Therapeutic Potential for RNA-Targeting Drugs

Drug candidates that target disease-related RNAs have potential where traditional small molecule and antibody approaches fall short.

Small molecule drug discovery is particularly challenging in the case of closely related gene families where it can be challenging to achieve sufficient selectivity to avoid undesirable off-target effects. In addition, although antibodies have demonstrated considerable potential as therapeutics, they are limited to extracellular therapeutic targets.

In contrast, oligonucleotide-based therapeutics use Watson-Crick base pairing rules to design drugs that are highly specific for disease-related mRNAs and proteins. As such, many more therapeutic targets, including intracellular proteins and members of closely-related gene families, are accessible to RNA-targeting drug candidates.

Because messenger RNA serves as the template for directing the synthesis of all proteins, modulating RNA function is a way to alter protein function. The link between RNA and protein means that, using RNA-targeting drugs, one can target RNAs as a means to modulate protein expression or even the nature of the protein.

Over the past two decades, considerable progress has been made toward the delivery of oligonucleotides to liver, and now there are multiple products that are either approved or in late-stage clinical development. However, delivery of oligonucleotide drug candidates to other cells and tissues has been limited to those tissues where drug can be administered locally, such as the eye and the central nervous system.

Avidity’s antibody oligonucleotide conjugate (AOCTM) technology utilizes antibodies to target cells and tissues of interest and facilitate the uptake and internalization of oligonucleotide payloads. By combining the cellular and tissue selectivity of antibodies with the selectivity and efficiency of oligo-based approaches, Avidity’s AOC technology has demonstrated modulation of disease-related RNAs in cell types and tissues including muscle, heart, liver, tumors and immune cells.

Advantages of Antibody-Oligonucleotide Conjugates (AOCs)

Avidity’s AOC technology exploits the specificity of antibodies for targeted delivery to cell surface receptors that are internalized along with their oligonucleotide payloads.

Once internalized, the antibody is digested, leaving the oligonucleotide free to exit the endosomal/lysosomal compartment.  Once inside the cytoplasm, depending on its structure and intended mechanism of action, the oligonucleotide payload can exert its effect either through RNA interface, exon skipping, antisense or microRNA-based approaches.

One of the key advantages of AOCs relative to unconjugated oligonucleotide therapeutics is their pharmacokinetic and biodistribution properties. Traditional, unconjugated oligonucleotide conjugates are rapidly cleared from circulation. In contrast, AOCs have enhanced pharmacokinetic and biodistribution properties similar to antibodies and therefore can dramatically improve the delivery of therapeutic oligonucleotides.

AOCs also avoid the lipid-based toxicities that have previously been observed in the context of first-generation therapeutic candidates that have utilized liposomes or other lipid-based delivery technologies.

Specificity of AOCs is achieved as a function of both the sequence of the oligonucleotide payload and the specificity of the monoclonal antibody to which the oligonucleotide payload is conjugated. The dual specificity of targeted delivery taken together with the inherent specificity of rationally-designed oligonucleotides offers unmatched precision.

As a pioneer in the field of oligonucleotide conjugates, Avidity has developed considerable intellectual property relating to antibody-oligonucleotide conjugates.

Application of AOC’s to Muscle Diseases

As one of the first therapeutic applications of its technology, Avidity is advancing a pipeline of therapeutic candidates for the treatment of serious muscle diseases including Duchenne muscular dystrophy, myotonic dystrophy and muscle atrophy.

Avidity has demonstrated both RNA interference and exon skipping in studies in vivo. As an example, shown below, Avidity has demonstrated gene specific knockdown of myostatin. Specifically, a single dose of 3 mg/kg of an siRNA targeting myostatin conjugated to an anti-transferrin receptor antibody reduced expression of the mRNA for myostatin more than 95%. Even 6 weeks after that single dose, myostatin levels in muscle remained reduced more than 50% versus controls. This reduction in myostatin mRNA levels was accompanied by reductions in circulating concentrations of myostatin protein and the phenotypic and functional changes associated with loss of myostatin: faster growth and increased strength, respectively.

Figure 1 Myostatin mRNA levels over time after a single dose of myostatin siRNA. Expression of myostatin mRNA is significantly reduced in mice after a single intravenous dose of 3 mg/kg siRNA against myostatin conjugated to a monoclonal antibody against the transferrin receptor. Controls groups consisting of the active siRNA or intravenously injected saline had little or no change in myostatin mRNA.

Therapeutic Programs

Duchenne Muscular Dystrophy

Duchenne Muscular Dystrophy is an x-linked muscular disease caused by a missense mutation in one of 79 exons in the dystrophin gene, which prevents the synthesis of a functional dystrophin protein because the mRNA for dystrophin is frame shifted. The dystrophin protein functions as a spring or shock absorber for the muscle cell membrane during contraction.

In the absence of functional dystrophin, repeated contractions of muscle lead to micro-tears in the muscle membrane and progressive loss of muscle function in all skeletal muscles as well as cardiac muscle. The affected boys lose ambulatory function in their teens from degeneration of the repeatedly damaged muscle and the exhaustion of supplies of stem cells.

Because dystrophin is spring-like with repeating ‘coil’ domains, it is possible to splice out the mutated exons using oligonucleotides that bind to the RNA transcript for dystrophin and force the RNA processing apparatus to skip over the exon with the missense mutation and begin reading the RNA transcript downstream in-frame. Exon skipping results in a shortened but functional dystrophin protein being synthesized. This approach has now been clinically validated.

Avidity has demonstrated that an AOC containing an exon skipping oligonucleotide demonstrates ~100-fold greater activity relative to the same oligonucleotide without targeted delivery.

Treatment of the mdx mouse (mouse model of muscular dystrophy) with a single dose of 10 mg/kg of the appropriate splice skipping oligonucleotide conjugated to an anti-transferrin receptor antibody produced a 10% increase in dystrophin concentration relative to control mice.

In contrast, a dose of 10 mg/kg of the unconjugated oligonucleotide produced no detectable dystrophin. Published data suggest that alternate day dosing of 100 mg/kg of the unconjugated oligonucleotide for 21 days (~1000 mg/kg total dose) would be required to equal what the Avidity conjugated oligonucleotide produced after a single dose.

Avidity is focused on developing AOCs to treat DMD patients with mutations in exons 44 and 45 of the dystrophin gene. Lead sequences have been identified and are currently undergoing optimization. In addition, we are advancing a human anti-transferrin receptor antibody, and we have begun bioconjugation CMC activities to enable clinical applications.

Myotonic Dystrophy Type 1 (DM1)

Myotonic Dystrophy Type 1 is a disease induced by a toxic RNA species that results from large numbers of CTG repeats in the gene for the protein DMPK. Although the RNA itself is non-toxic, the expansion of CTGs in the gene (and CUGs in the RNA) creates a high-affinity sponge for an important protein MBNL.

As a result of the presence of this high affinity attractor for MBNL, MBNL is sequestered and cannot perform its normal function, which is to assist with the processing of the mRNAs for a number of important proteins.

Ultimately it is the malfunctioning of these downstream RNAs and their cognate proteins that gives rise to the disease. For example, causes of the pathophysiology of the disease include reduced expression of a chloride channel protein (ClC1), which is associated with myotonia, and a mis-splicing of the insulin receptor, which correlates well with insulin resistance in the affected population.

If the expression of the toxic DMPK RNA can be reduced effectively, then the RNA processing effects on the downstream RNAs will be reversed and the disease symptoms can be managed. With Avidity’s AOC technology, it is possible to deliver siRNA effectively to muscle. Avidity has identified highly potent siRNA that reduce the expression of the target gene, DMPK.