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Product News | Leader in RNAi Therapeutics: Alnylam Pharmaceuticals

May.05.2023

Once a Nobel Prize winner, Alnylam was once rejected by pharmaceutical companies and investors because of safety concerns. At this moment, Alnylam's lipid nanoparticle delivery system (LNP) has successfully brought RNA interference therapy from the clinic to the market, demonstrating the strength of this innovative therapy.

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RNA interference (RNAi) therapy marks milestone

RNA interference therapy extends the range of 'drug-ready' targets.

 

Modern drug discovery is 'target-based' and finding drug-ready targets is no easy task.

 

RNA interference (RNAi) was first discovered in plants in the 1990s, and RNA interference quickly became an important tool in the development of gene-targeted drugs.

 

RNAi is a gene silencing phenomenon in which small interfering RNA (siRNA) mediates the degradation of target mRNAs through the RNA-induced silencing complex (RISC). Using this mechanism, RNAi can be used to silence specific genes associated with disease.

 

The advances in siRNA design algorithms and chemical synthesis techniques, combined with the sequencing of the human genome, have opened up the prospect of siRNA therapies, which are typically double-stranded oligonucleotides consisting of 19-25 base pairs.

 

siRNAs can be precisely designed to target mRNA transcripts of most genes, or one or more genes simultaneously, greatly expanding the range of druggable targets.

 

Unlike small molecules and antibodies, siRNAs can be used to treat disease by blocking the production of disease-causing proteins, enabling intervention and control of disease at the source.

 

In 2006, two pioneers of RNAi, Andrew Fire of Stanford University School of Medicine and Craig Mello of the University of Massachusetts Medical School, were awarded the Nobel Prize in Physiology or Medicine for their discovery of RNA interference in eukaryotes.

 

 

See also: Alnylam official website

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RNAi therapeutic challenges - the problem of targeted delivery

 

The safety of siRNAs has caused a number of setbacks in clinical applications.

 

"Naked" siRNAs are highly unstable, are subject to rapid degradation in the circulation, do not accumulate easily in target tissues and do not cross target cell membranes efficiently to reach their sites of action in the cytoplasm.

 

Therefore, the development of safe and effective delivery systems is key to the implementation of siRNA technology.

 

In the process, several large pharmaceutical companies have abandoned RNAi therapies, a number of research programmes and collaborations have been terminated, and investors have lost confidence in the therapies because of the lack of promising results.

 

Alnylam has remained committed to the RNAi therapeutic field, developing two siRNA delivery technologies - a lipid nanoparticle delivery platform and an N-acetylgalactosamine-siRNA delivery platform.

 

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Alnylam-RNAi a warrior in the field of therapeutics

 

Development of a second-generation lipid nanoparticle delivery system.

 

The Lipid Nanoparticle (LNP) delivery system is one of the most effective proven delivery methods for siRNA, primarily for intravenous drug delivery.

 

The LNP delivery system consists of four main components: PEG-lipid (e.g. PEG-DMG); Ionizable amino-lipid (e.g. DLin-MC3-DMA); Distearoyl phosphatidylcholine (DSPC) and Cholesterol.

 

 

See: Chen S. Doctoral dissertation, University of British Columbia.

 

The LNP delivery system consists of four main components: PEG-lipid (e.g. PEG-DMG); Ionizable amino-lipid (e.g. DLin-MC3-DMA); Distearoyl phosphatidylcholine (DSPC) and Cholesterol.

 

The PEG on the outer layer of the LNP acts as a 'cloaking' agent to reduce the clearance of the nanoparticles by macrophages, thereby prolonging the cycle time and achieving passive targeting.

 

Ideally, ionisable aminolipids play multiple roles in drug delivery: they self-assemble with negatively charged siRNAs to form anti-micellar structures; at physiological pH, the aminolipid surface is uncharged to prolong circulation time and increase uptake by target cells; and when the LNP is endocytosed into the acidic endonucleosomes of target cells, the siRNAs escape the endonucleosomes and enter the cytoplasm to function.

 

Alnylam, in collaboration with scientists at AlCana Technologies and the University of British Columbia (UCB), identified an important relationship between the dissociation constant pKa value of ionisable lipids and LNP activity (i.e. the ability of LNP to deliver siRNA into target cells and trigger gene silencing), and conducted a systematic study.

 

They found that aminolipids with pKa values between 6.2 and 6.5 could be used to design ideal LNP carriers. One of the most efficient lipids was the DLin-MC3-DMA-based LNP, which they published in Angewandte Chemie (DOI: 10.1002/anie.201203263), prior to filing a patent application.

 

In 2010, Alnylam filed both a US patent application (Grant No. US8158601B2) and a PCT patent application (Publication No. PCT/US2010/038224) protecting DLin-MC3-DMA.

