Therapeutic nucleotides: targeting genes

While very few nucleotide drugs have thus far reached the market, a huge amount of research is underway. Dr Sarah Houlton reports from the recent TIDES conference on a few nucleotide technologies with great therapeutic potential

The unravelling of the human genome provided the potential for a huge number of new drug targets. While much of the talk has proved to be more hype than hope, much research is going on into nucleotide drugs for genetic targets. Very few have thus far reached the market, but many potential nucleotide-based treatments are in development.

Many exploit antisense technology, where a short fragment of single-stranded RNA, complementary to a messenger RNA (mRNA) transcribed in a cell, binds to the mRNA, thus blocking translation and, essentially, turning the gene off. The potential of this approach was first shown in the late 1970s, and the first antisense drug – Isis’s fomivirsen (Vitravene) – was licensed by the FDA in 1998 for cytomegalovirus retinitis. This remained the only approved antisense drug until the start of 2013, when the company’s mipomersen (Kynamro) got the go-ahead.

Mipomersen is a second-generation antisense oligonucleotide designed to treat the rare genetic disorder homozygous familial hypercholesterolaemia, a condition that affects only around 300 people in the US. According to Mary McGowan, who was at the time of the trials at the Concord Hospital Cholesterol Treatment Center in New Hampshire and is now a senior medical director at Genzyme (which collaborated with Isis on the project), people with the disorder can take the maximum possible dose of statins in combination with other cholesterol-lowering agents, and still have a low-density lipoprotein cholesterol level in excess of 400. This rises to 1000 or more if untreated.

Some affected children have a myocardial infarction before they are two years old; the average age of death is 33, even with LDL lowering treatment

This is in stark contrast to the normal target healthy LDL cholesterol level in the low 100s. ‘Some affected children have a myocardial infarction before they are two years old; normally untreated they will have one by the time they are 16, and be dead by 18,’ McGowan said. ‘The average age of death is 33, even with LDL lowering treatment.’

Patients typically have a mutation in both LDL receptor alleles, which leads to decreased LDL cholesterol clearance, and increased production of apolipoprotein-B, the protein associated with LDL-C. This was the target the company went after: using antisense technology to inhibit the production of this disease-associated protein by binding to specific mRNA molecules.

Mipomersen is a 20-nucleotide antisense oligonucleotide that binds to only one target, and crosses the hepatocyte cell membrane, enabling it to inhibit apo-B synthesis. Phase I trials in healthy volunteers were started within nine months of its discovery, and showed that both LDL and apo-B levels were reduced significantly in a dose-dependent manner, with a 30-day half-life and a two to three month duration of activity, and the only substantial adverse events being injection site reactions.

Phase II trials were completed in 2005, with 300 and 400mg weekly doses proving very efficacious but with higher incidence of adverse events, so 200mg weekly doses were selected for Phase III. These established its effectiveness as monotherapy, and also in combination with lipid lowering agents, typically giving LDL-C reductions of 25%. It was approved earlier this year for patients with this rare disease.

‘While statins have lowered LDL-C and improved cardiovascular outcomes, these drugs are less effective in HoFH patients,’ McGowan concluded. ‘Patients with HoFH lack functional LDL receptors, and overproduce apo-B. Mipomersen directly reduces apo-B-containing lipoprotein production, and its action is independent of the LDL receptor.’

Advancing antisense

Isis remains extremely active in the antisense area, with around 30 potential drugs in the pipeline in multiple therapeutic areas. Its Senior Vice-President for Development, Richard Geary spoke about the company’s programme to discover second generation 2’-O-methoxyethyl chimeric antisense drugs. These have 10 to 15 times higher potency than the first generation, plus a 10–20 fold increased duration of action. This allows 50–100 fold lower doses to be administered, with a concomitant reduction in side-effects.

Improved screening also enhances tolerability – for example, fewer injection site reactions

‘Improved screening gives more potent second generation antisense drugs,’ he said. ‘We have found nucleotides that are more potent than mipomersen which, at the time, was the most potent antisense compound taken forward into humans. Improved screening also enhances tolerability – for example, fewer injection site reactions.’

One of these developmental drugs, ISIS-TTRRx, is designed to treat transthyretrin amyloidosis. This is a rare and severe genetic disease, in which an inherited mutant gene produces a misfolded form of TTR. This gradually accumulates in the tissues, including the heart, peripheral nerves and gastrointestinal tract, interfering with their normal function. The condition progresses through neurodegeneration and, ultimately, death; around 10,000 people around the world are affected. Current therapy options are limited.

