PEG-variant surface modified Lipid Nanoparticles for genome editing in retina.

Can lipid nanoparticles help treat blindness?

Approximately 1 out of every 2000 individuals are affected by inherited retinal diseases globally due to mutations in nearly 300 genes. The current treatments for such diseases are dependent on adenovirus-associated virus-based CRISPR-Cas9 delivery. However, the major limitation associated with AAVs is their limited gene-carrying capacity (<4.8Kb). Furthermore, safety issues like: High AAV vector integration into CRISPR-induced DNA breaks and episome formation of the delivered DNA in retinal cells lead to constitutive activity of Cas-9 nucleases. That’s why there is a need for an efficient and safer gene delivery system for the treatment of inherited retinal diseases.

Lipid nanoparticles (LNPs) have emerged as a promising safe and efficient tool for delivering genetic material for precise genome editing in the delicate environment of the retina. The approval of Onpattro, the first FDA-approved LNP-RNAi drug for hereditary transthyretin amyloidosis, followed by the approval of the LNP-mRNA COVID-19 vaccine in 2021, has spurred progress in LNP-based mRNA delivery systems. In comparison to the virus-based gene therapies, LNPs significantly reduce the off-target activity owing to their efficient encapsulation of the payload (i,e nucleic acids), endosomal release, and cellular delivery. These LNP-based gene therapy formulations can be effectively tailored to have reduced renal clearance, and extended blood circulation while eliciting no significant immune response. Moreover, LNPs are a versatile tool for targeted delivery since they may be encapsulated with a range of payloads, including different drugs, DNA, RNA, and genetic materials.

LNPs exhibit a core-shell structure: the core contains ionizable lipids, sterols, and nucleotide payload, whereas the shell consists of helper lipid (DSPC), ionizable lipid, and PEG-lipid (DMG-PEG2k). While ionizable lipids are crucial for endosomal escape, the other components contribute to stability and cellular targeting (tropism). The cellular tropism can be further altered/enhanced by modifying LNPs with different Polyethylene-glycol (PEG) variants.

In a recent study focusing on LNPs with PEG-variant surface modifications, researchers have made significant strides in mediating genome editing within the mouse retina. The researchers developed LNPs with three different surface modifications to deliver mRNA into the retina with high cellular tropism. The LNPs were surface modified by amine-modified PEG lipids (LNPs), carboxyl, and carboxyl ester-modified PEG (LNPz & LNPx respectively), and an unmodified PEG-lipids (LNP). Moreover, the LNPx was subjected to further modification to allow encapsulation of Cas9 encoded as mRNA and the guide RNA (sgRNA) through a process called microfluidic mixing.

These components are transported to the cell's cytoplasm, where Cas9 mRNA is synthesized into a protein that binds to the guide RNA. The guide RNA-Cas9 ribonucleoprotein complex then enters the nucleus to locate the target location in the genome. This LNP-mRNA method allows for temporary CRISPR-Cas9 enzyme activity, reducing the risk of constitutive off-target activity and the likelihood of gene editing machinery integration in the genome, which are the two fundamental constraints of AAV-based gene therapy.

Figure 1: The structure of LNP

The main findings of the study revealed the potential of LNPs with PEG-variant surface alterations as an efficient, and more precise method of Cas9-based gene editing in photoreceptors cells. Furthermore, the study gives insight into the safety and efficacy of LNPs, as demonstrated by in vitro cell survival assays.

In a field where accuracy and safety are so critical, the utilization of LNPs with PEG-variant surface modifications is a big step forward in retinal therapy. LNPs, which have the ability to correct genetic abnormalities underlying hereditary retinal diseases, provide a light of hope for people suffering from vision impairment.

Reference: Gautam M, Jozic A, Su GL, Herrera-Barrera M, Curtis A, Arrizabalaga S, Tschetter W, Ryals RC, Sahay G. Lipid nanoparticles with PEG-variant surface modifications mediate genome editing in the mouse retina. Nat Commun. 2023 Oct 13;14(1):6468. doi: 10.1038/s41467-023-42189-3. PMID: 37833442; PMCID: PMC10575971.

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