 

 

Claim 1 of US8158601B2 protects a cationic lipid or a pharmaceutically acceptable salt thereof with the following structural formula:

 

 

Next, Alnylam filed several continuation cases of US8158601B2, of which the protected subject matter of US8802644B2 relates to lipid formulations and nucleic acid-lipid particles, among others, where claim 1 protects a lipid formulation comprising: DLin-MC3-DMA, a neutral lipid, a sterol and a PEG-modified lipid; claim 33 protects a nucleic acid-lipid particle comprising a lipid formulation as protected by claim 1 and a therapeutic siRNA encapsulated therein.

 

Continuation case US9394234B2 Protects a method for the preparation of DLin-MC3-DMA.

 

PCT/US2010/038224 was entered in EP, CN, JP and other countries, of which China's granted patent CN102625696B and its sub-case CN104873464B protect the cationic lipid DLin-MC3-DMA; nucleic acid-lipid particles and a method for preparing cationic lipids, respectively.

 

The PCT/US2010/038224 patent family is expected to expire after 2030.

 

With this in mind, Alnylam has developed a second generation lipid nanoparticle delivery platform containing the ionised aminolipid DLin-MC3-DMA.

 

A distinctive feature of neutral LNPs is their ease of enrichment in hepatocytes via the Apo E mediated endocytosis pathway (so-called 'endogenous targeting'), making LNPs more suitable for the treatment of hepatocyte-associated diseases.

 

Development of ESC+ GalNAc-siRNA subcutaneous drug delivery platform.

 

Alnylam splices siRNA to N-Acetylgalactosamine (GalNAc) to form a GalNAc-siRNA splice, in which the GalNAc ligand binds to the hepatocyte-expressed Asialoglycoprotein receptor (ASGPR) and delivers the siRNA. ASGPR) and target siRNA to hepatocytes. GalNAc-siRNA can therefore be used to develop a variety of liver-targeted delivery nucleic acid drugs, and clinical studies have shown that GalNAc-siRNA is more suitable for subcutaneous delivery.

 

To improve the efficiency of gene silencing, Alnylam has also developed the ESC+ GalNAc-siRNA platform technology, using enhanced stabilization chemistry (ESC) to modify the RNA chain.

 

 

See: Alnylam official website

 

Alnylam has filed a family of patents covering GalNAc-siRNA, such as claim 1 of US8106022B2 protecting an iRNA agent.

The general formula is:

 

 

US8106022B2 defines the individual substituents and has a broad scope of protection covering GalNAc-siRNA.

In addition, Alnylam filed a PCT international application - PCT/US2012/065601 - in 2012 involving modified RNA.

 

PCT/US2012/065601 enters the US national phase of the granted patent US9796974B2 protecting a double-stranded RNAi therapeutic capable of inhibiting the expression of a target gene, comprising a sense strand and an antisense strand, each having between 14 and 30 nucleotides, and limiting the modification to a number of sites. (CN104105790A).

 

Launch of the "Alnylam 5x15" strategic plan

 

Alnylam launched the 'Alnylam 5x15' strategic programme in 2011 and has developed a number of drug candidates with the development of the second generation LNP platform and ESC+ GalNAc-siRNA platform technologies.

 

 

See also: Alnylam official website

 

According to Alnylam, all candidates are developed using ESC+ GalNAc-siRNA technology, with the exception of the approved patisiran, which is based on a second-generation LNP delivery system. It is clear that Alnylam has shifted its focus to subcutaneous delivery of GalNAc-siRNA conjugates.

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First RNA interference therapy approved

 

On 10 August 2018, the FDA approved ONPATTRO® (patisiran) lipid nanoparticle complex for the treatment of a rare disease, adult hereditary transthyretin (TTR)-mediated polyneuropathy caused by amyloidosis.

 

ONPATTRO® delivers patisiran (a double-stranded siRNA that specifically binds TTR mRNA) to the liver via intravenous infusion, causing degradation of TTR mRNA through RNA interference, thereby reducing TTR protein in serum and TTR protein deposits in tissues.

 

 

ONPATTRO® is the first siRNA drug approved by the FDA, a milestone event in the RNAi field.

 

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Delivery platform fuels adoption of RNA interference therapies

 

From 1998, when RNA interference was first observed in nematodes, to 2018, when the first RNAi drug was approved, it took 20 years for delivery platforms to evolve and move from the clinic to the marketplace.

In the future, the development of advanced RNA delivery platforms and the improvement of gene silencing efficiency will bring even greater promise for RNA interference therapies.

 

* The above text is intended to facilitate discussion and communication only and does not constitute legal advice or consultancy.

 

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