The drug is being developed in collaboration with GlaxoSmithKline, and is designed to prevent the production of the TTR protein, Geary says. It reduces all known mutations, preventing the accumulation of TTR in the nerves. Phase I trials in healthy subjects were completed last year, with weekly subcutaneous doses causing rapid and dose-dependent reductions in plasma TTR protein levels – more than 80% reductions being observed in many subjects. It was generally well tolerated, and caused no flu-like symptoms. A Phase II/III study in patients with familial amyloid polyneuropathy was initiated in February this year.

Another project that is advancing well is in spinal muscular atrophy. This motor neurone disease is the leading genetic cause of infant mortality, affecting at least 30,000 patients in the US alone, with one in 50 people being carriers of the survival motor neuron 1, or SMN1, gene. Carriers are asymptomatic, but if both parents are carriers there is a one-in-four chance that their child will inherit both copies, and thus the disease. The survival motor neuron protein this gene codes for is vital for the health and survival of spinal cord nerve cells.

ISI-SMNRx is being developed in conjunction with Biogen-Idec to treat all forms of childhood SMA. The antisense drug alters the splicing of a closely related gene, SMN2, increasing production of functional SMN protein. This gene normally produces only small amounts of SMN protein because of inappropriate splicing; SMNRx increases the production of SMN protein by promoting appropriate RNA processing.

After success in a mouse model, where it had a profound effect on survival and also had an acceptable half-life in the central nervous system, the drug moved into an open label Phase I trial in children with SMA aged between 2 and 14, who had sufficient SMN protein to stay alive, but not sufficient for walking. Single intrathecal doses were given, and after three months, those given the highest dose had a significant increase in muscle function. A Phase Ib/IIa multiple dose, dose-escalation study in children with SMA is now underway to establish the optimal dose for Phase III trials, and the first babies have now been dosed in a Phase II/III trial in infant onset SMA.

Studying siRNAs

Where antisense drugs are single strands of RNA, siRNA-based therapies comprise short double strands of RNA. These small interfering RNAs stop the expression of genes with the complementary nucleotide sequence, creating gene knock-downs. Delivering the siRNA to the cells of interest is a challenge and, if they get there, there is no guarantee that they will cause the desired knockdown. But if these issues can be overcome, they have potential.

Delivering the siRNA to the cells of interest is a challenge and, if they get there, there is no guarantee that they will cause the desired knockdown

Mano Manoharan, Senior Vice-President for Drug Discovery at Alnylam, spoke about his company’s conjugate programme. The idea is to modify siRNA by chemical means to confer stability and improve activity, potency and uptake. ‘Our technology works on multiple targets and therapeutic areas,’ he says. There are numerous ways modifications can be made, including sugar modifications, internal or end caps, backbone modifications, nucleotide base modifications, and creating siRNA conjugates. Alnylam has pioneered the conjugation of strands of siRNA with N-acetylgalactosamine, or GalNac, which enables the nucleotide to be delivered to hepatocytes, and facilitates subcutaneous dosing.

Manoharan described several of the company’s projects that use this technology, including one in haemophilia, the blood clotting disorder normally treated via replacement clotting factors. However, many patients with the haemophilia A form of the disease, which is caused by loss of function mutations in Factor VIII, develop an antibody to the replacement factor, becoming refractory to treatment. This could be overcome by using a siRNA to silence the gene. ALN-AT3 is in the late stages of preclinical development, and the company hopes it will move into Phase I in late 2013.

This siRNA silences antithrombin III (AT3), which inactivates both factor Xa and thrombin, two of the essential components on the coagulation cascade. AT3 is expressed in the liver, and circulates in the plasma. It has a long duration of action, he says, so subcutaneous doses of 0.5mg/kg once or twice a month should be sufficient to give 80% suppression. Trivalent GalNAc carbohydrate clusters have high affinity for the asialoglyoprotein receptor in the liver, with the GalNAc ligand conjugated to a chemically modified, AT-targeting siRNA to mediate targeted delivery. The company presented preclinical data at the Congress of the International Society on Thrombosis and Haemostasis in July, which showed that ALN-AT3 gave a normalisation of thrombin generation and improvement of haemostasis in models of haemophilia.

Mimicking miRNAs

Another form of nucleotide with therapeutic potential is microRNA, or miRNA. First discovered in the 1990s, these are naturally occurring, small – typically 22 nucleotide – non-coding pieces of RNA that regulate the genome, interacting with messenger RNAs with complementary sequences. More than 1,500 genes are known to encode for miRNAs. These are estimated to control about a third of all human genes, as each one regulates the expression of many different genes to co-ordinate cellular pathways in functions such as immune responses, cell growth and proliferation. They may, therefore, prove useful targets in autoimmune diseases and cancers.

