Polysarcosine-lipid liposomal formulations for mRNA delivery targeting the brain

The global situation regarding disorders of the nervous system is dire. As of 2016, neurological disorders were the leading cause of disability-adjusted life years (DALY) and the second-leading cause of death globally. The top five contributors of neurological DALYs in 2016 were stroke, migraine, dementia, meningitis, and epilepsy. RNA-therapeutics represent a promising treatment method for genetic diseases, neurodegenerative diseases, brain tumors, behavioral disorders, and stroke but for many of the most common neurological diseases few treatments have been developed, and none that take advantage of RNA-based therapies.

As of January 2022, there were only 10 FDA-approved RNA-based therapies for neurological diseases. These include therapies for cytomegalovirus retinitis, hereditary transthyretin amyloidosis, CNL Batten disease, age-related macular degeneration, and several therapies for Duchenne muscular dystrophy. So, what issues and challenges are keeping RNA-based therapeutics targeting neurological diseases out of development and the clinic?

Two of the major challenges are inherently associated with mRNA – its instability and immunogenicity. These challenges are not unique to neurological disorders but rather come with any RNA-based therapeutic no matter the target. The third challenge, one unique to treating neurological disorders, is our brain’s natural defense mechanism, the Blood Brain Barrier (BBB). The same filter used to protect the brain from toxins carried via blood also prevents many therapies from efficiently crossing into the central nervous system (CNS).

A recent publication from researchers at the Leiden Academic Centre for Drug Research in the Netherlands (Dongdong, B. et.al., 2023) addressed these challenges by administering mRNA via non-PEG liposomes directly to the CNS of embryonic zebrafish. Keep reading to see how non-PEG liposomal formulations performed compared to their traditional PEG-liposome counterparts.

The PEG-dilemma

To solve mRNA related challenges, the right delivery method must be used to encapsulate mRNA while also safely and effectively delivering it to the desired location. With the success of the mRNA-based Covid-19 vaccines, lipid nanoparticles have become the premier delivery method for RNA-based therapeutics, but other lipid-based systems such as cationic liposomes and lipoplexes are also being investigated for various applications.

Lipid-based formulations typically include a PEGylated lipid to act as a stability enhancer and to increase systemic circulation time. There are currently five FDA approved therapeutics containing PEGylated lipids. The advantageous characteristics provided by PEGylated lipids also come with at least one noticeable disadvantage – also known as the PEG-dilemma. PEGylated lipids do pose some toxicological and immunological concerns due to hypersensitivity reactions in some patients. Furthermore, repeated administration of PEG containing formulations can lead to production of anti-PEG IgM antibodies which then leads to the rapid clearance of PEG containing therapeutics, also called the accelerated blood clearance (ABC) phenomenon. Rapid clearance means that a higher therapeutic dose or repeated administration is required which can lead to further adverse effects.

Alternatives to PEGylated lipids have been investigated for several years. Some examples include polyglycerols, poly(lactic acid-co-glycolic acid) (PLGA), and polysarcosines (pSar). pSar jumps out as one alternative with great potential due to its known “stealth” properties like those of PEG conjugates. In addition, other studies have shown that pSars tend to be non-immunogenic.

In 2020, Noguera, et.al reported pSar-lipids as an alternative to PEGylated lipids in lipid nanoparticle formulations capable of robust mRNA transfection potency while also displaying an improved safety profile.

So, with all of this previous data showing the potential of pSar-lipids in lipid-based drug delivery systems, researchers at Leiden University designed a liposome using pSar-lipids to sequester and administer mRNA to the central nervous system of zebrafish embryos. Four pSar-lipids were created using polymeric average molecular weights of 2000 or 5000 and attaching lipid tails of either C14 or C18. The pSar-lipids were then used to create four liposomes and compared to liposomes prepared with DSPE-PEG2K.

There are two primary notes to make regarding this study and delivery of liposomes to the CNS: 1) this study uses embryonic zebrafish as a model and 2) the intracranial (IT) injection method used in this study bypasses the BBB.

The BBB in zebrafish

Now, you might be wondering “Why use an embryonic zebrafish as a model? Are those even similar to humans?” And the answer to that question is that embryonic zebrafish are a surprisingly good model particularly with regards to the CNS and the BBB.

Zebrafish have emerged as an ideal animal model to study complex biological processes. One major advantage is that embryonic zebrafish are optically transparent. So, tracking functional roles of cell types in vivo becomes significantly easier. They are also the smallest vertebrate species with a functioning BBB and endothelial cell-based vasculature. Analyses of vertebrates over the years have revealed the necessity for evolutionarily conserved functionality of a BBB. Humans, rodents, and zebrafish have histological and ultrastructural similarities (Eliceiri, et al, 2014). Studying drug delivery in zebrafish is not exactly the same as in humans but it can provide incredible insight at early stages of development.

IT injection as a drug administration method

There are several methods for CNS drug delivery – drug or transport modification, increase plasma drug concentration circulated to the brain, barrier bypass, and BBB disruption. IT injection falls under the barrier bypass approach and is considered an invasive technique. IT injection allows the drug to be administered directly to the cerebrospinal fluid and exploits the smaller volume of distribution compared to plasma administration (Partridge, et. al, 2022).

There are advantages and disadvantages of the IT injection approach. The primary disadvantage is associated with the invasive nature of the technique. As you could imagine, if a therapy needs repeated administration, injecting the drug into the patient’s cerebrospinal fluid poses significant concerns. The drug distribution in the parenchyma of the brain is difficult to set to the desired range. Even with these disadvantages, IT injection is a way to get around the BBB and is a network-specific treatment approach that can be utilized if necessary. Roughly 1% of a drug dose administered systemically reaches the brain so, while there is a lot of work that needs to be done to improve therapeutic delivery across the BBB, using IT injection to develop therapies targeting neurological diseases is currently very insightful (Gernert, M. and Malte, F., 2020).

Outcome of the study

Several parameters were assessed to determine how pSar-lipids affected the liposomal formulations and here were the major observations:

• pSar-lipids and PEGylated lipids affected physicochemical properties of liposomal formulations similarly.
• In vitro analysis showed promising mRNA transfection data for pSar-liposomes.
• Both pSar- and PEGylated lipids exhibited similar circulation behavior in vivo.
• pSar-liposomes are capable of releasing mRNA in embryonic zebrafish.
• C14-pSar2k liposomes led to the most robust protein expression of the liposomal formulations tested

Check out our full portfolio of pSar-lipids to use in your nucleic acid delivery research. People do amazing things with our lipids. We can’t wait to see what you will do!