-penetrating peptides fit in, for anyone interested...

Last week Phylogica revealed that it has chosen antisense oligonucleotides (ASOs) as its flagship program and is currently working on the question of what will be the target tissue/s for the different ASOs that form part of its program. The company intends to provide therapeutic read outs for multiple cargoes in multiple tissues during 2019.

ASOs were first discovered to be able to alter RNA and reduce, restore, or modify protein expression over two decades ago. However, challenges faced by this class of drug, including inadequate target engagement, insufficient biological activity, and off-target toxic effects, has meant that only a few ASO therapies have successfully made it through to market. It is considered that further advancement of ASO therapies will require optimization of ASO delivery, target engagement, and safety profile.

Intracellular delivery is recognized as the major barrier to effective ASO activity within target cells. The trafficking of ASOs from endosomes into the cytoplasm is increasingly being recognized as an important rate-limiting step for ASO therapeutics. Consequently, interventions that enhance endosome escape have the potential to improve ASO therapeutic activity in the clinic.

Neuromuscular diseases have been at the forefront of ASO drug development, with two ASO-mediated splice-switching therapies for SMA and DMD having gained FDA approval in recent years. Eteplirsen (Exondys 51, Sarepta) was approved by the FDA in September 2016 for patients with Duchenne muscular dystrophy (DMD) gene mutation amenable to exon 51 skipping, and the ASO nusinersen (Spinraza, Biogen), for the treatment of Spinal muscular atrophy (SMA) was approved a few months later. Spinraza has quickly become a $1 bn+ drug for Biogen.

In its work to date with ASOs, Phylogica has validated the ability of a proprietary CPP to deliver an ASO in an in vivo DMD disease model. The Phylomer CPP, 1746c27, was conjugated to the PMO, M23D (+7–18), which targets exon 23 of the dystrophin gene transcript. As described in the paper “A platform for discovery of functional cell-penetrating peptides for efficient multi-cargo intracellular delivery”, which was published in Nature - Scientific Reports in August

The Phylomer CPP 1746c27-delivered PMO cargo in vivo, and induced production of functional dystrophin in distal muscles. While the major impact of the absence of dystrophin is on muscle (striated, smooth and cardiac muscles), various dystrophin isoforms are expressed in many tissues. Therefore, global distribution of the cargo as shown here is preferable. The low-level expression of dystrophin in the heart muscle is also encouraging, as cardiac muscle has proved refractory to uptake of nucleic acid analogue therapeutics. As dystrophin levels in excess of 3–5% of normal are expected to confer substantial therapeutic benefit in DMD, this study provides strong evidence for the power of Phylomers to deliver high-potency therapeutics in vivo.

Duchenne muscular dystrophy (DMD) is a fatal genetic disorder diagnosed in childhood and affecting mainly boys. It occurs in approximately 1 in every 3,500 live male births (about 20,000 new cases each year).  DMD is characterized by an absence of dystrophin (a protein that helps maintain muscle cell structure) caused by a mutation in the gene that encodes for dystrophin. There are several mutations of the dystrophin gene that can lead to DMD. Without dystrophin, muscles progressively weaken, leading to serious medical problems. During late adolescence, serious heart and respiratory conditions become more prevalent (shortness of breath, fluid in the lungs, swelling in the feet and lower legs, enlarged heart), typically resulting in death before the age of 30. Death is typically caused by heart failure but, unfortunately, current antisense chemistries, including Sarepta’s approved Exondys 51, have very low tissue penetration into the heart.

According to the FDA in its review of eteplirsen, major challenges for ASOs in the treatment of DMD include

• difficulty in delivering sufficient amounts of ASOs to tissues,
• the high variability in dystrophin expression both between muscles and within the same muscle following treatment,
• expression is undetectable or low in cardiac muscle compared to skeletal muscle, and
• the difficulty in identifying a minimum amount of dystrophin expression anticipated to have a functional effect.

Sarepta’s earlier ASO therapies, including eteplirsen, were based on phosphorodiamidate morpholino oligomer (PMO) chemistries. More recently, the company has initiated a program in which the PMOs are conjugated to cell-penetrating peptides (PPMOs) to improve delivery. PPMOs tested to date in preclinical models have resulted in higher levels of exon skipping and expression of dystrophin protein in muscle.

Just months prior to eteplirsen’s approval by the FDA, Biomarin was unsuccessful in gaining approval for its ASO exon 51 skipping drug, drisapersen (Kyndrisa). Drisapersen was one of four exon-skipping ASOs (the others targeting 44, 45 and 53) that Biomarin purchased from the Dutch company, Prosensa for up to $840 m in 2014.

The FDA rejected drisapersen due partly to safety concerns, but primarily because clinical trials of the drug had not effectively demonstrated a clear increase in levels of dystrophin. In June 2016, Biomarin announced that it was discontinuing further clinical and regulatory development of drisapersen and its other pipeline ASO drugs.

A year later, following an ongoing patent dispute between Biomarin and Sarepta related to the use of exon-skipping ASOs in the treatment of DMD, Sarepta agreed to pay BioMarin a one-time $35 million payment, plus regulatory and commercial milestone payments and royalties for exons 51, 45, 53, and possibly on future exon-skipping products. Sarepta currently has therapies targeting exons 45 and 53 in Phase 3 development. Under the settlement terms, BioMarin retains rights to convert that license to a co-exclusive right should it decide to proceed with its own exon-skipping therapy for DMD (its ASO therapies targeted exons 51, 45, 53 and 44).

Although exon-skipping ASOs have disappeared from Biomarin’s pipeline on its company website, its scientists, together with researchers from Leiden University, authored a paper (submitted December 2016 and published in Molecular Therapy in January 2018), titled “Cyclic Peptides to Improve Delivery and Exon Skipping Antisense Oligonucleotides in a Mouse Model for Duchenne Muscular Dystrophy”. The authors describe previous mouse model experiments aimed at improving ASO delivery, including use of the TAT-peptide. In seeking an alternative approach the researchers used phage display screening of a cyclic peptide library to identify candidate muscle-homing peptides suitable for conjugation. Peptides for the experiment were sourced from New England Biolabs. The researchers then conjugated the lead peptide to an ASO for their DMD mouse model experiment, achieving a 2-fold increase in delivery and exon skipping in all analyzed skeletal and cardiac muscle. While selected as a muscle-homing peptide, uptake was also increased in the liver and kidney. The compound was observed to be well tolerated. The authors concluded that the identified peptide had the potential to facilitate delivery of AONs (ASOs) and perhaps other compounds to skeletal and cardiac muscle.

Biomarin was revealed as one of a number of “target customers” for Phylogica at last year’s AGM. It will be interesting to see if the therapeutic read outs from Phylogica’s flagship program of CPP ASO conjugates in DMD will spark interest from Biomarin in resurrecting its ASO in DMD program.