More than 1,500 genes are known to encode for miRNAs. These are estimated to control about a third of all human genes

The first company to advance miRNA-based treatments into the clinic is Mirna Therapeutics, with an initial focus on cancer. As the company’s Director of Discovery, David Brown, explained, cancer is very heterogeneous, even within the tumour itself. ‘miR-34 is the mater-regulator of oncogenes, controlling the expression of more than 20 oncogenes,’ he said. ‘It is downregulated in most solid tumours and, apparently, is critical for the development of cancer stem cells. Our strategy is to try and replace it.’

Various cancer-important genes such as Met, Myc and Wnt are upregulated in the absence of miR-34 so, in theory, if a miR-34 mimic were introduced into the cells it should downregulate all these genes. Mirna’s miR-Rx34 does just this, and has been formulated into liposomal particles with a size of about 120nm for delivery. ‘Intravenous delivery of miR-Rx34 causes a significant increase in a variety of solid tumours and tissues in mouse models,’ Brown said. ‘A single dose reduces the expression of multiple oncogenes in the mouse cancer model. For example, it causes liver tumour regression – doses of 3mg/kg and 0.3mg/kg caused the regression of orthotopic liver cancer xenografts. Repeated dosing eliminates liver tumours without affecting normal liver morphology.’

Phase I trials are now underway, with a first-in-human dose of 0.3mg/kg, in patients with liver cancer or solid cancers with liver involvement. As miRNAs are natural molecules, the company anticipates non-specific side-effects are less likely.

Numerous other companies are working on potential miRNA treatments, including San Diego-based biotech Regulus Therapeutics, a joint venture between Isis and Alnylam. For example, RG-101 is targeting miR-122 in hepatitis C. It is a GalNAc-conjugated anti-miR targeting hepatocytes. miR-122 is the most abundant miRNA within hepatocytes, and HCV uses it as a viral replication factor, so inhibiting it may have therapeutic potential. By targeting a host factor in this way, rather than the virus itself, the company believes resistance is less likely to develop.

As the company’s Chief Scientific Officer Neil Gibson explained, RG101 is a carbohydrate conjugate that is at least 20 times more potent than the naked oligonucleotide. ‘It is rapidly cleared from the plasma, and distributes preferentially to the liver,’ he said. ‘Increased potency is achieved at a significantly lower tissue concentration of total anti-miR. The active anti-miR is released from the parent conjugate in the liver, and it is important to pay attention to the chemistry of the linker to get maximal release.’

Its pharmacological activity is sustained for more than 28 days after a single dose in mouse models, and there was a significant lowering of viral load. The company hopes to start Phase I trials of RG-101 as monotherapy in both healthy subjects and HCV patients next year; ultimately, the aim would be to develop it as a component of combination therapy.

New nucleic acid polymers

A completely different approach is being taken by Canadian biotech Replicor in its search for a treatment for hepatitis B. It has developed phosphorothioated oligonucleotide based amphipathic polymers, which operate independently of the antisense mechanism and have a broad spectrum antiviral activity against enveloped viruses. REP 9AC’ is a HBV surface antigen release inhibitor, which prevents the release of subviral particles within an infected liver cell by interfering with the biochemical processes involved in their formation.

These subviral particles sequester the antibodies the body makes against HBV, while also suppressing innate immunity, T-cell proliferation and cytokine signalling. Apolipoprotein H is involved in the formation of these subviral particles, and the nucleic acid polymers interact with apo-H, thus blocking the release of the particles. The result is a recovery in the immune system’s own ability to fight off hepatitis B infection. In a study in ducks infected with duck hepatitis B, more than half were cured by a four-week daily dosing regimen.

Initial Phase I/II trial results look promising. ‘After 15 weeks in patients, all the surface antigen is gone, and this can be maintained with chronic exposure,’ said Andrew Vaillant, Replicor’s Chief Scientific Officer. However, only about a quarter of patients had an off-treatment sustained viral response after three years. By adding a course of immunotherapy with Pegasys or Zadaxin immunotherapy after treatment with REP 9AC’ treatment, this led to a significant increase in immune function in all patients, and in eight of nine patients the viral infection remained under control 12–24 weeks after treatment ceased. ‘Immunotherapy requires cytokine signalling to achieve an immune response,’ Vaillant says. ‘All patients had a rapid increase in antibody production after treatment with immunotherapy, often similar to the strong vaccine response seen in healthy individuals,’ he said. ‘This is maintained even after the patients come off therapy.’

Nucleic acid polymers appear to effectively clear surface antigens in the blood, he concluded. ‘This clearance allows the restoration of immunological function, especially when combined with immunotherapy,’ he said. ‘I think the combination should be able to achieve permanent immunological control in some patients with chronic hepatitis B infection.’

Much of this article is based on the TIDES conference, held in Boston, MA, US in May 2013.